WO2020072857A1 - Methods and compositions related to plod3 - Google Patents

Abstract

A solution to the problem of HNSCC identification and treatment are provided, embodiments described provide methods for identifying or treating HNSCC. In particular aspects, overexpression of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLODS) in HNSCC has been discovered and inhibition or down regulation of PLODS can treat HNSCC. In particular, inhibitory nucleic acids, such as siRNAs, are identified for down regulating PLODS expression.

Description

DESCRIPTION

METHODS AND COMPOSITIONS RELATED TO PLOD3

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 62/741,081, filed October 4, 2018, the contents of which is incorporated into the present application by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH [0002] None.

REFERENCE TO SEQUENCE LISTING

[0003] A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference.

BACKGROUND

[0004] Head and neck cancer (HNC) is the sixth most common cancer worldwide, accounting for approximately 630,000 new cases diagnosed and 350,000 cancer deaths every year¹. Head and neck squamous cell carcinoma (HNSCC) is by far the most common type of HNC². It encompasses epithelial malignancies that arise in the mucosal linings of the upper airway and food passages (paranasal sinuses, nasal cavity, oral cavity, pharynx, and larynx)^3,4. The incidence rates of HNSCC increase with age. Males generally have a higher incidence rate than females, with the ratio ranges from 2: 1 to 4: l⁵. Wide geographical variation exists in incidence around the world. High risk region of HNSCC includes Indian subcontinent, Australia, France, Brazil, and Southern Africa⁶. The decline in tobacco product use has contributed to the decreasing incidence of oral squamous cell carcinoma and laryngeal SCC in the developed countries⁷. However, in developed world there is a huge surge in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) which is caused by human papilloma virus (HPV) infection^{8 10}. HNSCC incidence rate is even various in the different ethnic groups in a specific geographical region. For instance, increased incidence of HNSCC was reported in black as compared to white male Americans. In addition, higher percentage of advanced staged, poor prognosis HNSCC was observed in black patients as compared to white patients ^11,12. [0005] Early detection of HNSCC is extremely important for improving the prognosis¹⁷. However, currently there is no proven screening method. A thorough history inquiry and combination of inspection, palpation, indirect mirror examination, or direct flexible laryngoscopy contribute to the initial assessment of the primary tumor. Computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PCT) and integrated PCT/CT are valuable for assessing the degree of local infiltration, involvement of regional lymph nodes, and presence of distant metastases or second primary malignancies. Confirmed diagnosis of HNSCC is based on histology of the biopsy from the primary site. However, suspicious lesions are often relatively inaccessible for histological assessment. Fine needle aspiration (FNA) biopsy might be performed instead. FNA biopsy is also often used for an initial tissue diagnosis of HNSCC when a patient presents with a neck mass without an obvious primary tumor.

[0006] Accurate staging is an important guidance for therapeutic decision making. The tumor, node, metastases (TNM) staging system of the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) is widely used to classify HNSCC¹⁸.“T” describes the extent of primary tumor (T),“N” refers to absence or presence and extent of metastatic regional lymph node(s), and“M” indicates the absence or presence of distant metastasis. One significant shortcoming of TNM system is that it does not include pathological or biological parameters. Therefore, molecular staging has become a relatively new concept in the past decade. Combination of TNM staging system and molecular staging might be helpful to guide precise medicine in the coming future.

[0007] Surgery is the major treatment option for primary and recurrent HNSCC. In most cases, various treatment methodologies include surgery, radiation, and chemotherapy are combined for the management of HNSCC⁴⁰. For early stage HNSCC, the patients are usually treated with surgery and radiotherapy. For the advanced stage HNSCC, chemotherapy is commonly required. Cisplatin remains the cornerstone for treating recurrent and metastatic HNSCC. Administration of cisplatin or other chemotherapeutic drugs such as 5-fluorouracil (5-FU) can significantly enhance the efficiency of radiotherapy⁴¹. Cetuximab (EGFR monoclonal antibody) in combination with platinum/5-FU has become a new alternative regimen for platinum refractory HNSCC⁴².

[0008] There remains a need for additional methods of identifying and/or treating HNSCC. SUMMARY

[0009] In providing a solution to the problem of identification and treatment of cancer, in particular cancers overexpressing PLOD3,, embodiments described herein provide additional methods for identifying or treating these PLOD3 overexpressing cancers. In particular aspects the inventors have identified the overexpression of procollagen-lysine, 2-oxoglutarate 5- dioxygenase 3 (PLOD3) in a variety of cancers, including HNSCC, and have discovered that the inhibition or down regulation of PLOD3 can treat cancer, e.g., HNSCC. In particular, inhibitory nucleic acids, such as siRNAs, were identified for down regulating PLOD3 expression.

[0010] Certain embodiments are directed to an inhibitory nucleic acid targeting a procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) mRNA comprising a 10, 11, 12,

13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37

38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, to 50 nucleotides corresponding to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, to 50 consecutive nucleotides from SEQ ID NO: l . In certain aspects the inhibitory nucleic acid is a small interfering RNA (siRNA). The siRNA can include, consist of, or consist essentially of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, to 25 consecutive nucleotides of SEQ ID NO: l, or may contain 1, 2, 3, or 4 nucleotide variants as compared to SEQ ID NO: l or a corresponding PLOD3 variant. In a particular aspect the inhibitory nucleic acid, e.g., siRNA, targets nucleotides 720 to 760 or to nucleotides 800 to 840 of human PLOD3 mRNA (SEQ ID NO: l). In a further aspect, the inhibitory nucleic acid can include, consist of, or consist essentially of the sequence AGAAGGAAAUGGAGAAAUA (SEQ ID NO:3) and/or its complement, and CCACAGAGCUGCUGAAGAA (SEQ ID NO:4) and/or its complement, and may include 1, 2, 3, or 4 nucleotide variants of the sequences. The inhibitory nucleic acid can further comprise one or more modifications. The one or more modifications can be a modification of a base moiety, a sugar moiety, a phosphate moiety, a phosphate-sugar backbone, or a combination thereof. In certain embodiment the inhibitory nucleic acid is an inhibitory RNA. In certain aspects, the inhibitory RNA is a siRNA, shRNA, or miRNA.

[0011] A small interfering RNA (siRNA) is an RNA molecule that decreases or interferes with or silences (prevents) the expression of a gene/mRNA of its endogenous cellular counterpart. The term is understood to encompass “RNA interference” (RNAi). RNA interference (RNAi) refers to the process of sequence-specific post transcriptional gene silencing in mammals mediated by small interfering RNAs (siRNAs). The RNA interference response may feature an endonuclease complex containing an siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA may take place in the middle of the region complementary to the antisense strand of the siRNA duplex.

[0012] A short hairpin RNA (shRNA) is a nucleic acid molecule comprising at least two complementary portions hybridized or capable of hybridizing to form a duplex structure sufficiently long to mediate RNAi (typically between 15-29 nucleotides in length), and at least one single-stranded portion, typically between approximately 1 and 10 nucleotides in length that forms a loop connecting the ends of the two sequences that form the duplex. The structure may further comprise an overhang. The duplex formed by hybridization of self-complementary portions of the shRNA has similar properties to those of siRNAs and, as described below, shRNAs are processed into siRNAs by the conserved cellular RNAi machinery. Thus shRNAs are precursors of siRNAs and are similarly capable of inhibiting expression of a target transcript. As is the case for siRNA, an shRNA includes a portion that hybridizes with a target nucleic acid, e.g., an mRNA transcript and is usually the perfectly complementary to the target over about 15-29 nucleotides, typically between 17-21 nucleotides, e.g., 19 nucleotides. However, one of ordinary skill in the art will appreciate that one or more mismatches or unpaired nucleotides may be present in a duplex formed between the shRNA strand and the target transcript.

[0013] Other embodiments are directed to a composition comprising an inhibitory nucleic acid described herein and a carrier and/or excipient. In certain aspects carrier is a liposomal carrier or a nanoparticle carrier.

[0014] Certain embodiments are directed to an expressing cassette encoding an inhibitory nucleic acid as described herein. The expression cassette can be further comprised in an expression vector or other delivery vehicle.

[0015] Other embodiments are directed to methods of reducing PLOD3 expression in a target cell by administering an inhibitory nucleic acid described herein. In certain aspects the target cell is a cancer cell overexpressing PLOD3. In a further aspect the cancer cell is a squamous cell carcinoma. The squamous cell carcinoma can be a head and neck squamous cell carcinoma. In other aspects the cancer cell is an acute myeloid leukemia (AML), bladder urothelial carcinoma (BLCA), breast cancer (BRCA), colon adenocarcinoma (COAD), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), glioblastoma (GBM), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), stomach and esophageal carcinoma (STES), or thyroid carcinoma (THCA).

[0016] In certain embodiments, the methods of treating can include the administration of an anti-PLOD3 antibody that inhibits the activity of PLOD3. Such an antibody can be administered in combination with the inhibitory nucleic acids described herein. In certain aspects, the anti-PLOD3 antibody can be a polyclonal antibody, e.g., PLOD3 Polyclonal ANTIBODY, Proteintech, Rosemont, IL.

[0017] Certain embodiments are directed to methods of treating a PLOD3 overexpressing cancer comprising administering an inhibitory nucleic acid as described herein. In certain aspects the method further comprises administering a miR-l24-3p nucleic acid or a miR-l24- 3p mimic. The miR-l24-3p nucleic acid or mimic can comprise the nucleic acid sequence UAAGGCACGCGGUGAAUGCC (SEQ ID NO:6) or a 1, 2, 3, or 4 nucleotide variant thereof. The methods can further comprise administering a second cancer therapy. The second cancer therapy can be surgery, chemotherapy, radiotherapy, or immunotherapy. In certain aspects the second cancer therapy comprises administering cisplatin and/or 5-fluorouracil (5-FU), and/or cetuximab or other anti -epidermal growth factor antibody. The subject being treated can have or suspected of having a squamous cell carcinoma. In certain aspects the squamous cell carcinoma is a head and neck squamous cell carcinoma. In other aspects, the subject can have acute myeloid leukemia (AML), bladder urothelial carcinoma (BLCA), breast cancer (BRCA), colon adenocarcinoma (COAD), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), glioblastoma (GBM), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), stomach and esophageal carcinoma (STES), or thyroid carcinoma (THCA) that over express PLOD3. The inhibitory nucleic acid can be incorporated into a liposomal carrier or a nanoparticle carrier.

[0018] Certain embodiments are directed to treating cancer comprising administering a cancer therapy to a subj ect having a cancer overexpressing PLOD3. Overexpression of PLOD3 can be determined by measuring PLOD3 protein levels in a sample from a subject. The measuring and/or detecting of PLOD3 overexpression can be used to identify a cancerous cell and idnentify a subject as a candidate for early treatment of a cancer.

[0019] Other embodiments are directed to methods for early detection, diagnosis, prognosis, or assessment of a subjected having or suspected of having, or at risk of having squamous cell carcinoma comprising measuring the level of PLOD3 in a sample from the subject comprising suspected cancer cells and determining the expression level of PLOD3 protein.

[0020] An inhibitory PLOD3 nucleic acid can target a PLOD3 mRNA or PLOD3 mRNA segment having at least, or about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 consecutive nucleotides of SEQ ID NO: l, the mRNA segments including a segment starting at nucleotide 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,

143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,

162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,

181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,

200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,

219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,

238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,

257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,

276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,

295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,

3214, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997,

998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012,

1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107, 2108, 2109, 2110, 2111, 2112, 2113, 2114, 2115, 2116, 2117, 2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127, 2128, 2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140, 2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164, 2165, 2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175, 2176, 2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188, 2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200, 2201, 2202, 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213, 2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225, 2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236, 2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320, 2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333, 2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345, 2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356, 2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368, 2369, 2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380, 2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404, 2405, 2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 2667, 2668, 2669, 2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680, 2681, 2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690, 2691, 2692, 2693, 2694, 2695, 2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704, 2705, 2706, 2707, 2708, 2709, 2710, 2711, 2712, 2713, 2714, 2715, 2716, 2717, 2718, 2719, 2720, 2721, 2722, 2723, 2724, 2725, 2726, 2727, 2728, 2729, 2730, 2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740, 2741, 2742, 2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752, 2753, 2754, 2755, 2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764, 2765, 2766, 2767, 2768, 2769, 2770, 2771, 2772, 2773, 2774, 2775, 2776, 2777, 2778, 2779, 2780, 2781, 2782, 2783, 2784, 2785, 2786, 2787, 2788, 2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800, 2801, 2802, 2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812, 2813, 2814, 2815, 2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824, 2825, 2826, 2827, 2828, 2829, 2830, 2831, 2832, 2833, 2834, 2835, 2836, 2837, 2838, 2839, 2840, 2841, 2842, 2843, 2844, 2845, 2846, 2847, 2848, 2849, 2850, 2851, 2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860, 2861, 2862, 2863, 2864, 2865, 2866, 2867, 2868, 2869, 2870, 2871, 2872, 2873, 2874, 2875, 2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884, 2885, 2886, 2887, 2888, 2889, 2890, 2891, 2892, 2893, 2894, 2895, 2896, 2897, 2898, 2899, 2900, 2901, 2902, 2903, 2904, 2905, 2906, 2907, 2908, 2909, 2910, 2911, 2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919, 2920, 2921, 2922, 2923, 2924, 2925, 2926, 2927, 2928, 2929, 2930, 2931, 2932, 2933, 2934, 2935, 2936, 2937, 2938, 2939, 2940, 2941, 2942, 2943, 2944, 2945, 2946, 2947, 2948, 2949, 2950, 2951, 2952, 2953, 2954, 2955, 2956, 2957, 2958, 2959, 2960, 2961, 2962, 2963, 2964, 2965, or 2966, and ending at nucleotide 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,

46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,

71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 3214, 315 , 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761,

762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780,

781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799,

800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,

819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837,

838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856,

857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875,

876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894,

895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913,

914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932,

933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951,

952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970,

971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989,

990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021,

1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036,

1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051,

1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066,

1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081,

1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096,

1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111,

1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126,

1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141,

1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156,

1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171,

1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186,

1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201,

1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216,

1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231,

1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246,

1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261,

1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276,

1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291,

1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107, 2108, 2109, 2110, 2111, 2112, 2113, 2114, 2115, 2116, 2117, 2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127, 2128, 2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140, 2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164, 2165, 2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175, 2176, 2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188, 2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200, 2201, 2202, 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213, 2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225, 2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236, 2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320, 2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333, 2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345, 2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356, 2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368, 2369, 2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380, 2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404, 2405, 2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 2667, 2668, 2669, 2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680, 2681, 2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690, 2691, 2692, 2693, 2694, 2695, 2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704, 2705, 2706, 2707, 2708, 2709, 2710, 2711, 2712, 2713, 2714, 2715, 2716, 2717, 2718, 2719, 2720, 2721, 2722, 2723, 2724, 2725, 2726, 2727, 2728, 2729, 2730, 2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740, 2741, 2742, 2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752, 2753, 2754, 2755, 2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764, 2765, 2766, 2767, 2768, 2769, 2770, 2771, 2772, 2773, 2774, 2775, 2776, 2777, 2778, 2779, 2780, 2781, 2782, 2783, 2784, 2785, 2786, 2787, 2788, 2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800, 2801, 2802, 2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812, 2813, 2814, 2815, 2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824, 2825, 2826, 2827, 2828, 2829, 2830, 2831, 2832, 2833, 2834, 2835, 2836, 2837, 2838, 2839, 2840, 2841, 2842, 2843, 2844, 2845, 2846, 2847, 2848, 2849, 2850, 2851,

2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860, 2861, 2862, 2863, 2864, 2865, 2866,

2867, 2868, 2869, 2870, 2871, 2872, 2873, 2874, 2875, 2876, 2877, 2878, 2879, 2880, 2881,

2882, 2883, 2884, 2885, 2886, 2887, 2888, 2889, 2890, 2891, 2892, 2893, 2894, 2895, 2896,

2897, 2898, 2899, 2900, 2901, 2902, 2903, 2904, 2905, 2906, 2907, 2908, 2909, 2910, 2911,

2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919, 2920, 2921, 2922, 2923, 2924, 2925, 2926,

2927, 2928, 2929, 2930, 2931, 2932, 2933, 2934, 2935, 2936, 2937, 2938, 2939, 2940, 2941,

2942, 2943, 2944, 2945, 2946, 2947, 2948, 2949, 2950, 2951, 2952, 2953, 2954, 2955, 2956,

2957, 2958, 2959, 2960, 2961, 2962, 2963, 2964, 2965, 2966, 2967, 2968, 2969, 2970, 2970,

2971, 2972, 2973, 2974, 2975, or 2976, including all values and ranges there between. The inhibitory nucleic acid can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 variations in nucleotides, as compared to SEQ ID NO: 1, at any position along its length as long as it maintains its inhibitory effect.

[0021] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0022] The use of the word“a” or“an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.”

[0023] Throughout this application, the term“about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0024] The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.”

[0025] As used in this specification and claim(s), the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as “have” and“has”),“including” (and any form of including, such as“includes” and“include”) or“containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0026] As used herein, the terms“comprises,”“comprising,”“includes,”“including,”“has,” “having,”“contains”,“containing,”“characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and/or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and/or method.

[0027] As used herein, the transitional phrases“consists of’ and“consisting of’ exclude any element, step, or component not specified. For example,“consists of’ or“consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase“consists of’ or“consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase“consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0028] The feature or features of one embodiment may be applied to other embodiments, even though not described or illustrated, unless expressly prohibited by this disclosure or the nature of the embodiments.

[0029] Any method or system of the present invention can consist of or consist essentially of— rather than comprise/include/contain/have— any of the described elements and/or features and/or steps. Thus, in any of the claims, the term“consisting of’ or“consisting essentially of’ can be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb. A composition“consisting essentially of’ the recited elements excludes any further active ingredients but does not exclude pharmaceutical excipients, buffers, structural components, etc.

[0030] Other obj ects, features and advantages of certain embodiments will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0032] FIG. 1 - PLOD3 was increased in HNSCC tissues based on GEO data and TCGA. The levels of PLOD3 were higher in HNSCC tissues compared with the normal tissues in various independent studies (GSE6631, GSE37991, GSE23558, GSE25099, GSE30784 and TCGA). In addition, PLOD3 levels were higher in HNSCC tissues compared to oral precancerous lesions (GSE85514 and GSE30784). HNSCC patients with higher PLOD3 suffered worse long term overall survival. [0033] FIG. 2 - PLOD3 was increased in various types of cancers. Based on the data available from TCGA database, it was found that the expression level of PLOD3 was significantly increased in cancer tissues including acute myeloid leukemia (AML), bladder urothelial carcinoma (BLCA), breast cancer (BRCA), colon adenocarcinoma (COAD), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), glioblastoma(GBM), kidney chromophobe (KICH), kidney renal clear cell carcinoma(KIRC) (FIG. 2A), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma(LIHC), lung adenocarcinoma (LET AD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), stomach and esophageal carcinoma (STES) and thyroid carcinoma (THCA) (FIG. 2B). [0034] FIG. 3 - PLOD3 upregulation were associated with unfavorable overall survival in various types of cancers. Based on the data available from TCGA database, higher PLOD3 expression was associated with poorer long-term overall survival rates in many other types of cancers including GBM, KIRC, LIHC, LUSC, low grade glioma (LGG), LUAD, ovarian cancer (OV), cervical squamous cell carcinoma (CESC), THCA and sarcoma (SARC). [0035] FIG. 4 - The expression level of PLOD3 in HNSCC cell lines and tissues. The expression levels of PLOD3 were significantly higher in all the HNSCC cell lines compared to the normal cells (FIG. 4A-4B). In addition, the expression levels of PLOD3 were upregulated in HNSCC tissues in comparison with the matched adjacent normal tissues (FIG. 4C-4D).

[0036] FIG. 5 - Immunohistochemical analysis of PLOD3 staining intensity in HNSCC. The results showed the expression of PLOD3 to increase gradually with the progression of carcinogenesis from normal epithelium, well differentiated HNSCC, moderately differentiated HNSCC to poorly differentiated HNSCC (FIG. 5A-5B).

[0037] FIG. 6 - Downregulation of PLOD3 suppressed the malignant phenotypes of HNSCC cells in vitro. The expression levels of PLOD3 mRNA and proteins were significantly downregulated following siPLOD3 (siRNAl and siRNA2) transfection (FIG. 6A-6C). Downregulation of PLOD3 inhibited the proliferation, migration and invasion capacity of HNSCC cells in vitro (FIG. 6D-6I).

[0038] FIG. 7 - Downregulation of PLOD3 suppressed tumor growth of HNSCC cells in vivo. The tumor volume and weight were both significantly lower in HNSCC cells with PLOD3 downregulation

[0039] FIG. 8 - Upregulation of PLOD3 promoted the malignant phenotypes of HNSCC cells in vitro. The expression levels of PLOD3 mRNA and proteins were significantly upregulated following PLOD3 overexpressing lentiviruses infection (FIG. 8A-8B). Downregulation of PLOD3 inhibited the proliferation, migration and invasion capacity of HNSCC cells in vitro (FIG 8C-8H).

[0040] FIG. 9 - Ectopic expression of PLOD3 promoted tumor growth of HNSCC cells in vivo. The tumor volume and weight were both significantly increased in HNSCC cells with PLOD3 overexpression.

[0041] FIG. 10 - MiR-l24-3p directly targeted PLOD3 by binding to 3'UTR region. PLOD3 3'UTR (SEQ ID NO: 5) was highly complementary to the seed sequence of miR-l24- 3p (SEQ ID NO:6) (FIG. 10A). In addition, the 3'UTR PLOD3 sequence that miR-l24-3p targeted was highly conserved across different species (FIG. 10B). Luciferase assays showed that the miR-l24-3p mimic significantly suppressed relative luciferase activity compared to the scramble control (FIG. 10C). MiR-l24-3p mimic remarkably reduced PLOD3 expression at the protein level (FIG. 10D-10E). ACCUGCCUGCCAUUGUGCCUUU (SEQ ID NO:5); UAAGGCACGCGGUGAAUGCC (SEQ ID NO:6); AUUGUGCCUUUUUAGG (SEQ ID NO: 7); ACUGUGCCUUGUUGGA (SEQ ID NO: 8); ACUGUGCCUUGUUGGG (SEQ ID NO: 9); AUGGUGCCUUCUCAGG (SEQ ID NO: 10); AUGGUGCCUU (SEQ ID NO: 11); AUCGUGCCUU (SEQ ID NO: 12); GCUGUGCCUU (SEQ ID NO: 13).

[0042] FIG. 11 - Eipregulation of miR-l24-3p suppressed the malignant phenotypes of HNSCC cells in vitro. The expression level of miR-l24-3p was significantly increased following miR-l24-3p mimic transfection (FIG. 11A). Eipregulation of miR-l24-3p inhibited the proliferation, migration and invasion capacity of HNSCC cells in vitro (FIG. 11B-11G). [0043] FIG. 12 - Downregulation of miR-l24-3p promoted the malignant phenotypes of

HNSCC cells in vitro. The expression level of miR-l24-3p was significantly decreased following miR-l24-3p inhibitor transfection (FIG. 12A). Downregulation of miR-l24-3p promoted the proliferation, migration and invasion capacity of HNSCC cells in vitro (FIG. 12B-12G). [0044] FIG. 13 - PLOD3 overexpression partially rescued the tumor suppressive effects of miR-l24-3p. WB results showed that the levels of PLOD3 were higher in HNSCC cells infected with PLOD3 overexpressing lentiviruses following miR-l24-3p mimic transfection (FIG. 13A). PLOD3 overexpression could partially increase HNSCC cell proliferation, migration and invasion capacity which were inhibited by miR-l24-3p upregulation (FIG. 13B- 13G).

[0045] FIG. 14 - RNA-seq analysis the DEGs following PLOD3 downregulation. Compared to the controls, a total of 3275 (1631 upregulated and 1644 downregulated) and 3146 (1497 upregulated and 1649 downregulated) DEGs were found siPLOD3 -treated SCC1 and SCC23 cells respectively (FIG. 14A). Heat map was used to visualize the distribution of DEGs following PLOD3 inhibition (FIG. 14B). In addition, most DEGs were overlapped between SCC1 and SCC23 (FIG. 14C).

[0046] FIG. 15 - PLOD3 inhibition significantly affected the morphology of HNSCC cells. The morphology of cancer cells became round and the cells were not able to reattach to the plates following PLOD3 downregulation. [0047] FIG. 16 - PLOD3 was an upstream modulator of FAK-PI3K-AKT signaling pathway. The expression levels of pFAK, pPI3K and pAKT was significantly downregulated following PLOD3 downregulation, while their levels were upregulated when PLOD3 was overexpressed.

[0048] FIG. 17 - The tumor promoting role of PLOD3 was mediated by phosphorylation of FAK in HNSCC cells. The expression levels of FAK and pFAK were both significantly reduced following siFAK transfection (FIG. 18A). FAK and pFAK downregulation could significantly inhibite HNSCC cell proliferation, migration and invasion capacity which were promoted by PLOD3 overexpression (FIG. 18B-18G).

DETAILED DESCRIPTION OF THE INVENTION

[0049] The following discussion is directed to various embodiments of the invention. The term“invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be exemplary of that embodiment, and not intended to intimate that the scope of the disclosure, including the claims, is limited to that embodiment.

[0050] Metabolic remodeling is now widely regarded as a hallmark of cancer. However, the role of metabolic enzymes in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC) is poorly known. The inventors have identified many novel differentially expressed genes/metabolic enzymes and metabolites between HNSCC and adjacent normal tissues by using transcriptomic and metabolomic analyses. In particular, PLOD3 was found to be significantly upregulated in HNSCC tumor tissues and cell lines. Patients with higher PLOD3 expression suffered poorer tumor differentiation and worse long term overall survival. This observation was also found in many other types of human cancers. PLOD3 downregulation inhibited cancer cell proliferation, migration and invasion in vitro and tumor growth in vivo, and ectopic expression of PLOD3 potentiated (promoted) cancer cell proliferation, migration and invasion in vitro and tumor growth in vivo.

[0051] In addition, the potential molecular mechanisms accounting for the pro-carcinogenic role of PLOD3 in HNSCC were explored. MiR-l24-3p was demonstrated to be an upstream regulator of PLOD3. Overexpression of miR-l24-3p suppressed the proliferation, migration and invasion capacity of HNSCC cells, whereas underexpression of miR-l24-3p led to the opposite results. More importantly, PLOD3 overexpression partially rescued the tumor suppressive effect of miR-l24-3p, indicating that miR-l24-3p was a functional modulator of PLOD3.

[0052] RNA sequencing (RNA-Seq) was performed to screen the potential downstream effectors of PLOD3. Functional analysis revealed the differentially expressed genes were enriched in actin cytoskeleton regulation and focal adhesion. Furthermore, it was observed that PLOD3 downregulation changed the morphology of cancer cells, which failed to attach the surface of the petri dish. Therefore, it was hypothesized that focal adhesion kinase (FAR) might be a potential downstream effector of PLOD3. PLOD3 downregulation suppressed the phosphorylation of FAR, PI3K and ART, and vice versa. Furthermore, FAR inhibition significantly suppressed the tumor-promoting role of PLOD3 in HNSCC. In conclusion, the results have revealed a miR-l24-3p-PLOD3-FAK/PI3K/ART regulatory axis that contributes to the tumorigenesis of HNSCC and PLOD3 might represent a therapeutic target for HNSCC treatment.

I. PLOD3 Polypeptide/Polynucleotide

[0053] Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) is a member of the 2- oxoglutarate-dependent dioxygenase family and is an enzyme that modifies lysyl residues in collagens (lysyl hydroxylase (LH)). PLOD3 is an isoenzyme encoded by encoded by the PLOD3 gene. In addition to lysyl hydroxylase activity, PLOD3 also has collagen galactosyltransferase (GT; EC 2.4.1.50) and glucosyltransferase (GGT; EC 2.4.1.66) activity (Salo et ak, Am J. Hum. Genet. 83 :495-503, 2008).

[0054] Location and structure o/PLOD3. Currently three members have been discovered in procollagen-lysine, 2-oxoglutarate 5-di oxygenase (PLOD) family, namely PLOD1, PLOD2 and PLOD3¹¹⁶. The gene encoding the PLOD3 is located in human 7q22 chromosome, which is distinct from the locations of PLOD1 (lp36) and PLOD2 (3q24)^{117 118}. The different location of the three PLODs isoforms indicate they are different metabolic enzymes and might not be able to compensate for one another. All three PLOD isoforms have similar structure of 19 exons. The introns of PLOD 3 contains many contain many Alu repeats, but it is markedly shorter than PLOD1 and PLOD2, indicating that PLOD1 and PLOD2 may have lengthened introns during vertebrate evolution¹¹⁹. Human PLOD3 has 738 amino acids and its molecular weight is 85 kDa. The amino acid sequence of three PLODs are similar and all contain a signal sequence at the amino-terminus. There are about 60% similarity for the amino acid sequence between PLOD1 and PLOD3 or PLOD2 and PLOD3 ¹²⁰.

[0055] The PLOD3 protein can be encoded by an mRNA having a nucleotide sequence of

TAGGCTTCCTCCCTCCCTGTCGCCGCCAGCCTCTCGCTCTCCCCGTCTTCCGCCCG

CACTCTCTGGGCAGGGCTGCGGCCAGGACCCGCCCCCGCGTCCCGCCCGACCCTC

CGCCAGGGGTCACTTCCCCTGTCCAGGTTTCAGCTTCCACATGTGTCAAGCGGCT

GGCTCAGCCCAGAGTCCCTGTCTCCCGCCCGCCGGCCCGAGCCGCCGCCCCTCCC

CCGCCTCCCGTGCGCCCGGGACAATCCTCGCCTTGTCTGTGGCGCCGGCATCTGG

AGCTTTCTGTAGCCTCCGGATACGCCTTTTTTTCAGGGCGTAGCCCCAGCCAAGC

TGCTCCCCGCGGCGGCCGCACAGCAGCCCGAGCGCCCCCTTTCCAGAGCTCCCCT

CCGGAGCTGGGATCCAGGCGCGTAGCGGAGATCCCAGGATCCTGGGTGCTGTCT

GGGCCCGCTCCCCACCATGACCTCCTCGGGGCCTGGACCCCGGTTCCTGCTGCTG

CTGCCGCTGCTGCTGCCCCCTGCGGCCTCAGCCTCCGACCGGCCCCGGGGCCGAG

ACCCGGTCAACCCAGAGAAGCTGCTGGTGATCACTGTGGCCACAGCTGAAACCG

AGGGGTACCTGCGTTTCCTGCGCTCTGCGGAGTTCTTCAACTACACTGTGCGGAC

CCTGGGCCTGGGAGAGGAGTGGCGAGGGGGTGATGTGGCTCGAACAGTTGGTGG

AGGACAGAAGGTCCGGTGGTTAAAGAAGGAAATGGAGAAATACGCTGACCGGG

AGGATATGATCATCATGTTTGTGGATAGCTACGACGTGATTCTGGCCGGCAGCCC

CACAGAGCTGCTGAAGAAGTTCGTCCAGAGTGGCAGCCGCCTGCTCTTCTCTGCA

GAGAGCTTCTGCTGGCCCGAGTGGGGGCTGGCGGAGCAGTACCCTGAGGTGGGC

ACGGGGAAGCGCTTCCTCAATTCTGGTGGATTCATCGGTTTTGCCACCACCATCC

ACCAAATCGTGCGCCAGTGGAAGTACAAGGATGATGACGACGACCAGCTGTTCT

ACACACGGCTCTACCTGGACCCAGGACTGAGGGAGAAACTCAGCCTTAATCTGG

ATCATAAGTCTCGGATCTTTCAGAACCTCAACGGGGCTTTAGATGAAGTGGTTTT

AAAGTTTGATCGGAACCGTGTGCGTATCCGGAACGTGGCCTACGACACGCTCCCC

ATTGTGGTCCATGGAAACGGTCCCACTAAGCTGCAGCTCAACTACCTGGGAAACT

ACGTCCCCAATGGCTGGACTCCTGAGGGAGGCTGTGGCTTCTGCAACCAGGACC

GGAGGACACTCCCGGGGGGGCAGCCTCCCCCCCGGGTGTTTCTGGCCGTGTTTGT

GGAACAGCCTACTCCGTTTCTGCCCCGCTTCCTGCAGCGGCTGCTACTCCTGGAC

TATCCCCCCGACAGGGTCACCCTTTTCCTGCACAACAACGAGGTCTTCCATGAAC

CCCACATCGCTGACTCCTGGCCGCAGCTCCAGGACCACTTCTCAGCTGTGAAGCT

CGTGGGGCCGGAGGAGGCTCTGAGCCCAGGCGAGGCCAGGGACATGGCCATGG ACCTGTGTCGGCAGGACCCCGAGTGTGAGTTCTACTTCAGCCTGGACGCCGACGC

TGTCCTCACCAACCTGCAGACCCTGCGTATCCTCATTGAGGAGAACAGGAAGGTG

ATCGCCCCCATGCTGTCCCGCCACGGCAAGCTGTGGTCCAACTTCTGGGGCGCCC

TGAGCCCCGATGAGTACTACGCCCGCTCCGAGGACTACGTGGAGCTGGTGCAGC

GGAAGCGAGTGGGTGTGTGGAATGTACCATACATCTCCCAGGCCTATGTGATCCG

GGGTGATACCCTGCGGATGGAGCTGCCCCAGAGGGATGTGTTCTCGGGCAGTGA

CACAGACCCGGACATGGCCTTCTGTAAGAGCTTTCGAGACAAGGGCATCTTCCTC

CATCTGAGCAATCAGCATGAATTTGGCCGGCTCCTGGCCACTTCCAGATACGACA

CGGAGCACCTGCACCCCGACCTCTGGCAGATCTTCGACAACCCCGTCGACTGGA

AGGAGCAGTACATCCACGAGAACTACAGCCGGGCCCTGGAAGGGGAAGGAATC

GTGGAGCAGCCATGCCCGGACGTGTACTGGTTCCCACTGCTGTCAGAACAAATGT

GTGATGAGCTGGTGGCAGAGATGGAGCACTACGGCCAGTGGTCAGGCGGCCGGC

ATGAGGATTCAAGGCTGGCTGGAGGCTACGAGAATGTGCCCACCGTGGACATCC

AC AT GAAGC AGGT GGGGT ACGAGGACC AGTGGCTGC AGCTGCTGCGGACGT AT G

TGGGCCCCATGACCGAGAGCCTGTTTCCCGGTTACCACACCAAGGCGCGGGCGG

TGATGAACTTTGTGGTTCGCTACCGGCCAGACGAGCAGCCGTCTCTGCGGCCACA

CCACGACTCATCCACCTTCACCCTCAACGTTGCCCTCAACCACAAGGGCCTGGAC

TATGAGGGAGGTGGCTGCCGCTTCCTGCGCTACGACTGTGTGATCTCCTCCCCGA

GGAAGGGCTGGGCACTCCTGCACCCCGGCCGCCTCACCCACTACCACGAGGGGC

TGCCAACGACCTGGGGCACACGCTACATCATGGTGTCCTTTGTCGACCCCTGACA

CTCAACCACTCTGCCAAACCTGCCCTGCCATTGTGCCTTTTTAGGGGGCCTGGCC

CCCGTCCTGGGAGTTGGGGGATGGGTCTCTCTGTCTCCCCACTTCCTGAGTTCATG

TTCCGCGTGCCTGAACTGAATATGTCACCTTGCTCCCAAGACACGGCCCTCTCAG

GAAGCTCCCGGAGTCCCCGCCTCTCTCCTCCGCCCACAGGGGTTCGTGGGCACAG

GGCTTCTGGGGACTCCCCGCGTGATAAATTATTAATGTTCCGCAGTCTCACTCTG

AAT AAAGGAC AGTTTGT AAGTCTTGAAA AAAAAAAA AAAAAAAA (SEQ ID

NO: l)(accession NM_00l084, wherein nucleotides 457-2673 encodes a PLOD3 polypeptide, with 457 to 528 encoding a signal peptide and the mature peptide encoded by 529 to 2670.

This mRNA sequence is representative of PLOD3 mRNA and may vary from individual to individual, thus 1, 2, 3, 4, 5, 6, 7, 8, ,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

25, 26, 27, 28, 29, 30 up to 200 nucleotides across the mRNA may differ but still be recognized as a PLOD3 mRNA. [0056] The amino acid sequence of a representative PLOD3 polypeptide is MTSSGPGPRFLLLLPLLLPPAASASDRPRGRDPVNPEKLLVITVATAETEGYLRFLRSA EFFNYT VRTLGLGEEWRGGD VART V GGGQKVRWLKKEMEK Y ADREDMIIMF VD S Y D VILAGSPTELLKKF VQSGSRLLF S AESF CWPEW GLAEQ YPEVGTGKRFLNSGGFIGF ATTIHQIVRQWKYKDDDDDQLFYTRLYLDPGLREKLSLNLDHKSRIFQNLNGALDEV VLKFDRNRVRIRNVAYDTLPIVVHGNGPTKLQLNYLGNYVPNGWTPEGGCGFCNQD RRTLPGGQPPPRVFLAVFVEQPTPFLPRFLQRLLLLDYPPDRVTLFLHNNEVFHEPHIA DSWPQLQDHFSAVKLVGPEEALSPGEARDMAMDLCRQDPECEFYFSLDADAVLTNL QTLRILIEENRK VI APML SRHGKLW SNF W GAL SPDE YY ARSED YVELV QRKRV GVW NVP YI S Q A Y VIRGDTLRMELPQRD VF S GSDTDPDM AF CK SFRDKGIFLHL SN QHEF G RLL AT SRYDTEHLHPDLW QIFDNP VDWKEQ YIHENY SRALEGEGIVEQPCPD VYWFP LL SEQMCDEL V AEMEH Y GQ W S GGRHED SRL AGGYEN VPT VDIHMKQ V GYEDQ WL QLLRT Y V GPMTESLFPGYHTK AR A VMNF VVRYRPDEQP SLRPHHD S S TF TLN V ALN HKGLDYEGGGCRFLRYDCVISSPRKGWALLHPGRLTHYHEGLPTTWGTRYIMVSFV DP (SEQ ID NO:2) (NP 001075.1). Amino acids 1-24 form a signal peptide and amino acids 25-738 provide for a mature form of the protein. In certain aspects the polypeptide can be glycosylated at various positions, e.g., amino acid 63 and/or 548.

[0057] Physiological functions of PLOD3. Collagen is a group of extracellular matrix proteins that play versatile roles in cellular physiology, ranging from structural support to mediating cell signaling¹²¹. Currently there are 28 known collagen types. All types of collagen contain at least one triple helical domain formed by repeating Gly-X-Y (where X and Y can be any amino acid) sequences¹²². Collagen is synthesized in endoplasmic reticulum as a precursor, then undergoes various post-translational modifications (PTMs) before being secreted out of the cell. One of the most important PTM for forming mature collagen is hydroxylation of many lysine residues into and hydroxylysine (Hyl)¹²³. Hyl can serve as an attachment site for carbohydrates, leading to the subsequent galactosylhydroxylysine (GHyl) or glucosylgalactosylhydroxylysine (GGHyl). PLODs are metabolic enzymes that play an important role in catalyzing the hydroxylation of specific lysine residues in proteins especially in collagen. Some of these Hyl residues play an essential role in forming certain covalent cross links in collagen, which predominate in bone, cartilage, ligament, most tendons, embryonic skin, and most major internal connective tissues of the body. In addition to collagen, PLODs can also modify the proteins which have the collagenous like structure (Gly-X-Y). All three PLODs need to form homodimers for lysyl hydroxylase activity. Interestingly, PLOD3 also can form a heterodimer with PLOD1. Dimerization is required for the LH activity of PLOD3, whereas it is dispensable for glycosyltransferase activities. The dimerization process is mainly mediated by the amino acids 541-547 in PLOD3¹²⁴. Mutations in PLOD3 led to a 30% lower Hyl levels of collagens IV and V, while no such decrease was observed in collagen I and III, indicating PLOD3 is important for the lysyl hydroxylation of collagens IV and V¹²⁵. In addition, loss of PLOD3 prevents the intracellular tetramerization of type VI collagen and leads to impaired secretion of type IV and VI collagens¹²⁶. The gene mutated in the Ehlers-Danlos syndromes (EDS) type VIA patients is PLOD1. The triple-helical regions of collagen I and III were hydroxylated abnormally in patients with EDS-VIA, suggesting that PLOD1 is important for hydroxylation of lysine in collagen I and III¹²⁷. Other studies also indicated that PLOD3 is important for the maturation of collagen I in osteoblast. PLOD3 is crucial for the formation of glucosylate galactosylhydroxylysine residues in type I collagen and its downregulation significantly affects type I collagen fibrillogenesis^{128 129}. PLOD2 is mainly involved in the lysyl hydroxylation of the N- and C-telopeptides¹³⁰. The difference of PLODs in hydroxylation of lysine in different types of collagen further support that their biological functions cannot be compensate for one another.

[0058] In addition to LH activity, PLOD3 also have collagen glucosyltransferase (GGT) and galactosyltransferase (GT) activities. These two enzymes are required for forming the unique 2-0-a-D-glucopyranosyl-0-p-D-galactopyranosyl hydroxylysyl residues typical of collagens and of other proteins with collagenous domains. Both PLOD3 knockout embryos and the fibroblastic cells from the PLOD3 knockout embryos showed a significant reduction in GGT activity, suggesting that PLOD3 is the key molecule accounting for the GGT activity¹³¹. As GGT activity is highly sensitive and specific for PLOD3, measuring the GGT activity in the biological samples can reflect the expression level of PLOD3. The GT activity is not exclusive to PLOD3, and many GTs have GT activities¹¹⁷. A conserved cysteine at position 144 and a leucine at position 208 as well as the aspartates of a DXD-like motif crucial are crucial for maintaining the GGT activity ¹³².

[0059] As mentioned above, the major target of PLOD3 in vivo is collagen type IV. PLOD3 was demonstrated to be an important regulator for the biosynthesis, secretion and activity of adiponectin, which has crucial roles in glucose and lipid metabolism and inflammation. Adiponectin has a collagenous region, and hydroxylation and glycosylation of the lysine residues of adiponectin can enhance its biological function¹³³. PLOD3 is important for catalyzing formation of the glucosylgalactosylhydroxylysines of mannan-binding lectin (MBL), which is a component of lectin pathway of complement activation. Mice lacking LH activity had reduced levels of circulating MBL¹³⁴.

[0060] As PLOD3 is an important enzyme, it is no wonder that PLOD3 is ubiquitously expressed in cells. PLOD3 is manly located in the ER, and it is found extracellularly in serum, the extracellular space, and on cellular membrane¹³⁵. It is the only secreted PLOD isoform. The glycosyltransferase domain of PLOD3 rather than the LH domain is responsible for secretion of PLOD3 into the cell medium. Two pathways have been proposed for the secretion of PLOD3 from the ER; one is Golgi dependent and the other is Golgi independent. PLOD3 secreted in the cell medium is Golgi dependent and relies on the glycosyltransferase activity. However, the PLOD3 expressed on the cellular membrane is secreted in a Golgi independent manner. The secretion of PLOD3 into extracellular space indicates that it might be important for the extracellular matrix (ECM) remodeling¹³⁶. PLOD3 is indispensable for development, as the PLOD3 knockout embryos leads to embryonic lethality at E9.5. Downregulation of GGT activity in PLOD3 knockout mice is the major reason responsible for the embryonic lethality, which can disrupts the type IV collagen location and subsequent basement membrane formation¹³⁷. Lack of PLOD3 also significantly affect the normal organization of the extracellular matrix (ECM) and cytoskeleton¹³¹. PLOD3 played a critical role in regenerative growth and guidance of axons of the dorsal nerve branch by partially regulated it functional substrate collagen alpha-5(IV) chain ¹³⁸. Neural stem cell with PLOD3 deletion failed to transition from a sheet to a stream, indicating that changes in modification and distribution of ECM components due to PLOD3 deletion can significantly influence the signals required for the migration capacity of neural crest cells¹³⁹. MMP-9 secreted by tumor cells and leukocytes can be recruited to the fibroblast surface through its FNII repeats or collagen-binding domain and PLOD3 is responsible for the docking of MMP-9 to fibroblast surface. In addition, PLOD3 mediated MMP-9 recruitment stimulate the activation of transforming growth factor beta 1 (TGF-b), which subsequently promote the differentiation of fibroblast into myofibroblasts¹⁴⁰.

[0061] Regulation of PLOD3. Currently the molecular mechanisms that regulate PLOD3 activities are poorly unknown. MiR-663 was demonstrated to be a direct regulator of PLOD3. Ectopic expression of miR-663 could inhibit the PLOD3 expression, leading to reduced type IV collagen biosynthesis, indicating miR-663 might be functional modulator of PLOD3¹⁴¹. In addition to miRNAs, transcriptional factors are the major potential regulators of PLOD3. Further studies are needed to reveal the upstream regulators and downstream effectors of PLOD3.

[0062] Deregulation of PLOD3 in diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is a disease caused by reduced or absent type VII collagen. The expression level of PLOD3 was significantly reduced in basement membrane of RDEB patient skin compared to the normal skin, indicating that PLOD3 at the basement membrane might be important for progression of RDEB¹⁴². Mutation of PLOD 3 leads to an inherited syndrome of congenital malformations with various connective tissues disorders, which significantly affects the normal function of many tissues and organs¹⁴³. The copy number of PLOD3 was increased in patients with gastric cancer, indicating PLOD3 might be a tumor promoter of gastric cancer¹⁴⁴. Another study demonstrated that the expression level of PLOD3 was significantly upregulated in colorectal cancer tissues in comparison with the normal tissues¹⁴⁵. The levels of PLOD3 were also found to be upregulated in the secretome from pancreatic cancer cell lines compared with the normal pancreatic cells¹⁴⁶, indicating PLOD3 might be important for the cell to cell communication in the tumor microenvironment. It was reported that PLOD3 was overexpressed in glioblastoma (GBM) tissues and cell lines. Downregulation of PLOD3 inhibited the oncogenic activities of GBM cells¹⁴⁷. Although there are some studies revealing the potential oncogenic role of PLOD3 in cancer, the concrete molecular mechanisms are poorly investigated. In addition, the role of PLOD3 in HNSCC is unknown. II. Modulation of PLOD3 expression/function

[0063] Certain embodiments are directed to inhibition of PLOD3 or negative modulation of PLOD3 levels in a cell, e.g, a cancer cell.

[0064] The expression of the cancer-associated genes, such as PLOD3, is elevated in human cancer tissue; therefore, certain aspects of the methods and composition described herein is modulate the function or expression of such genes. In certain embodiments the expression of the cancer-associated gene can be modulated by an antisense oligonucleotide, a ribozyme, a siRNA or the like. Other nucleotides or oligonucleotides can be used as a probe or a primer for detecting the cancer-associated gene.

[0065] A. PLOD3 Polynucleotides and Oligonucleotides [0066] The polynucleotide or the oligonucleotides can be single stranded or double stranded, and may be DNA, RNA, or a mixture thereof or a derivative of PNA or the like. Such a polynucleotide or oligonucleotide may be chemically modified at the internucleoside linkage, the base moiety and/or the sugar moiety, or may have a modifier at the 5' end and/or 3' end.

[0067] Polynucleotides described herein can have a nucleotide sequence represented SEQ ID NOs: 1, including segments thereof and SEQ ID NO:3 and SEQ ID NO:4 or a nucleotide sequence complementary thereof. The polynucleotides or oligonucleotides described herein may be provided as a nucleic acid construct to be introduced into a cell to produce a desired polynucleotide, oligonucleotide, antisense, ribozyme or siRNA in the cell.

[0068] When the polynucleotide or the oligonucleotide is used as an antisense, ribozyme, siRNA or the like, the polynucleotide or the oligonucleotide may have a chain length of preferably at least 12 nucleotides or more, more preferably 12 to 50 nucleotides, particularly preferably 12 to 25 nucleotides. Such a polynucleotide or oligonucleotide may be a variant in which one or more nucleotides are substituted, added or deleted from the nucleotide sequence as provided herein, as long as it has a desired antisense, ribozyme or siRNA activity. Such a variant may have a nucleotide sequence with an identity of at least 70%, preferably 90% or more, more preferably 95% or more with a nucleotide sequence provided herein.

[0069] The polynucleotide or the oligonucleotide of the present invention can be synthesized with using a DNA synthesizer ( e.g ., 394 synthesizer, Applied Biosystems). Alternatively, it can be produced based on the sequence information disclosed herein by PCR amplification or expression cassettes or vectors.

[0070] The polynucleotide or oligonucleotide of the present invention can silence a cancer- associated gene using an antisense molecule that binds to mRNA encoded by the cancer- associated gene and inhibits its expression, or a ribozyme or siRNA that cleaves mRNA. The compositions may be administered together with an appropriate carrier, or a vector encoding an antisense, ribozyme or siRNA may be administered to induce its expression in vivo.

[0071] The term“ribozyme” means a nucleic acid molecule having an enzymatic activity of cleaving mRNA. The ribozyme generally shows an endonuclease, ligase or polymerase activity. Various types of trans-acting ribozymes such as hammerhead type and the hairpin type ribozymes are known in the art. [0072] The term“antisense” means a nucleic acid molecule or a derivative thereof that hybridizes specifically to genomic DNA and/or mRNA and inhibits its transcription and/or translation to inhibit the expression of the protein. The binding may occur through general base pair complementation, or in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. The target site of the antisense nucleic acid is preferably the 5' end of mRNA, for example, the 5 '-untranslated sequence up to and including the AUG initiation codon. However, it is known that the 3 '-untranslated sequence of mRNA or the sequence of the coding region is also effective in inhibiting the translation of mRNA.

[0073] The term“small interfering RNA” or“siRNA” means a double-stranded nucleic acid that can effect RNA interference (RNAi) (see, for example, Bass, 2001 , Nature, 4.11, 428-429; Elbashir et al., 2001, Nature , 411, 494-498). The siRNA can degrade mRNA in a sequence- specific manner, thereby inhibiting the expression of a gene. The siRNA is typically a double- stranded RNA with a length of 20 to 25 base pairs containing a sequence complementary to a target sequence. The siRNA molecule may contain a chemically modified nucleotide or non nucleotide moiety. The siRNA may include short overhangs at each end. The overhangs can be 1-6 nucleotides in length at the 3' end. The siRNAs can be chemically synthesized, or derived from a longer double-stranded RNA or a hairpin RNA. The siRNAs have significant sequence similarity to a target RNA so that the siRNAs can pair to the target RNA and result in sequence-specific degradation of the target RNA through an RNA interference mechanism. The siRNAs are understood to, but not necessarily limited to, recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex. In a particular embodiment, the siRNA molecules comprise a 3' hydroxyl group.

[0074] Certain embodiments are directed to a siRNA targeting nucleotides of human PLOD3 mRNA (SEQ ID NO: l). Particular embodiments are directed to siRNA having a nucleic sequence of AGAAGGAAAUGGAGAAAUA (SEQ ID NO:3); CCACAGAGCUGCUGAAGAA (SEQ ID NO:4); and/or a variant or complement thereof. The siRNA molecules may include one or more modifications ( e.g ., to the base moiety, sugar moiety, phosphate moiety, phosphate-sugar backbone, or a combination thereof). For example, the phosphodiester linkages may be modified to include at least one heteroatom other than oxygen, such as nitrogen or sulfur. In this case, for example, the phosphodiester linkage may be replaced by a phosphothioester linkage. Similarly, bases may be modified to block the activity of adenosine deaminase. Other examples of useful modifications are morpholino modifications and LNA. Where the siRNA molecule is produced synthetically, or by in vitro transcription, a modified ribonucleoside may be introduced during synthesis or transcription.

[0075] Non-limiting examples of modified base moieties include inosine, 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, l-methylguanine, l-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3 -(3 -amino-3 -N-2- carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[0076] Non-limiting examples of modified sugar moieties include arabinose, 2- fluoroarabinose, xylulose, and hexose. Modified siRNAs may contain substituted sugar moieties comprising one of the following at the 2' position: OH, SH, SCH₃, F, OCN, 0(CH₂)nNH₂ or 0(CH₂)_nCH₃ where n is from 1 to about 10; Cl to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O; S- or N-alkyl; 0-, S-, or N-alkenyl; SOCH₃; S0₂CH₃; ON0₂; N0₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted sialyl; a fluorescein moiety; a reporter group; a group for improving the pharmacokinetic properties; or a group for improving the pharmacodynamic properties, and other substituents having similar properties. Modified siRNAs may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group.

[0077] Non-limiting examples of modifications of phosphate backbone include a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, a phosphotriester, an alkyl phosphotriester, and a formacetal or analog thereof, as well as chimeras between methylphosphonate and phosphodiester, short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Specific non-limiting examples include those with CH2- NH-0-CH2, CH2-N(CH₃)-0-CH2, CH₂-0-N(CH₃)-CH₂, CH₂-N(CH₃)-N(CH₃)-CH₂ and O- N(CH₃)-CH2-CH2 backbones (where phosphodiester is O-PO2-O-CH2). Nitrogen linkers or groups containing nitrogen can also be used to prepare oligonucleotide mimics.

[0078] Certain embodiments can include modified siRNA molecules having morpholino backbone structures in which the bases are linked to 6-membered morpholine rings, which are connected to other morpholine-linked bases via non-ionic phosphorodiamidate intersubunit linkages. Morpholino siRNAs are highly resistant to nucleases and have good targeting predictability (U.S. Patent 5,034,506; Summerton, Biochim. Biophys. Acta 1999, 1489: 141- 158; Summerton and Weller, Antisense Nucleic Acid Drug Dev. 1997, 7: 187-195; Arora et ak, J. Pharmacol. Exp. Ther. 2000, 292:921-928; Qin et al., Antisense Nucleic Acid Drug Dev. 2000, 10: 11-16; Heasman et al., Dev. Biol. 2000, 222: 124-134; Nasevicius and Ekker, Nat. Genet. 2000, 26:216-220).

[0079] Another type of a useful modification is the peptide-nucleic acid (PNA) backbone: the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al., Science 1991, 254: 1497).

[0080] In other embodiments, locked nucleic acids (LNA) can be used (reviewed in, e.g. , Jepsen and Wengel, Curr. Opin. Drug Discov. Devel. 2004, 7: 188-194; Crinelli et al., Curr. Drug Targets 2004, 5:745-752). LNA are nucleic acid analog(s) with a 2'-0, 4'-C methylene bridge. This bridge restricts the flexibility of the ribofuranose ring and locks the structure into a rigid C3-endo conformation, conferring enhanced hybridization performance and exceptional biostability.

[0081] Modified siRNAs can include appending groups such as, e.g. , peptides, or agents facilitating transport across the cell membrane (see, e.g. , Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86:6553-6556; Lemaitre et al., Proc. Natl. Acad. Sci. USA 1987, 84:648-652; PCT Publication WO 88/09810) or blood-brain barrier (see, e.g., PCT Publication WO 89/10134).

[0082] siRNAs may be introduced to a target cell as an annealed duplex siRNA, or as single stranded sense and antisense nucleic acid sequences that, once within the target cell, anneal to form the siRNA duplex. Alternatively, the sense and antisense strands of the siRNA may be encoded on an expression construct that is introduced to the target cell. Upon expression within the target cell, the transcribed sense and antisense strands may anneal to reconstitute the siRNA. [0083] The siRNA molecules can, but do necessarily contain nucleotide sequences that are fully complementary to nucleotides 346 to 364 or to nucleotides 430 to 448 of human PLOD3 mRNA (SEQ ID NO: 1) or corresponding nucleotides in PLOD3 orthologs in other species (e.g, other mammalian species), 100% sequence complementarity between the siRNA and the target nucleic acid is not required to practice the invention.

[0084] siRNA molecules can be synthesized by using an automated synthesizer. RNAs produced by such methodologies tend to be highly pure and to anneal efficiently to form siRNA duplexes. Following chemical synthesis, single stranded RNA molecules are deprotected, annealed to form siRNAs, and purified (e.g, by gel electrophoresis or HPLC). Alternatively, in vitro transcription of RNA from DNA templates carrying RNA polymerase promoter sequences can be used to produce RNA (e.g. , T7 or SP6 RNA polymerase promoter sequences). Efficient in vitro protocols for preparation of siRNAs using T7 RNA polymerase have been described (Donze and Picard, Nucleic Acids Res. 2002, 30:e46; and Yu et al., Proc. Natl. Acad. Sci. USA 2002, 99:6047-6052). The sense and antisense transcripts may be synthesized in two independent reactions and annealed later, or may be synthesized simultaneously in a single reaction.

[0085] siRNA molecules may be formed within a cell by transcription of RNA from an expression construct introduced into the cell. For example, both a protocol and an expression construct for in vivo expression of siRNAs are described in Yu et al., supra.

[0086] The expression constructs for in vivo production of siRNA molecules comprise siRNA encoding sequences operably linked to elements necessary for the proper transcription of the siRNA encoding sequence(s), including promoter elements and transcription termination signals. Preferred promoters for use in such expression constructs include the polymerase-III HI-RNA promoter and the U6 polymerase-III promoter. The siRNA expression constructs can further comprise vector sequences that facilitate the cloning of the expression constructs. Standard vectors that maybe used in practicing the current invention are known (e.g, pSilencer 2.0-U6 vector, Ambion Inc., Austin, Tex.).

[0087] B. Polynucleotide or Oligonucleotide Carriers.

[0088] Certain embodiments are directed to a composition comprising an inhibitory nucleic acid described herein and a carrier and/or excipient. In certain aspects carrier is a liposomal carrier or a nanoparticle carrier. [0089] Liposomal Carrier. In certain embodiments, liposomes are used to deliver an polynucleotide or oligonucleotide to a subject. Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half- life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are herein incorporated by reference.

[0090] The liposomes can be modified so as to avoid clearance by the mononuclear macrophage system (“MMS”) and reticuloendothelial system (“RES”). Such modified liposomes have opsonization-inhibition moieties on the surface or incorporated into the liposome structure.

[0091] Opsonization-inhibiting moieties for use in preparing the liposomes are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization inhibiting moiety is“bound” to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer that significantly decreases the uptake of the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference.

[0092] In some embodiments, opsonization inhibiting moieties suitable for modifying liposomes are water-soluble polymers with a number-average molecular weight from about 500 to about 40,000 daltons, or from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N- vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization inhibiting polymer can be a block copolymer of PEG and either a poly amino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups. In some embodiments, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called“PEGylated liposomes.”

[0093] Nanoparticle Carrier. The term“nanoparticles” generally refers to particles in the range of between 500 nm to less than 0.5 nm, preferably having a diameter that is between 50 and 500 nm, more preferably having a diameter that is between 50 and 300 nm. Cellular internalization of polymeric particles is highly dependent upon their size, with nanoparticulate polymeric particles being internalized by cells with much higher efficiency than micoparticulate polymeric particles. Nanoparticles also have a greater ability to diffuse deeper into tissues in vivo.

[0094] The polymer that forms the core of the nanoparticle may be any biodegradable or non-biodegradable synthetic or natural polymer. In a preferred embodiment, the polymer is a biodegradable polymer. Nanoparticles are ideal materials for the fabrication of inhibitory RNA delivery vehicles: (1) control over the size range of fabrication, down to 100 nm or less, an important feature for passing through biological barriers; (2) reproducible biodegradability without the addition of enzymes or cofactors; (3) capability for sustained release of encapsulated, protected inhibitory RNAs over a period in the range of days to months by varying factors such as the monomer ratios or polymer size, for example, poly(lactic acid) (PLA) to poly(glycolic acid) (PGA) copolymer ratios, potentially abrogating the booster requirement (Gupta, et al., Adv. Drug Deliv. Rev., 32(3):225-246 (1998); Kohn, et al., J Immunol. Methods, 95(l):31-8 (1986); Langer, et al., Adv. Drug Deliv. Rev., 28(l):97-l 19 (1997); Jiang, et al., Adv. Drug Deliv. Rev., 57(3):391-410 (2005)), well-understood fabrication methodologies that offer flexibility over the range of parameters that can be used for fabrication, including choices of the polymer material, solvent, stabilizer, and scale of production; and (5) control over surface properties facilitating the introduction of modular functionalities into the surface.

[0095] Examples of biodegradable polymers include synthetic polymers that degrade by hydrolysis such as poly(hydroxy acids), such as polymers and copolymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), and poly(lactide-co- caprolactone).

[0096] Natural polymers include alginate and other polysaccharides, collagen, albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo , by surface or bulk erosion.

[0097] In some embodiments, non-biodegradable polymers can be used, especially hydrophobic polymers. Examples of preferred non-biodegradable polymers include ethylene vinyl acetate, poly(meth) acrylic acid, copolymers of maleic anhydride with other unsaturated polymerizable monomers, poly(butadiene maleic anhydride), polyamides, copolymers and mixtures thereof, and dextran, cellulose and derivatives thereof.

[0098] Other suitable biodegradable and non-biodegradable polymers include, but are not limited to, polyanhydrides, polyamides, polycarbonates, polyalkylenes, polyalkylene oxides such as polyethylene glycol, polyalkylene terepthalates such as poly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyethylene, polypropylene, poly(vinyl acetate), poly vinyl chloride, polystyrene, polyvinyl halides, polyvinylpyrrolidone, polymers of acrylic and methacrylic esters, polysiloxanes, polyurethanes and copolymers thereof, modified celluloses, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, and polyacrylates such as poly(methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate). These materials may be used alone, as physical mixtures (blends), or as co-polymers.

[0099] In certain embodiments, the nanoparticles are formed of polymers fabricated from polylactides (PLA) and copolymers of lactide and glycolide (PLGA). These have established commercial use in humans and have a long safety record (Jiang, et ah, Adv. Drug Deliv. Rey, 57(3):39l-4l0); Aguado and Lambert, Immunobiology, 184(2-3): 113-25 (1992); Bramwell, et ah, Adv. Drug Deliv. Rev., 57(9): 1247-65 (2005)). These polymers have been used to encapsulate siRNA (Yuan, et al., Jour. Nanosocience and Nanotechnology, 6:2821-8 (2006); Braden, et al., Jour. Blamed. Nanotechnology, 3 : 148-59 (2007); Khan, et al., Jour. Drug Target, 12:393-404 (2004)).

[0100] In a certain embodiments, the inhibitory RNAs can be complexed to polycations to increase the encapsulation efficiency of the inhibitory RNAs into the nanoparticles. The term “polycation” refers to a compound having a positive charge, preferably at least 2 positive charges, at a selected pH, preferably physiological pH. Polycationic moieties have between about 2 to about 15 positive charges, preferably between about 2 to about 12 positive charges, and more preferably between about 2 to about 8 positive charges at selected pH values.

[0101] Many polycations are known in the art. Suitable constituents of polycations include basic amino acids and their derivatives such as arginine, asparagine, glutamine, lysine and histidine; cationic dendrimers; and amino polysaccharides. Suitable polycations can be linear, such as linear tetralysine, branched or dendrimeric in structure.

[0102] Examples of polycations include, but are not limited to, synthetic polycations based on acrylamide and 2-acrylamido-2-methylpropanetrimethylamine, poly(N-ethyl-4- vinylpyridine) or similar quartemized polypyridine, diethylaminoethyl polymers and dextran conjugates, polymyxin B sulfate, lipopolyamines, poly(allylamines) such as the strong polycation poly(dimethyldiallylammonium chloride), polyethyleneimine, polybrene, and polypeptides such as protamine, the histone polypeptides, polylysine, polyarginine and polyornithine.

[0103] The polycation can be a polyamine. Polyamines are compounds having two or more primary amine groups. In a preferred embodiment, the polyamine is a naturally occurring polyamine that is produced in prokaryotic or eukaryotic cells. Naturally occurring polyamines represent compounds with cations that are found at regularly-spaced intervals and are therefore particularly suitable for complexing with nucleic acids. Polyamines play a major role in very basic genetic processes such as DNA synthesis and gene expression. Polyamines are integral to cell migration, proliferation and differentiation in plants and animals. The metabolic levels of polyamines and amino acid precursors are critical and hence biosynthesis and degradation are tightly regulated. Suitable naturally occurring polyamines include, but are not limited to, spermine, spermidine, cadaverine and putrescine. In a preferred embodiment, the polyamine is spermidine. [0104] The polycation can be a cyclic polyamine. Cyclic polyamines are known in the art and are described, for example, in U.S. Patent 5,698,546, PCT publications WO 1993/012096 and WO 2002/010142. An example of cyclic polyamines include, but are not limited to, cyclen.

[0105] Coupling Agents or Ligands. The external surface of the polymeric nanoparticles may be modified by conjugating to, or incorporating into, the surface of the nanoparticle a coupling agent or ligand.

[0106] In certain embodiments, the coupling agent is present in high density on the surface of the nanoparticle. As used herein,“high density” refers to polymeric nanoparticles having a high density of ligands or coupling agents, which is preferably in the range of 1,000 to 10,000,000, more preferably 10,000-1,000,000 ligands per square micron of nanoparticle surface area. This can be measured by fluorescence staining of dissolved particles and calibrating this fluorescence to a known amount of free fluorescent molecules in solution.

[0107] Coupling agents associate with the polymeric nanoparticles and provide substrates that facilitate the modular assembly and disassembly of functional elements to the nanoparticles. Coupling agents or ligands may associate with nanoparticles through a variety of interactions including, but not limited to, hydrophobic interactions, electrostatic interactions and covalent coupling.

[0108] Coupling agents can also be attached to polymeric nanoparticles through covalent interactions through various functional groups. Functionality refers to conjugation of a molecule to the surface of the particle via a functional chemical group (carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls) present on the surface of the particle and present on the molecule to be attached.

[0109] Functionality may be introduced into the particles in two ways. The first is during the preparation of the nanoparticles, for example during the emulsion preparation of nanoparticles by incorporation of stablizers with functional chemical groups. Suitable stabilizers include hydrophobic or amphipathic molecules that associate with the outer surface of the nanoparticles.

[0110] A second is post-particle preparation, by direct crosslinking particles and ligands with homo- or heterobifunctional crosslinkers. This second procedure may use a suitable chemistry and a class of crosslinkers (CDI, EDAC, glutaraldehydes, etc. as discussed in more detail below) or any other crosslinker that couples ligands to the particle surface via chemical modification of the particle surface after preparation. This second class also includes a process whereby amphiphilic molecules such as fatty acids, lipids or functional stabilizers may be passively adsorbed and adhered to the particle surface, thereby introducing functional end groups for tethering to ligands.

[0111] Another coupling method involves the use of 1 -ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDAC) or “water-soluble CDF in conjunction with N- hydroxylsulfosuccinimide (sulfo NHS) to couple the exposed carboxylic groups of polymers to the free amino groups of molecules in a totally aqueous environment at the physiological pH of 7.0. Briefly, EDAC and sulfo-NHS form an activated ester with the carboxylic acid groups of the polymer which react with the amine end of a molecule to form a peptide bond. The resulting peptide bond is resistant to hydrolysis. The use of sulfo-NHS in the reaction increases the efficiency of the EDAC coupling by a factor of ten-fold and provides for exceptionally gentle conditions that ensure the viability of the molecule-polymer complex.

[0112] A useful coupling procedure for attaching molecules with free hydroxyl and carboxyl groups to polymers involves the use of the cross-linking agent, divinylsulfone. This method would be useful for attaching sugars or other hydroxylic compounds with bioadhesive properties to hydroxylic matrices. Briefly, the activation involves the reaction of divinylsulfone to the hydroxyl groups of the polymer, forming the vinylsulfonyl ethyl ether of the polymer. The vinyl groups will couple to alcohols, phenols and even amines. Activation and coupling take place at pH 11. The linkage is stable in the pH range from 1-8 and is suitable for transit through the intestine.

[0113] Any suitable coupling method known to those skilled in the art for the coupling of molecules and polymers with double bonds, including the use of EiV crosslinking, may be used for attachment of molecules to the polymer.

[0114] In certain embodiments, coupling agents can be conjugated to affinity tags. Affinity tags are any molecular species which form highly specific, noncovalent, physiochemical interactions with defined binding partners. Affinity tags which form highly specific, noncovalent, physiochemical interactions with one another are defined herein as “complementary”. Suitable affinity tag pairs are well known in the art and include epitope/antibody, biotin/avidin, biotin/streptavidin, biotin/neutravidin, glutathione-S- transferase/glutathione, maltose binding protein/amylase and maltose binding protein/maltose. Examples of suitable epitopes which may be used for epitope/antibody binding pairs include, but are not limited to, HA, FLAG, c-Myc, glutatione-S-transferase, His6, GFP, DIG, biotin and avidin. Antibodies (both monoclonal and polyclonal and antigen-binding fragments thereof) which bind to these epitopes are well known in the art.

[0115] Affinity tags that are conjugated to coupling agents allow for highly flexible, modular assembly and disassembly of functional elements which are conjugated to affinity tags which form highly specific, noncovalent, physiochemical interactions with complementary affinity tags which are conjugated to coupling agents. Adaptor elements may be conjugated with a single species of affinity tag or with any combination of affinity tag species in any ratio. The ability to vary the number of species of affinity tags and their ratios conjugated to adaptor elements allows for exquisite control over the number of functional elements which may be attached to the nanoparticles and their ratios.

[0116] In another embodiment, coupling agents are coupled directly to functional elements in the absence of affinity tags, such as through direct covalent interactions. Coupling agents can be covalently coupled to at least one species of functional element. Coupling agents can be covalently coupled to a single species of functional element or with any combination of species of functional elements in any ratio.

[0117] The coupling agents are preferably provided on, or in the surface of, nanoparticles at a high density. This high density of coupling agents allows for coupling of the polymeric nanoparticles to a variety of species of functional elements while still allowing for the functional elements to be present in high enough numbers to be efficacious.

[0118] Targeting Molecules. One class of functional elements that can be attached to the nanoparticles is targeting molecules. Targeting molecules can be proteins, peptides, nucleic acid molecules, saccharides or polysaccharides that bind to a receptor or other molecule on the surface of a targeted cell. The degree of specificity and the avidity of binding to the graft can be modulated through the selection of the targeting molecule. For example, antibodies are very specific. These can be polyclonal, monoclonal, fragments, recombinant, or single chain, many of which are commercially available or readily obtained using standard techniques.

[0119] Engineered nanoparticles, e.g., PLGA nanoparticles, can be used to deliver target molecules for anti-cancer treatment. This can be targeted towards all solid tumors. The target molecules include, but are not limited to (1) siRNAs targeting PLOD3 (to directly inhibit the expression of PLOD3); (2) anti-PLOD3 antibodies (to specifically inhibit PLOD3 function); (3) inhibitors targeting miR-l24-3p towards PLOD3 (to inhibit PLOD3 expression and function); (4) miR-l24-3p mimics targeting PLOD3 (to inhibit PLOD3 expression and function). It is possible that two or more of these molecules are combined for targeting PLOD3. In certain embodiments, these target molecules can be used in combination with agents that inhibit HIF-la and pFAK in combination with PLOD3 inhibitors. In certain aspects, HIF-la and pFAK can include siRNAs targeting HIF-la or FAK, or pFAK phosphorylation inhibitors.

[0120] miR-124-3p and related pathways. MicroRNAs (miRNAs) are an abundant class of evolutionarily conserved, short non-coding RNAs of 20-24 nucleotides that play important roles in virtually all biological pathways throughout the plant and animal kingdoms¹⁵⁷. The first miRNA, lin4, was discovered in 1993 on Caenorhabditis elegans ^{158 159} Currently there are more than 2600 human mature miRNAs based on data from the miRBase (www.mirbase.org/). The main function of miRNAs is to negatively regulate protein expression by binding of fully or partially complementary sequences to the 3'- untranslated region (UTR) of target messenger RNAs (mRNAs), resulting in translation inhibition or/and mRNA degradation^{160 161}. The microRNA-mRNA binding site is relatively short (6-8 base pairs), and therefore each mature miRNA can control the expression of a number of target mRNAs¹⁶². In addition, each mRNA might be regulated by various miRNAs.

[0121] Mechanism of miRNAs regulation. MiRNAs can be originated from either intergenic or coding-intronic^{163 164}. Intergenic miRNAs are transcribed by RNA polymerase II or III to generate a primary transcript named pri-miRNA ¹⁶⁵. Then pri-miRNA is cleaved into pre- miRNA in the nucleus by the core components (Drosha and DGCR8) of the microprocessor complex¹⁶⁶. MiRNA within introns depends on the RNA splicing machinery for their biogenesis. Pre-miRNAs are exported to the cytoplasm by Exportin 5 and Ran-GTP. The pre- miRNAs are cleaved by a complex including the RNase Dicer, AG02 (Argonaute 2), and TRBP (trans-activation-responsive RNA-binding protein) to form mature miRNAs. These mature miRNAs are loaded into the RNA-induced silencing complex (RISC), then bind to the 3' UTR of the targeted mRNAs^167,168. The fate of miRNAs is determined by the binding. Perfect complementarity between miRNA and target mRNA leads to targeting the mRNA transcript degradation, while imperfect complementarity results in translation inhibition ¹⁶¹. [0122] miRNAs in cancer. MiRNAs have been demonstrated to potentially regulate every aspect of cellular activities including cell growth, proliferation, differentiation, and motility^{169 170}. Deregulation of miRNAs has been reported in many human diseases such as cardiovascular diseases, inflammatory diseases neurodevelopmental diseases and cancer^{171 172}. It is now well documented that miRNAs are aberrantly expressed in various human cancers. MiRNAs may function as either oncogene or tumor suppressor during the tumorigenesis. MiRNAs have shown great promise for clinical application. MiRNA expression pattern is associated with tumor subtypes, which might help facilitate tumor classification. For instance, only 48 miRNA markers can discriminate the cancers from various tumor origin with an overall accuracy of approximately 90%, indicating miRNA expression signature is effective fortracing the tissue of origin of cancers¹⁷³. MiRNAs are also closely associated with the chemoresi stance of cancer cells, indicating that miRNAs can be used for monitoring therapeutic response. For instance, overexpression of miR-449b in nasopharyngeal carcinoma increased cisplatin resistance by targeted transforming growth factor beta-induced (TGFBI) directly¹⁷⁴. Interestingly, single-nucleotide polymorphisms (SNPs) is existed in the miRNA binding site, which might be closely associated with cancer risk and susceptibility¹⁷⁵. MiRNAs are highly stable in the biofluids such as serum, plasma, urine and saliva. Thus, they serve as promising non-invasive biomarkers for early detection and prognostic prediction of various diseases including cancer^{176 177}. For instance, the expression level of miR-31 was significantly increased in the saliva samples from oral cancer patients compared with those from patients with oral verrucous leukoplakia or healthy controls. In addition, its levels dropped significantly following excision¹⁷⁸.

[0123] MiRNAs have been shown to play a crucial role in tumorigenesis of HNSCC. The expression level of miR-l34 was significantly upregulated in HNSCC compared to the normal mucosa. In addition, high miR-l34 expression was associated with adverse clinicopathological parameters and worse survival. Furthermore, ectopic expression of miR-l34 promoted the oncogenic phenotypes of cancer cells both in vitro and in vivo , and opposite results were observed when miR-l34 was downregulated. These data indicated that miR-l34 might serve as a tumor promoter in HNSCC¹⁷⁹. MiR-l35b overexpression promoted the proliferation, migration, and colony formation in vitro and enhanced tumor growth in vivo. In addition, hypoxia-inducible factor- la (HIF-la) was demonstrated to be a downstream target of miR- l35b¹⁸⁰. Most miRNAs play a tumor suppressive role in the carcinogenesis. For instance, the expression level of miR-874 was significantly downregulated in HNSCC. In addition, miR- 874 overexpression remarkably inhibited proliferation and induced G2/M arrest and cell apoptosis. Histone deacetylase 1 (HDAC1) was a downstream target of miR-874¹⁸¹. Based on miRNA profiling, miR-l25b was found to be decreased in HNSCC tissues and cell lines. Loss of miR-l25b might be partially result from the hyperm ethylation of its promoter. In addition, miR-l25b might exert its tumor suppressive role by deregulating TACSTD2 and mitogen- activated protein kinase pathway¹⁸². Taken together, MiRNAs are powerful molecules that play an essential role in the tumorigenesis of various types of cancers including HNSCC. They are attractive as biomarkers for the cancer detection, diagnosis, and prognosis assessment, which is very important for therapeutic guidance. However, currently there are a number of candidate miRNAs emerging as promising biomarkers. From a clinical and practical view, it is impossible to test these miRNAs simultaneously. Therefore, well designed prospective clinical trials with large patient groups are urgently needed to explore the most powerful miRNAs for clinical applications.

III. Pharmaceutical Formulations and Administration

[0124] In certain embodiments, the invention also provides compositions comprising 1, 2, 3 or more anti-cancer agents (e.g., an anti-PLOD3 agent) with one or more of the following: a pharmaceutically acceptable diluent; a carrier; a solubilizer; an emulsifier; a preservative; and/or an adjuvant. Such compositions may contain an effective amount of at least one anti cancer agent. Thus, the use of one or more anti-cancer agents that are provided herein in the preparation of a pharmaceutical composition of a medicament is also included. Such compositions can be used in the treatment of a variety of cancer s. In certain embodiments the treatment is for squamous cell carcinoma such as HNSCC.

[0125] The anti-cancer agents may be formulated into therapeutic compositions in a variety of dosage forms such as, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, nanoparticles, and/or injectable or infusible solutions. The preferred form depends upon the mode of administration and the particular disease targeted. The compositions also preferably include pharmaceutically acceptable vehicles, carriers, or adjuvants, well known in the art.

[0126] Acceptable formulation components for pharmaceutical preparations are nontoxic to recipients at the dosages and concentrations employed. In addition to the anti- cancer agents that are provided, compositions may contain components for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable materials for formulating pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as acetate, borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethyl enedi amine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counter ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants (see Remington's Pharmaceutical Sciences , 18 th Ed., (A. R. Gennaro, ed.), 1990, Mack Publishing Company), hereby incorporated by reference.

[0127] Formulation components are present in concentrations that are acceptable to the site of administration. Buffers are advantageously used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 4.0 to about 8.5, or alternatively, between about 5.0 to 8.0. Pharmaceutical compositions can comprise TRIS buffer of about pH 6.5-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.

[0128] The pharmaceutical composition to be used for in vivo administration is typically sterile. Sterilization may be accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization may be conducted either prior to or following lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle, or a sterile pre-filled syringe ready to use for injection.

[0129] The above compositions can be administered using conventional modes of delivery including, but not limited to, intravenous, intraperitoneal, oral, intralymphatic, subcutaneous administration, intraarterial, intramuscular, intrapleural, intrathecal, and by perfusion through a regional catheter. Local administration to a tumor in question is also contemplated by the present invention. When administering the compositions by injection, the administration may be by continuous infusion or by single or multiple boluses. For parenteral administration, the anti -metastatic agents may be administered in a pyrogen-free, parenterally acceptable aqueous solution comprising the desired anti-cancer agents in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which an one or more anti-cancer agents are formulated as a sterile, isotonic solution, properly preserved.

[0130] Once the pharmaceutical composition of the invention has been formulated , it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready -to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0131] The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

[0132] For the therapeutic agents described herein, alone or as part of a pharmaceutical composition, such doses are between about 0.001 mg/kg and 1 mg/kg body weight, preferably between about 1 and 100 pg/kg body weight, most preferably between 1 and 10 pg/kg body weight. Therapeutically effective doses will be easily determined by one of skill in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, the patient's age, weight, height, sex, previous medical history and the judgment of the treating physician. [0133] In some methods of the invention, the cancer cell is a PLOD3 overexpressing cancer cell. The cancer can be a tumor cell. The cancer cell may be in a patient. The patient may have a solid tumor. In such cases, embodiments may further involve performing surgery on the patient, such as by resecting all or part of the tumor. Compositions may be administered to the patient before, after, or at the same time as surgery. In additional embodiments, patients may also be administered directly, endoscopically, intratracheally, intratumorally, intravenously, intralesionally, intramuscularly, intraperitoneally, regionally, percutaneously, topically, intrarterially, intravesically, or subcutaneously. Therapeutic compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times, and they may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

[0134] Methods of treating cancer may further include administering to the patient chemotherapy or radiotherapy, which may be administered more than one time. Chemotherapy includes, but is not limited to, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, taxotere, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate, gemcitabine, oxaliplatin, irinotecan, topotecan, or any analog or derivative variant thereof. Radiation therapy includes, but is not limited to, X-ray irradiation, UV- irradiation, g-irradiation, electron-beam radiation, or microwaves. Moreover, a cell or a patient may be administered a microtubule stabilizing agent, including, but not limited to, taxane, as part of methods of the invention. It is specifically contemplated that any of the compounds or derivatives or analogs, can be used with these combination therapies.

[0135] In some embodiments, the PLOD3 overexpressing cancer that is administered the composition(s) described herein may be a bladder, blood, bone, bone marrow, brain, breast, colorectal, esophagus, gastrointestine, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testicular, tongue, or uterus cell. In certain aspects the cancer is HNSCC. In certain embodiments, a subject can have acute myeloid leukemia (AML), bladder urothelial carcinoma (BLCA), breast cancer (BRCA), colon adenocarcinoma (COAD), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), glioblastoma (GBM), kidney chromophobe (RICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), stomach and esophageal carcinoma (STES), or thyroid carcinoma (THCA).

IV. Examples

[0136] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Role of PLOD3 in Head And Neck Squamous Cell Carcinoma (HNSCC)

A. Materials and Methods

[0137] Cell culture. SCC1, SCC23, SCC17, SCC5, SCC6, UM1 and UM2 HNSCC cell lines were cultured in the Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 pg/mL). Normal human oral keratinocytes (NHOKs) and normal human epidermal keratinocytes (NHEKs) were cultured in EpiLife media supplemented with the human keratinocyte growth supplement (Invitrogen, Carlsbad, CA, USA). The cells were maintained at 37°C, 5% CCk in a humidified cell culture incubator and passaged when they reached 90-95% confluence.

[0138] Immunohistochemistry. For immunohistochemistry analyses, formalin-fixed paraffin-embedded sections were deparaffmized by sequential washing with xylene, 100% ethanol, 95% ethanol, 80% ethanol and PBS. The sections were incubated with 0.3% H2O2 in methanol for 5 min to quench the endogenous peroxidase activity. The slides were blocked in PBS with 5% BSA for 30 min and then incubated overnight with a 1 : 100 dilution of anti- PLOD3 primary antibody (Proteintech, Chicago, IL, USA) at 4 °C. After sections were rinsed with PBS, they were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen) for 2 h at room temperature. [0139] MTT assay. After 24 h of serum starvation, the cells were seeded into a 96-well plate at a density of 3000 cells/well. At the indicated time points, 20pL of MTT (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS at 5 mg/ml was added to each culture well. Followed by incubation for 4 h at 37 °C, the supernatant was then discarded and the precipitate dissolved in 200 pl of dimethyl sulfoxide (DMSO, Sigma). The absorbance of each well was measured using a Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA) at 570 nm.

[0140] 5-ethynyl-2’-deoxyuridine assay. The 5-ethynyl-2'-deoxyuridine (EdU) detection kit (Invitrogen) was used to evaluate cell proliferation. According to the manufacturer's instructions, the cells were treated with 10 pmol/L EdU for 2 h at 37 °C and fixed with 3.7% formaldehyde for 15 min. After cells were washed with 3% BSA in PBS, they were treated with 0.5% Triton X-100 (Sigma-Aldrich) for 20 min and stained with 1 x Click-iT reaction cocktail for 30 minutes at room temperature. After PBS wash, Hoechst 33342 dye was used to stain the cell nucleus at room temperature for 30 min. Images were captured under a confocal laser scanning microscope (Carl Zeiss, Jena, Germany).

[0141] Cell colony formation assay. The cells were seeded into a 6-well plate at a density of 3000 cells/well in 2 mL medium. After incubation at 37 °C for 14 days, the cells were washed three times with PBS and stained with 0.5% crystal violet for 30 min.

[0142] Soft agar assay. Soft agar assays were performed in six-well plates. Each well contained a 2 ml 0.6% low-gelling temperature agarose base layer on which cancer cells were suspended in 2 ml of 0.35% low-gelling temperature agarose in medium. Anchorage- independent growth was assessed by counting colonies after two weeks.

[0143] Migration assay. The migration assays were performed with the Transwell™ Chambers (BD Biosciences. Bedford, MA, USA). Following 24 h serum starvation, trypsinized cells (1 x 10⁵ cells /well) were re-suspended in DMEM containing 0.1% FBS and added to upper chamber of transwell inserts. DMEM supplemented with 10% FBS was used in the lower chamber to act as a chemoattractant. After 24 h, cells that had migrated through the membrane were fixed and stained with the crystal violet. The percentage of migrated area per field under light microscopy (Carl Zeiss) in four random fields were calculated by using Image J (FijiVersion, National Institutes of Health, Bethesda, MD, USA).

[0144] Transwell Matrigel invasion assay. The invasion assays were performed with the Transwell Matrigel Invasion Chambers (BD Biosciences. Bedford, MA, USA). Following 24 h serum starvation, trypsinized cells (5 x 10⁵ cells /well) were resuspended in DMEM containing 0.1% FBS and added to upper chamber of transwell inserts. DMEM supplemented with 10% FBS was used in the lower chamber to act as a chemoattractant. After 48 h, cells that had invaded through the membrane were fixed and stained with the crystal violet. The percentage of invaded area per field under light microscopy in four random fields were calculated by using Image J.

[0145] Real-time PCR. Total RNA was isolated from cancer cells using the Quick-RNA™ kit (Zymo Research Corp, Irvine, CA, ETSA) according to the manufacturer's instructions. First- strand complementary DNA synthesis was performed using the Superscript III Reverse Transcriptase (Invitrogen). The complementary DNA levels were amplified with Light Cycler 480@ SYBR Green I MasterMix (Roche, Applied Science, Indianapolis, IN, ETSA) using the CFX96 Real-Time PCR detection system (Bio-Rad Laboratories Inc., Hercules, CA, ETSA). GADPH and U6 were used as the internal controls for mRNA and miRNA analysis respectively.

[0146] Western blotting. The protein samples were loaded and separated on a 4-12% Bis- Tris NuPAGE gel (Invitrogen) and transferred onto a nitrocellulose membrane using a Trans- blot SD semi-dry transfer cell (Bio-Rad). The membranes were blocked for 2 h at room temperature in TBST buffer containing 5% nonfat milk (Santa Cruz Biotech), and incubated with first primary antibodies overnight at 4 °C, followed by HRP-linked secondary antibodies (GE Healthcare). Signal detection was performed with the ECL-Plus Western blotting reagent kit (GE Healthcare).

[0147] siRNA transfection. Cancer cells were transfected with double-stranded siRNAs using the RNAiMAX transfection regent (Invitrogen) according to the manufacturer's instruction. siPLOD3 (siRNAl (SEQ ID NO:3) and siRNA2 (SEQ ID NO:4)) were mixed with the transfection reagent respectively and then added to the cell culture. siRNA control (siCTRL) was used as negative control. After overnight incubation, the siRNAs were removed and the cells were further cultured in fresh media for 48 h before any additional experiments.

[0148] Production of PLOD3 recombinant lentiviral vectors. PLOD3 was cloned into the pGCL-GFP vector and constructs were confirmed by sequencing. Recombinant lentiviral vectors and packaging vectors were then transfected into 293T cells. The supernatant liquor containing lentiviruses was harvested 72 h after transfection. The lentiviruses were then purified by ultracentrifugation, and the titer of lentiviruses was determined. The empty vector was packaged as a negative control. The cancer cells were infected with the lentiviruses at a multiplicity of infection of 40.

[0149] Analysis of PLOD 3 gene expression in tumor samples from existing tumor databases. The normalized datasets comparing the gene expression profiles between HNSCC and normal controls were downloaded from NCBI GEO databases. The accession number was GSE663, GSE37991, GSE23558, GSE25099, GSE85514 and GSE30784 respectively.

[0150] The clinical information and RNASeq V2 datasets of cancer patients were obtained from The Cancer Genome Atlas (TCGA) database (available at EIRL tcga- data.nci.nih.gov/tcga) to determine the clinical significance of PLOD3 in cancers. Briefly the mRNA expression levels were log2-transformed and X tile software (EIRL medicine.yale.edu/lab/rimm/research/software.aspx) was used to find out the best cutoff point to divide the cancer patients into high/low PLOD3 expression groups. Kaplan-Meier overall survival curves were generated for patients whose follow-up data were available. The log-rank test was used to analyze survival differences between the two groups.

[0151] Xenograft mouse model. Male athymic nude mice (BALB/C-nu/nu, 4-5 weeks old) were used for the in vivo studies. Before injection, cancer cells were re-suspended in a 1 : 1 mixture of Matrigel (BD Biosciences) and PBS. A 100-m1 cell suspension containing 1 x 10⁶ PLOD3 knockdown or PLOD3 overexpression cells was subcutaneously injected into the dorsal flank of each mouse. For the control groups, mice received 100 mΐ injections of the respective control cells in corresponding concentrations. The tumor diameters and weight were measured and recorded.

B. Results

[0152] PLOD3 is overexpressed in HNSCC tissues and cell lines. Seven publicly available expression profiling data sets downloaded from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) Data Portal were used. The results revealed that the expression of PLOD3 was significantly increased in HNSCC cancer tissues compared with normal control tissues. Interestingly, in GSE85514 and GSE30784, the expression level of PLOD3 was significantly higher in tumor tissues than that in oral precancerous lesions. In addition, HNSCC patients in the high PLOD3 expression group suffered a significantly lower overall survival than those in the low PLOD3 expression group (FIG. 1). [0153] PLOD3 expression was analyzed in other types of cancers by using the data from TCGA database. The expression level of PLOD3 was significantly increased in cancer tissues including acute myeloid leukemia (AML), bladder urothelial carcinoma (BLCA), breast cancer (BRCA), colon adenocarcinoma (COAD), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), glioblastoma (GBM), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), stomach and esophageal carcinoma (STES) and thyroid carcinoma (THCA) compared to their respective adjacent normal tissues (FIG. 2A-2B). In addition, the cancer patients with higher expression of PLOD3 had poorer long-term overall survival rates in many other types of cancers including GBM, KIRC, LIHC, LUSC, low grade glioma (LGG), LUAD, ovarian cancer (OV), cervical squamous cell carcinoma (CESC), THCA and sarcoma (SARC) (FIG.

3)·

[0154] To assess the potential involvement of PLOD3 in HNSCC development and progression, the expression levels of PLOD3 in the HNSCC cell lines UM1, UM2, SCC5, SCC6, SCC17, SCC1 and SCC23 were investigated as well as in normal cell lines, NHOK and NHEK.

[0155] In comparison with the control normal cell lines, the results of western blot analysis showed that the expression of PLOD3 was upregulated in all HNSCC cell lines. In addition, PLOD3 was higher in high invasive oral cancer cell lines UM1 and UM5 compared to low invasive oral cancer cell lines UM2 and UM6 (FIG. 4A-4B). Then, the protein expression levels of PLOD3 were examined in fourteen paired tumor and non-tumor tissue samples. As predicted, PLOD3 expression levels were significantly increased in tumor tissues compared to non-tumor tissues (FIG. 4C-4D). PLOD3 expression was further examined in paraffin-embedded sections of 144 HNSCC and 20 normal specimens by IHC. The results revealed the staining intensity of PLOD3 was gradually increased in normal tissues, well-differentiated tumor, moderately differentiated tumor and poorly differentiate tumor (FIG. 5A-5B).

[0156] PLOD3 plays pro-carcinogenic roles in HNSCC. The significant upregulation of PLOD3 in HNSCC cell lines and tissues implied that PLOD3 may have a pro-carcinogenic role in HNSCC tumorigenesis. To test this concept, depletion of PLOD3 was achieved by transfecting siRNA oligos (siRNAl (SEQ ID NO:3) and siRNA2 (SEQ ID NO:4)) against PLOD3 into SCC1 cells (a cell line derived from tongue squamous cell carcinoma and SCC23 cells (a cell line derived from laryngeal squamous cell carcinoma). Significant losses ofPLOD3 at both mRNA and protein levels were achieved by siPLOD3 -treated cells with respect to control (siCTRL) transfection (FIG. 6A-6C). Cell proliferation was then assessed by MTT assay, which showed a drastic decrease of proliferative capacity after siPLOD3 -treatment with respect to siCTRL treatment (FIG. 6D). This was supported by the results of the colony formation and soft agar assay: as significantly less numbers and smaller size of colonies formed in siPLOD3 -treated group than those in siCTRL treated group (FIG. 6E-6F). In addition, the percentage of EdU positive cells was significantly lower in the cells with siPLOD3 transfection when compared to those with siCTRL transfection (FIG. 6G). The mobility of cancer cells was significantly decreased upon PLOD3 knockdown as indicated by a transwell migration assay, demonstrating significantly fewer number of cells penetrated the pores of the membrane than siCTRL treated group (FIG. 6H). Matrigel invasion assay measurement showed that knockdown of PLOD3 also significantly suppressed the invasive ability of HNSCC cells (FIG. 61). Notably, it was observed that the knockdown of PLOD3 in SCC1 and SCC23 significantly decreased xenograft tumor volume and weight compared with the controls (FIG. 7).

[0157] PLOD3 overexpressing lentiviruses (PLOD3 OV) and control lentiviruses (CTRL) were consructed. Cancer cells were successfully infected with lentiviral particles and the expression level of PLOD3 protein was remarkably increased after lenti-PLOD3 infection (FIG. 8A-8B). The results showed that the cells with PLOD3 overexpression had higher proliferative capacity and stronger colony forming ability compared with cells infected with the control lentiviruses (FIG. 8C-8E). In addition, PLOD3 overexpression led to a higher percentage of EdU positive cells (FIG. 8F). SCC1 and SCC23 cells with PLOD3 overexpression were more proficient than control lentivirus-infected cells at migrating through the membrane (FIG. 8G- 8H). PLOD3 upregulation also significantly enhanced the invasive capacity of cancer cells. Overexpression of PLOD3 in SCC1 and SCC23 significantly increased xenograft tumor volume and weight compared with the controls (FIG. 9).

Example 2

Tumorigenesis of HNSCC and the MiR-124-3p/PLOD3/FAK/PI3K/AKT Pathway A. Materials and Methods

[0158] MiRNA mimic/inhibitor transfection. Cancer cells were transfected with miR-l24- 3p mimic/inhibitor using the RNAiMAX transfection regent (Invitrogen) according to the manufacturer's instruction. miR-l24-3p mimic/inhibitor was mixed with the transfection reagent respectively and then added to the cell culture. Scrambled miRNA control was used as negative control. After overnight incubation, the medium was removed, and the cells were further cultured in fresh media for 48 h before any additional experiments.

[0159] 3’- UTR luciferase reporter assay. For dual luciferase reporter assays, cancer cells were co-transfected with 3'-UTR-PLOD3 vector constructs (SwitchGear Genomics, Carlsbad, CA, USA), and either miR-l24-3p mimic or scramble miRNA using RNAiMAX reagent. Luciferase activity was measured at 48 h after transfection using the Dual-Luciferase Reporter Assay System (SwitchGear Genomics), according to the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

B. Results

[0160] miR-124-3p directly targets PLOD 3 by binding to 3' UTR region. Multiple miRNA target prediction programs showed that miR-l24-3p was a conserved and powerful regulator of PLOD3 (data not shown). The direct interaction between miR-l24-3p and 3'-UTR PLOD3 was investigated by luciferase assay. The PLOD3 3'-UTR shows high complementarity to the seed sequence of miR-l24-3p. In addition, the 3'-UTR PLOD3 sequence that miR-l24-3p targeted was highly conserved across different species. Luciferase assays showed that the miR- l24-3p mimic significantly suppressed relative luciferase activity compared to the scramble control, suggesting that miR-l24-3p directly targets 3'-UTR mRNA of PLOD3. In addition, miR-l24-3p mimic remarkably reduced PLOD3 expression at the protein level.

[0161] miR-124-3p plays a tumor suppressive role in HNSCC. The qRT-PCR analysis confirmed that transfection of miR-l24-3p mimic resulted in miR-l24-3p upregulation in SCC1 and SCC23 cells (FIG. 11 A). MTT, soft agar, colony formation and EdU assays indicated that miR-l24-3p overexpression suppressed proliferation of both SCC1 and SCC23 cells (FIG. 11B-11E). Migration assay also displayed that miR-l24-3p upregulation markedly prohibited CRC cell migration (FIG. 11F). Additionally, overexpression of miR-l24-3p led to decrease of invasion ability in both SCC1 and SCC23 cells (FIG. 11G).

[0162] Conversely, downregulation of miR-l24-3p was successfully achieved by transfecting the cells with miR-l24-3p inhibitor (FIG. 12A). The results showed downregulation of miR-l24-3p promoted the proliferation, migration and invasion capacity of both SCC1 and SCC23 cells (FIG. 12B-12G). [0163] Overexpression of PLOD3 partially rescues the suppression of miR-124-3p. To elucidate whether the suppressive effect of miR-l24-3p was mediated by repression of PLOD3 in HNSCC cells, the inventors evaluated whether ectopic expression of PLOD3 could rescue the suppressive effect of miR-l24-3p. miR-l24-3p mimic was transfected into cancer cells infected with PLOD3 overexpressing lentiviruses or control lentiviruses and western blot results showed that the levels of PLOD3 were higher in cells infected with PLOD3 overexpressing lentivirus following miR-l24-3p mimic transfection (FIG. 13A). MTT, soft agar, colony formation and EdU assays showed that PLOD3 overexpression could partially abrogate the effects mediated by miR-l24-3p in both SCC1 and SCC23 cells (FIG. 13B-13E). At the same time, the migration and invasion assays showed that PLOD3 upregulation could partially restore the migration and invasion activity compared with the cells infected with control lentivirus (FIG. 13F-13G).

[0164] RNA-seq analysis reveals potential downstream pathways regulated by PLOD 3. To understand the PLOD3 -mediated molecular events in HNSCC cells, a genome-wide analysis was conducted to globally characterize PLOD3 -regulated transcriptome changes. Total RNAs of SCC1 and SCC23 cells treated with siCTRL or siPLOD3 oligos were subjected to transcriptomic sequencing. Compared with the siCTRL-treated cells, a total of 3275 (1631 upregulated and 1644 downregulated) and 3146 (1497 upregulated and 1649 downregulated) transcripts were found to be significantly aberrantly expressed in siPLOD3 -treated SCC1 and SCC23 cells respectively (FIG. 14A-14B). In addition, most deregulated genes were overlapped between SCC1 and SCC23 (FIG. 14C). GSEA showed that the differentially expressed genes in both cell lines were enriched in pathways including cell cycle, homologous recombination, purine and pyrimidine metabolism, focal adhesion, regulation of actin cytoskeleton, chemokine signaling pathway, spliceosome and ubiquitin mediated proteolysis. Interestingly, the morphology of cancer cells became round following PLOD3 downregulation (FIG. 15). As focal adhesion kinase (FAR) is closely involved in cellular adhesion and spreading processes, it was hypothesized that FAR might be deregulated following PLOD3 suppression.

[0165] FAK phosphorylation (pFAK) is important for the pro-oncogenic role PLOD 3 in HNSCC. Western blot analysis showed that the expression level of pFAK, pPI3K and pAKT was significantly downregulated following PLOD3 downregulation, while their levels were upregulated when PLOD3 was overexpressed (FIG. 16). To explore whether the tumor promoting role of PLOD 3 was mediated by phosphorylation of FAK in HNSCC cells, the inventors evaluated whether suppression of FAK phosphorylation could inhibited the tumor promoting of PLOD3. siFAK or siCTRL was transfected into cancer cells infected with PLOD3 overexpressing lentivirus. The expression levels of FAK and pFAK were both significantly reduced following siFAK transfection. MTT, soft agar, colony formation and EdU assays showed that knockdown of FAK could significantly inhibited the proliferation of PLOD3 overexpression cancer cells (FIG. 17B-17E). Furthermore, the migration and invasion arrays showed that FAK silencing repressed the migration and invasion capacities of PLOD3 overexpression cancer cells (FIG. 17F-17G).

[0166] It was demonstrated that miR-l24-3p directly bound to the 3' LTTR region of PLOD3. LTpregulation of miR-l24-3p significantly suppressed the proliferation, migration and invasion capacity of HNSCC cells, while downregulation of miR-l24-3p promoted the oncogenic activities of cancer cells, indicating miR-l24-3p might play a tumor suppressive role in HNSCC. Moreover, overexpression of PLOD3 could partially restore the proliferation, migration and invasion capacity of cancer cells following miR-l24-3p upregulation, suggesting that miR-l24-3p is a powerful and functional upstream regulator of PLOD3. RNA seq analysis revealed that PLOD3 downregulation affected many important pathways such as cell cycle, regulation of the actin cytoskeleton and focal adhesion. In addition, the morphology of cancer cells became round following PLOD3 inhibition. Moreover, FAK-PI3K-AKT pathway was demonstrated to be an important downstream effector for the oncogenic role of PLOD3 in HNSCC.

[0167] The results showed that miR-l24-3p was an upstream regulator of PLOD3, and overexpression of PLOD3 partially rescued the tumor suppressive effects of miR-l24-3p, indicating that PLOD3 is a functional downstream effector of miR-l24-3p. As demonstrated PLOD3 played a tumor promoting role in HNSCC, the downregulation of miR-l24-3p in HNSCC might result in the PLOD3 overexpression, which subsequently enhance carcinogenesis.

[0168] RNA-seq was performed to investigate the downstream pathways regulated by PLOD3. It was observed that cell cycle was one of the most significantly deregulated pathways following PLOD3 suppression in both HNSCC cell lines, which was in line with phenotype studies. Other interesting affected pathways included homologous recombination, purine and pyrimidine metabolism, focal adhesion, regulation of actin cytoskeleton, chemokine signaling pathway, spliceosome and ubiquitin mediated proteolysis. Homologous recombination (HR) might exert a fundamental role in carcinogenesis. HR might serve as an errant DNA repair mechanism that can lead to loss of heterozygosity or genetic rearrangements ²¹¹. Purines and pyrimidines serve as the building blocks of DNA and RNA. In addition, they are important for energy conservation and transport, coenzymes synthesis and phospholipid and carbohydrate metabolism ^{212 213} Therefore, defective in purine and pyrimidine metabolism significantly affect any system in a cell. Adhesion of cells to the ECM is crucial for maintaining the normal cellular morphology, migration, proliferation, survival, and differentiation. One interesting finding was that the cells with PLOD3 knockdown gradually became round and died. In addition, most of the siPLOD3 treated cancer cells failed to re-attach to the plates when they were passaged. As PLOD3 is indispensable for the collagen synthesis, it was reasonable to observe thatPLOD3 deletion affected the adhesion of cancer cells. Furthermore, the functional analysis showed that focal adhesion and regulation of actin cytoskeleton were the most influenced pathways following PLOD3 downregulation. Focal adhesions are multi-protein complexes that are essential for cell contact with the extracellular matrix (ECM) and cellular communication between the ECM and the cell cytoplasm ^{2 I4 2 L}\ Chemokines are soluble factors which are critical for regulating immune cell recruitment during inflammatory responses and defense against foreign pathogens. Deregulated chemokine signaling pathways have been implicated in cancer development ²¹⁶.

[0169] FAR is a non-receptor tyrosine kinase that acts as a central regulator in signaling networks arising from focal adhesions. As focal adhesion and regulation of actin cytoskeleton were significantly affected after PLOD3 suppression, it is contemplated that FAR might be a downstream target of PLOD3. The results showed that PLOD3 inhibition suppressed the expression level of pFAK in both cell lines, while pFAK was increased when PLOD3 was overexpressed, indicating PLOD3 was an upstream regulator of pFAK. In addition, targeted inhibition of FAK significantly abrogated the tumor promoting effects of PLOD3. Activation of FAK has been demonstrated to play a pivotal role in promoting cancer progression and metastasis. PI3K-AKT is a downstream signaling pathway of FAK. Interestingly, the expression levels of pPI3K-pAKT were significantly decreased following PLOD3 inhibition, and vice versa , indicating PLOD3 downregulation significantly suppressed FAK-PI3K-AKT signaling pathway. The PI3K-AKT signaling pathway is frequently deregulated in almost all types of cancers including HNSCC ²²⁰²²¹.

Claims

1. An inhibitory nucleic acid that interferes with a procollagen-lysine, 2-oxoglutarate 5- di oxygenase 3 (PLOD3) mRNA, the inhibitory nucleic comprising 10 to 50 nucleotides that are complementary to the PLOD3 mRNA.

2. The inhibitory nucleic acid of claim 1, wherein the inhibitory nucleic is an shRNA, siRNA, or miRNA.

3. The inhibitory nucleic acid of claim 1, wherein the inhibitory nucleic is a siRNA.

4. The inhibitory nucleic acid of claim 3, wherein the siRNA comprises 15 to 25 nucleotides that are complementary to nucleic acid of SEQ ID NO: 1.

5. The inhibitory nucleic acid, of claim 2, wherein the siRNA targets nucleotides 720 to 760 or nucleotides 800 to 840 of human PLOD3 mRNA (SEQ ID NO: l).

6. The inhibitory nucleic acid of any one of claims 1 to 5, wherein the inhibitory nucleic acid comprises the nucleotide sequence AGAAGGAAAUGGAGAAAUA (SEQ ID NO:3) or CCACAGAGCUGCUGAAGAA (SEQ ID NO:4).

7. The inhibitory nucleic acid of any one of claims 1 to 6, wherein the inhibitory nucleic acid is a double stranded or hairpin inhibitory nucleic acid.

8. The inhibitory nucleic acid of any one of claims 1 to 7, further comprising one or more modifications.

9. The inhibitory nucleic acid of any one of claims 1 to 8, wherein the one or more modifications is a modification of a base moiety, a sugar moiety, a phosphate moiety, a phosphate-sugar backbone, or a combination thereof.

10. A composition comprising the inhibitory nucleic acid of any one of claims 1 to 9 and a carrier and/or excipient.

11. The composition of claim 10, wherein the carrier is a liposomal carrier or a nanoparticle carrier.

12. An expressing cassette encoding an inhibitory nucleic acid of any one of claims 1 to 9.

13. The expression cassette of claim 12, further comprised in an expression vector.

14. A method of reducing PLOD3 expression comprising administering an interfering nucleic acid of any one of claims 1 to 9 to a cell.

15. A method of treating a PLOD3 overexpressing cancer comprising administering an interfering nucleic acid of any one of claims 1 to 9.

16. The method of claim 15, further comprising administering a miR-l24-3p nucleic acid or a miR-l24-3p mimic.

17. The method of claim 16, wherein miR-l24-3p nucleic acid comprises the nucleic acid sequence UAAGGCACGCGGUGAAUGCC (SEQ ID NO:6).

18. The method of claim 15, further comprising administering a second cancer therapy.

19. The method of claim 18, wherein the second cancer therapy is surgery, a

chemotherapy, radiotherapy, or immunotherapy.

20. The method of claim 18, wherein the second cancer therapy comprises administering cisplatin and/or 5-fluorouracil (5-FU).

21. The method of claim 18, wherein the second cancer therapy comprises administering cetuximab or other anti-epidermal growth factor antibody.

22. The method of claim 19, wherein the immunotherapy comprises administering an anti-PLOD3 antibody that inhibits the activity of PLOD3.

23. The method of claim 15, wherein the subject has or suspected of having a squamous cell carcinoma.

24. The method of claim 23, wherein the squamous cell carcinoma is a head and neck squamous cell carcinoma (HNSCC).

25. The method of claim 15, wherein the subject has acute myeloid leukemia (AML), bladder urothelial carcinoma (BLCA), breast cancer (BRCA), colon adenocarcinoma (COAD), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), glioblastoma (GBM), kidney chromophobe (RICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), stomach and esophageal carcinoma (STES), or thyroid carcinoma (THCA).

26. The method of claim 15, wherein the siRNA is incorporated into a liposomal carrier or nanoparticle carrier.

27. A method of treating cancer comprising administering a cancer therapy to a subject having a cancer overexpressing PLOD3.

28. The method of claim 27, further comprising determining the levels of PLOD3 in the a subject’s cancer.

29. A method for assessment of a subjected of having squamous cell carcinoma comprising measuring the level of PLOD3 in a sample from the subject comprising suspected cancer cells and determining the expression level of PLOD3 protein.