TW202231280A - Method for inhibiting tumor progression or determining tumor progression state in gastric cancer - Google Patents

Method for inhibiting tumor progression or determining tumor progression state in gastric cancer Download PDF

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TW202231280A
TW202231280A TW110104309A TW110104309A TW202231280A TW 202231280 A TW202231280 A TW 202231280A TW 110104309 A TW110104309 A TW 110104309A TW 110104309 A TW110104309 A TW 110104309A TW 202231280 A TW202231280 A TW 202231280A
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phf8
gastric cancer
expression
inhibitor
pkcα
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TW110104309A
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王雯靜
曾琳蘆
鄭欣弘
喻秋華
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國立清華大學
財團法人國家衛生研究院
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Abstract

The present invention provides use of a composition for preparing a pharmaceutical composition for inhibiting tumor progression in a subject suffering from gastric cancer, wherein the composition comprises an inhibitor of targeting PHF8-c-Jun-PKCα-Src-PTEN axis, or a pharmaceutically acceptable salt thereof. Furthermore, the presents also provides a method of determining a tumor progression state in a subject suffering from gastric cancer, which comprises providing a sample from the subject; comparing the PHF8 expression level of the sample with a predetermined threshold value; and determining the tumor progression state of the subject, wherein the PHF8 expression level of the sample is higher than the predetermined threshold value indicating the subject suffering from a late stage of gastric cancer.

Description

抑制胃癌腫瘤生長或判斷胃癌腫瘤生長階段之方法 Method for inhibiting the growth of gastric cancer tumor or judging the growth stage of gastric cancer tumor

本發明涉及組合物在製備用於抑制患有胃癌的受試者的腫瘤進展的藥物組合物中的用途，並且還涉及抑制或確定患有胃癌的受試者的腫瘤進展的方法。 The present invention relates to the use of a composition in the manufacture of a pharmaceutical composition for inhibiting tumor progression in a subject with gastric cancer, and also to methods of inhibiting or determining tumor progression in a subject with gastric cancer.

胃癌(Gastric cancer，GC)是全世界所有惡性腫瘤中第二大死亡原因。超過50%的新胃癌病例發生在WHO西太平洋地區。胃癌分期系統通常遵循由美國癌症聯合委員會(American Joint Committee on Cancer，AJCC)的TNM系統，該系統包括三個關鍵特徵：腫瘤的範圍(大小)(T)；擴散到附近的淋巴結(N)；和遠處擴散(轉移)(M)。根據TNM系統分為五個階段：階段零(0)和階段I至IV(1至4)，該階段提供了描述癌症的常用方法。 Gastric cancer (GC) is the second leading cause of death among all malignant tumors worldwide. More than 50% of new gastric cancer cases occur in the WHO Western Pacific Region. The gastric cancer staging system generally follows the TNM system by the American Joint Committee on Cancer (AJCC), which includes three key features: the extent (size) of the tumor (T); spread to nearby lymph nodes (N); and distant spread (metastasis) (M). There are five stages according to the TNM system: stage zero (0) and stages I to IV (1 to 4), which provide a common way of describing cancer.

外科切除仍然是胃癌治療的黃金標準，尤其是對於早期的胃癌。然而，胃癌通常在早期沒有症狀。因此，超過40%的胃癌患者在 初診時被診斷出患有轉移性疾病。轉移性胃癌的預後仍然很差，接受最佳支持治療的患者中位生存期為4.3個月，接受聯合化療的患者中位生存期為11個月。最近二十年來接受化療的胃癌患者的生存一直保持穩定，是由於新細胞毒性藥物開發使得死亡具有重大突破。最近的治療趨勢是結合了標靶療法與化學療法。在一項多中心ToGA研究中，抗HER2標靶治療組(trastuzumab)的中位總生存期為13.8個月，而單純化療組為11.1個月，顯示出了HER2陽性胃癌患者(12-20%)可能會從這種方法中受益。然而，目前仍無有效的標靶治療用於晚期HER2陰性病例。 Surgical resection remains the gold standard for gastric cancer treatment, especially for early gastric cancer. However, stomach cancer usually has no symptoms in its early stages. Therefore, more than 40% of gastric cancer patients are Diagnosed with metastatic disease at initial visit. The prognosis for metastatic gastric cancer remains poor, with median survival of 4.3 months for patients receiving best supportive care and 11 months for patients receiving combination chemotherapy. Survival of gastric cancer patients treated with chemotherapy has remained stable for the last two decades due to a major breakthrough in death due to the development of new cytotoxic drugs. A recent treatment trend is a combination of targeted therapy and chemotherapy. In a multicenter ToGA study, the median overall survival in the anti-HER2-targeted therapy group (trastuzumab) was 13.8 months compared with 11.1 months in the chemotherapy-only group, showing that patients with HER2-positive gastric cancer (12-20% ) may benefit from this approach. However, there is still no effective targeted therapy for advanced HER2-negative cases.

以基因體為主的分子表徵分析提供病患分層、預後和制定治療的一新途徑。未進行化學療法和放射療法的295個主要胃癌組織的癌基因體圖譜數據揭示了DNA甲基化、體細胞基因改變、基因表達和蛋白質體事件的差異化模式。關鍵的基因改變主要存在於癌基因/抑癌基因中，包括TP53、KRAS、ARID1A、PIK3CA、ERBB3、PTEN和HLA-B。亞洲癌症研究小組對300個胃癌樣品進行的全球基因表達譜分析和標靶測序證實了常見驅動復發的基因突變，包括TP53、ARID1A、PIK3CA、KRAS、PTEN和ERBB3的突變。 Genome-based molecular characterization provides a new avenue for patient stratification, prognosis, and treatment planning. Oncogenome profiling data from 295 major gastric cancer tissues untreated with chemotherapy and radiation revealed differential patterns of DNA methylation, somatic gene alterations, gene expression, and proteosome events. Key genetic alterations were primarily found in oncogenes/tumor suppressor genes, including TP53, KRAS, ARID1A, PIK3CA, ERBB3, PTEN, and HLA-B. Global gene expression profiling and targeted sequencing of 300 gastric cancer samples by the Asian Cancer Research Group confirmed common mutations driving recurrence, including those in TP53, ARID1A, PIK3CA, KRAS, PTEN and ERBB3.

除了先前研究中揭示的突變模式之外，不能忽視促進疾病進 展、甚至會與染色體重塑和增加不穩定性的表觀遺傳異常。在表觀遺傳調節劑中，組蛋白離胺酸脫甲基酶(histone lysine demethylases，KDM)已引起了廣泛的關注，因為它們催化從組蛋白中去除關鍵甲基，這可能極大地影響基因表達和染色質的空間組織，甚至能重新組合具有增強的惡性能力的致瘤程序。 In addition to the mutational patterns revealed in previous studies, promoting disease progression cannot be ignored. development, and even epigenetic abnormalities associated with chromosomal remodeling and increased instability. Among epigenetic regulators, histone lysine demethylases (KDMs) have attracted considerable attention because they catalyze the removal of key methyl groups from histones, which can greatly affect gene expression and the spatial organization of chromatin, even recombining tumorigenic programs with enhanced malignant capacity.

植物同源域(Plant homeodomain，PHD)指蛋白8(PHF8，也稱為KDM7B)是組蛋白脫甲基酶家族的成員。多項研究顯示出PHF8在某些惡性腫瘤中過度表現，包括***癌、食道癌、肺癌、喉和下嚥鱗狀細胞癌、急性淋巴細胞白血病和胃癌，這表明PHF8可能是致癌的表觀遺傳調控因子。但是，PHF8的潛在機制參與調節胃癌進程仍需要研究。 Plant homeodomain (PHD) finger protein 8 (PHF8, also known as KDM7B) is a member of the histone demethylase family. Multiple studies have shown that PHF8 is overexpressed in certain malignancies, including prostate cancer, esophageal cancer, lung cancer, laryngeal and hypopharyngeal squamous cell carcinoma, acute lymphoblastic leukemia, and gastric cancer, suggesting that PHF8 may be an oncogenic epigenetic regulator factor. However, the underlying mechanism of PHF8 involved in regulating gastric cancer progression still needs to be studied.

本發明提供了在一患有胃癌的受試者中抑制腫瘤進展的方法，其包括向所述受試者施用一藥物組合物，所述藥物組合物包含靶向PHF8-c-Jun-PKCα-Src-PTEN軸的抑製劑或可接受的鹽。 The present invention provides a method of inhibiting tumor progression in a subject with gastric cancer, comprising administering to the subject a pharmaceutical composition comprising targeting PHF8-c-Jun-PKCa- Inhibitors or acceptable salts of the Src-PTEN axis.

一方面，本發明提供了一組合物在製備用於抑制一患有胃癌的受試者的腫瘤進展的藥物組合物中的用途，其中所述組合物包含靶向PHF8-c-Jun-PKCα-Src-PTEN軸的抑製劑或其可藥用鹽。 In one aspect, the present invention provides the use of a composition in the manufacture of a pharmaceutical composition for inhibiting tumor progression in a subject with gastric cancer, wherein the composition comprises targeting PHF8-c-Jun-PKC alpha - An inhibitor of the Src-PTEN axis or a pharmaceutically acceptable salt thereof.

本發明還提供一種確定一患有胃癌的受試者中的腫瘤進展狀態的方法，其包括：(a)從該患有為癌的受試者提供一樣品；(b)檢測該樣品中PHF8的表達水平；(c)將該樣品的PHF8表達水平與一預定閾值進行比較；以及(d)確定該患有胃癌的受試者的腫瘤進展狀態，其中該樣品的PHF8表達水平高於該預定閾值，表明該患有胃癌的受試者為一胃癌晚期。 The present invention also provides a method of determining the state of tumor progression in a subject with gastric cancer, comprising: (a) providing a sample from the subject with gastric cancer; (b) detecting PHF8 in the sample (c) comparing the PHF8 expression level of the sample to a predetermined threshold; and (d) determining the tumor progression status of the subject with gastric cancer, wherein the PHF8 expression level of the sample is higher than the predetermined threshold Threshold, indicating that the subject with gastric cancer is an advanced gastric cancer.

【詳細發明內容】[Detailed content of the invention]

在本發明中PHD指狀蛋白8(PHF8或KDM7B)與HER2陰性胃癌中差的存活率具有顯著相關。耗盡PHF8的表現顯著降低了胃癌細胞和小鼠異種移植物中的癌症進展。基於微陣列分析，PHF8調節參與細胞遷移/運動的基因。此外，PHF8在編碼PKCα的PRKCA啟動子上與c-Jun相互作用。耗損PHF8或PKCα的表現極大地上提高PTEN表現並減少了Src活化，這可以通過異位表達活性Src來恢復。用Midostaurin或Bosutinib處理的MKN28在體外和斑馬魚模型中均顯著抑制了遷移。PHF8、PKCα和PTEN的免疫組織化學分析顯示PHF8和PKCα之間呈正相關。此外，PHF8-PKCα的高表達與不良預後顯著相關。因此，本發明表明了PHF8-PKCα-Src-PTEN途徑是HER2陰性的晚期胃癌中的預後/治療標靶。 In the present invention PHD finger protein 8 (PHF8 or KDM7B) was significantly associated with poor survival in HER2 negative gastric cancer. Depletion of PHF8 expression significantly reduced cancer progression in gastric cancer cells and mouse xenografts. Based on microarray analysis, PHF8 regulates genes involved in cell migration/motility. Furthermore, PHF8 interacts with c-Jun on the PRKCA promoter encoding PKCα. Depleting expression of PHF8 or PKCα greatly enhanced PTEN expression and reduced Src activation, which could be restored by ectopic expression of active Src. MKN28 treated with midostaurin or bosutinib significantly inhibited migration both in vitro and in zebrafish models. Immunohistochemical analysis of PHF8, PKCα and PTEN showed a positive correlation between PHF8 and PKCα. Furthermore, high expression of PHF8-PKCα was significantly associated with poor prognosis. Thus, the present invention demonstrates that the PHF8 -PKCα-Src-PTEN pathway is a prognostic/therapeutic target in HER2-negative advanced gastric cancer.

除非本文另有定義，否則本發明中採用的科學和技術術語應具有本領域普通技術人員通常理解和使用的含義。另外，除非上下文另外要求，否則應理解，單數術語應包括相同的複數形式，而複數術語應包括單數。具體地，如本文和權利要求書中所使用的，單數形式“一個”和“一種”包括複數形式，除非上下文另外明確指出。 Unless otherwise defined herein, scientific and technical terms employed in the present invention shall have the meanings commonly understood and used by those of ordinary skill in the art. In addition, unless otherwise required by context, it should be understood that singular terms shall include the same plural and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms "a" and "an" include the plural forms unless the context clearly dictates otherwise.

因此，本發明提供了在患有胃癌的一受試者中抑制一腫瘤進展的方法，其中包括向該受試者施用一藥物組合物，其中該藥物組合物包含靶向PHF8-c-Jun-PKCα-Src-PTEN軸的一抑製劑或其可接受的鹽。 Accordingly, the present invention provides a method of inhibiting the progression of a tumor in a subject having gastric cancer, comprising administering to the subject a pharmaceutical composition, wherein the pharmaceutical composition comprises targeting PHF8-c-Jun- An inhibitor of the PKCα-Src-PTEN axis or an acceptable salt thereof.

一方面，本發明提供了組合物在製備用於抑制患有胃癌的受試者的腫瘤進展的藥物組合物中的用途，其中所述組合物包含靶向PHF8-c-Jun-PKCα-Src-PTEN軸的抑製劑或其可藥用鹽。 In one aspect, the present invention provides the use of a composition in the manufacture of a pharmaceutical composition for inhibiting tumor progression in a subject with gastric cancer, wherein the composition comprises targeting PHF8-c-Jun-PKCa-Src- An inhibitor of the PTEN axis or a pharmaceutically acceptable salt thereof.

在一個優選的實施方案中，其中該腫瘤進展包括腫瘤生長、癌症擴散和轉移。 In a preferred embodiment, wherein the tumor progression includes tumor growth, cancer spread and metastasis.

在另一個實施方案中，其中所述胃癌是HER2陰性胃癌。 In another embodiment, wherein the gastric cancer is HER2 negative gastric cancer.

在一個優選的實施中，其中該抑製劑是一PHF8抑製劑。在一個優選的實施方案中，其中該PHF8抑製劑是用於抑制PHF8表達 的一核苷酸抑製劑。在一個更優選的實施方案中，其中該核苷酸抑製劑是RNA寡核苷酸抑製劑，其包含小干擾RNA(siRNA)，短髮夾RNA(shRNA)或微小RNA寡核苷酸(miRNA)。 In a preferred implementation, wherein the inhibitor is a PHF8 inhibitor. In a preferred embodiment, wherein the PHF8 inhibitor is used to inhibit PHF8 expression of a nucleotide inhibitor. In a more preferred embodiment, wherein the nucleotide inhibitor is an RNA oligonucleotide inhibitor comprising small interfering RNA (siRNA), short hairpin RNA (shRNA) or microRNA oligonucleotide (miRNA ).

在另一個實施方案中，其中該抑製劑能夠破壞PHF8與c-Jun之間的相互作用，進而抑制激活PRKCA表達。 In another embodiment, wherein the inhibitor is capable of disrupting the interaction between PHF8 and c-Jun, thereby inhibiting activation of PRKCA expression.

在另一個實施方案中，其中該抑製劑是一PKCα抑製劑。在一個優選的實施方案中，其中該PKCα抑製劑是Midostaurin。 In another embodiment, wherein the inhibitor is a PKCa inhibitor. In a preferred embodiment, wherein the PKCa inhibitor is Midostaurin.

在另一個優選的實施方案中，其中該抑製劑是一Src抑製劑。在一個優選的實施方案中，該Src抑製劑是Bosutinib。 In another preferred embodiment, wherein the inhibitor is a Src inhibitor. In a preferred embodiment, the Src inhibitor is Bosutinib.

在另一個實施例中，其中該抑製劑是PKCα的抑製劑和Src抑製劑的組合。在一個優選的實施方案中，其中該抑製劑是Midostaurin和Bosutinib的組合。 In another embodiment, wherein the inhibitor is a combination of an inhibitor of PKCa and a Src inhibitor. In a preferred embodiment, wherein the inhibitor is a combination of Midostaurin and Bosutinib.

本發明還提供一種確定一患有胃癌的受試者中的腫瘤進展狀態的方法，其包括：(a)從該患有胃癌的受試者提供一樣品；(b)檢測該樣品中PHF8的表達水平；(c)將該樣品的PHF8表達水平與一預定閾值進行比較；以及(d)確定該患有胃癌的受試者的腫瘤進展狀態，其中該樣品的PHF8表達水平高於該預定閾值，表明該患有胃 癌的受試者為一胃癌晚期。 The present invention also provides a method of determining tumor progression status in a subject with gastric cancer, comprising: (a) providing a sample from the subject with gastric cancer; (b) detecting the level of PHF8 in the sample expression level; (c) comparing the PHF8 expression level of the sample to a predetermined threshold; and (d) determining the tumor progression status of the subject with gastric cancer, wherein the PHF8 expression level of the sample is above the predetermined threshold , indicating that the patient with gastric The cancer subject was an advanced gastric cancer.

在一個實施例中，其中該預定閾值為基於對早期和晚期胃癌患者中PHF8表達的預測分析來確定。 In one embodiment, wherein the predetermined threshold value is determined based on predictive analysis of PHF8 expression in patients with early and advanced gastric cancer.

在一個實施例中，其中該預定閾值是通過受試者工作特徵曲線(ROC)方法獲得，其中該預定閾值對應於當達到其最大AUC(ROC曲線下的面積)值。在一個優選的實施方案中，其中該AUC值高於0.8。 In one embodiment, wherein the predetermined threshold is obtained by a receiver operating characteristic (ROC) method, wherein the predetermined threshold corresponds to when its maximum AUC (area under the ROC curve) value is reached. In a preferred embodiment, wherein the AUC value is higher than 0.8.

在一個實施方案中，根據早期和晚期胃癌患者中PHF8的表達確定受試者工作特徵曲線(ROC)方法。 In one embodiment, the receiver operating characteristic (ROC) method is determined based on the expression of PHF8 in patients with early and advanced gastric cancer.

在一個實施方案中，其中該PHF8表達水平是一PHF8基因表達或一PHF8蛋白表達。在一個優選實施例中，PHF8表達水平是PHF8基因表達水平。在一個實施例中，其中該PHF8基因表達水平為透過定量即時聚合酶連鎖反應或原位雜交法來確定。 In one embodiment, wherein the PHF8 expression level is a PHF8 gene expression or a PHF8 protein expression. In a preferred embodiment, the PHF8 expression level is the PHF8 gene expression level. In one embodiment, wherein the PHF8 gene expression level is determined by quantitative real-time polymerase chain reaction or in situ hybridization.

在一個實施例中，其中該PHF8表達水平是一PHF8蛋白表達。在一個更優選的實施例中，其中該PHF8蛋白表達水平係透過過免疫印跡，免疫組織化學或免疫磁性還原來確定。在一個實施例中，其中該PHF8蛋白表達水平係通過免疫組織化學測定來確定。 In one embodiment, wherein the PHF8 expression level is a PHF8 protein expression. In a more preferred embodiment, wherein the PHF8 protein expression level is determined by immunoblotting, immunohistochemistry or immunomagnetic reduction. In one embodiment, wherein the PHF8 protein expression level is determined by immunohistochemical assay.

在一個實施例中，其中該PHF蛋白表達水平是根據一免疫反應評分(IRS)來確定，其中該免疫反應評分是通過將染色強度乘以陽性細胞的比例來計算。免疫反應評分(IRS)是基於強度等級(評分：1-3)和陽性腫瘤細胞比例(評分：1-4)的評分。在一個實施例中，其中該IRS值在1-4之間表示弱表達，IRS值在6-8之間表示中等表達，而IRS值在9-12之間表示強表達。 In one embodiment, wherein the PHF protein expression level is determined according to an immune response score (IRS), wherein the immune response score is calculated by multiplying the staining intensity by the proportion of positive cells. The Immune Response Score (IRS) is a score based on the intensity scale (score: 1-3) and the proportion of positive tumor cells (score: 1-4). In one embodiment, wherein the IRS value between 1-4 indicates weak expression, an IRS value between 6-8 indicates moderate expression, and an IRS value between 9-12 indicates strong expression.

在一個實施例中，其中該胃癌的腫瘤進展狀態由PHF8表達水平決定，其中該PHF8表達水平為中等表達到強表達，表明該患有胃癌的受試者為一胃癌晚期。 In one embodiment, the tumor progression status of the gastric cancer is determined by the expression level of PHF8, wherein the expression level of PHF8 is moderate to strong expression, indicating that the subject with gastric cancer is an advanced gastric cancer.

根據以上描述，在一優選實施例中，步驟(c)中的該預定閾值基於一IRS值，其中該IRS值高於5，表明該患有胃癌的受試者為一胃癌晚期。 According to the above description, in a preferred embodiment, the predetermined threshold in step (c) is based on an IRS value, wherein the IRS value is higher than 5, indicating that the subject suffering from gastric cancer is an advanced gastric cancer.

在本發明的另一方面，進一步包括檢測PKCα表達水平，其中該PKCα表達水平高於一預定閾值，則該該患有胃癌的受試者具有不良預後。在一個實施例中，該預定閾值基於一IRS值，其中該IRS值高於5。 In another aspect of the present invention, further comprising detecting the expression level of PKCα, wherein the expression level of PKCα is higher than a predetermined threshold, the subject with gastric cancer has a poor prognosis. In one embodiment, the predetermined threshold is based on an IRS value, wherein the IRS value is higher than five.

在一個實施例中，其中該胃癌的晚期是從第II期到第IV期。 在另一個實施例中，其中該胃癌晚期是指具有淋巴結轉移或遠處轉移的腫瘤。 In one embodiment, wherein the advanced stage of the gastric cancer is from stage II to stage IV. In another embodiment, the advanced gastric cancer refers to a tumor with lymph node metastasis or distant metastasis.

圖1、在HER2陰性胃癌中KDMs與具有較差臨床結果的相關分析結果。圖1A顯示了5年總生存期的Kaplan-Meier曲線以及圖1B顯示了以KDM1B表達水平分層的HER2陰性胃癌患者首次進展的Kaplan-Meier曲線。根據作為臨界值的最佳表現閾值(FDR=0.1)，將患者分為高表達組(高，實線)和低表達組(低，虛線)。通過對數秩檢驗確定統計學顯著性。 Figure 1. Correlation analysis results of KDMs and poor clinical outcomes in HER2-negative gastric cancer. Figure 1A shows Kaplan-Meier curves for 5-year overall survival and Figure 1B shows Kaplan-Meier curves for first progression of HER2-negative gastric cancer patients stratified by KDM1B expression levels. Patients were divided into high expression group (high, solid line) and low expression group (low, dashed line) according to the best performance threshold (FDR=0.1) as the cut-off value. Statistical significance was determined by the log-rank test.

圖2、PHF8的高表達與胃癌轉移有關。圖2A顯示了來自Oncomine數據庫的PHF8在胃粘膜(N)、原發部位腫瘤(P)和轉移部位腫瘤(M)中的表達。** p<0.01.(雙尾學生t檢驗)。圖2B顯示了使用Oncomine，從所選擇的研究中進行腫瘤(T)和相鄰正常組織(N)之間的PHF8表達的比較。** p<0.01(配對t檢驗)。 Figure 2. High expression of PHF8 is associated with gastric cancer metastasis. Figure 2A shows the expression of PHF8 from the Oncomine database in gastric mucosa (N), primary site tumors (P) and metastatic site tumors (M). **p<0.01. (Two-tailed Student's t-test). Figure 2B shows a comparison of PHF8 expression between tumor (T) and adjacent normal tissue (N) from selected studies using Oncomine. **p<0.01 (paired t-test).

圖3、PHF8對於MKN28細胞在體外和體內的增殖和遷移的重要性。圖3A顯示了以西方墨點法分析了攜帶對照pLKO或shPHF8構建體(# 1或# 2)的慢病毒感染的MKN28中PHF8表達水平的分析。肌動蛋白作為內部控制。圖3B顯示了在MKN28中耗盡PHF8顯示出降低細胞增殖的程度。圖3C顯示了在注射6週後對照和shPHF8 MKN28-luc異種移植的影像。圖3D顯示每週測量直至6週的腫瘤體積。數據表示為平均值±SD，SD來自五隻小鼠。**P<0.01(單因子變異數分析)。圖3E顯示了耗盡MKN28中PHF8顯示降低細胞遷移的程度。在斑馬魚遷移實驗中，耗盡PHF8的表現顯示出在MKN28中具有遷移行為的胚胎比例顯著降低。圖3F顯示出了斑馬魚胚胎的代表性螢光圖像，在3dpi(比例尺，100μm)下顯示細胞散佈(上圖)或無遷移(下圖)，並且圖3G示出了定量結果。括號中顯示了胚胎總數。* P<0.05(雙尾學生t檢驗)。來自圖3B-3G的數據表示為三個重複的平均值±SD。* P<0.05，** P<0.01(雙尾學生t檢驗)。 Figure 3. Importance of PHF8 for proliferation and migration of MKN28 cells in vitro and in vivo. Figure 3A shows the Western blot analysis of PHF8 expression levels in lentiviral infected MKN28 carrying control pLKO or shPHF8 constructs (#1 or #2). Actin as an internal control. Figure 3B shows the extent to which depletion of PHF8 in MKN28 was shown to reduce cell proliferation. Figure 3C shows images of control and shPHF8 MKN28-luc xenografts 6 weeks after injection. Figure 3D shows tumor volumes measured weekly up to 6 weeks. Data are presented as mean ± SD, SD from five mice. **P<0.01 (one-way ANOVA). Figure 3E shows the extent to which depletion of PHF8 in MKN28 was shown to reduce cell migration. In zebrafish migration experiments, depletion of PHF8 showed a significant reduction in the proportion of embryos with migratory behavior in MKN28. Figure 3F shows representative fluorescence images of zebrafish embryos at 3 dpi (scale bar, 100 μm) showing cell spreading (upper panel) or no migration (lower panel), and FIG. 3G shows quantitative results. The total number of embryos is shown in parentheses. *P<0.05 (two-tailed Student's t-test). Data from Figures 3B-3G are presented as the mean ± SD of three replicates. *P<0.05, **P<0.01 (two-tailed Student's t-test).

圖4、PHF8對於MKN45細胞在體外和體內的增殖和遷移的重要性。圖4A顯示了攜帶對照pLKO或shPHF8構建體(# 1或# 2)的慢病毒感染的MKN45中PHF8含量分析，然後進行嘌呤黴素 的篩選。Lysates were analyzed by western blotting.裂解物通過西方墨點法進行分析。圖4B顯示在MKN45中耗盡PHF8顯示出降低細胞增殖的程度。圖4C顯示了使用跨孔細胞遷移測定法分析在MKN45中耗盡PHF8所降低細胞遷移的程度。在1dpi和3dpi下進行螢光顯微鏡分析。圖4D顯示了斑馬魚胚胎代表性的螢光圖像，在3dpi下顯示細胞散佈(上圖)或無遷移(下圖)。比例尺，100μm。圖4E顯示出在MKN45中耗盡PHF8所減少具有遷移行為的胚胎的比例。括號中顯示了胚胎總數。數據來自3項獨立研究。* p<0.05，** p<0.01(雙尾學生t檢驗)。圖4B-C的數據表示為3次重複±SD的平均值。** p<0.01(雙尾學生t檢驗)。 Figure 4. Importance of PHF8 for proliferation and migration of MKN45 cells in vitro and in vivo. Figure 4A shows analysis of PHF8 content in lentiviral-infected MKN45 carrying control pLKO or shPHF8 constructs (#1 or #2) followed by puromycin filter. Lysates were analyzed by western blotting. Lysates were analyzed by western blotting. Figure 4B shows the extent to which depletion of PHF8 in MKN45 was shown to reduce cell proliferation. Figure 4C shows the extent to which depletion of PHF8 in MKN45 reduces cell migration using a transwell cell migration assay. Fluorescence microscopy analysis was performed at 1dpi and 3dpi. Figure 4D shows representative fluorescence images of zebrafish embryos at 3 dpi showing cell spread (upper panel) or no migration (lower panel). Scale bar, 100 μm. Figure 4E shows that depletion of PHF8 in MKN45 reduces the proportion of embryos with migratory behavior. The total number of embryos is shown in parentheses. Data are from 3 independent studies. *p<0.05, **p<0.01 (two-tailed Student's t-test). The data of Figures 4B-C are presented as the mean of 3 replicates ± SD. **p<0.01 (two-tailed Student's t-test).

圖5、PHF8調控與細胞遷移和細胞運動有關的基因表達。圖5A顯示了減弱PHF8表現的MKN28的微陣列分析結果，其中以DAVID功能註釋分析具有兩倍或更少改變的基因。圖5B顯示了在細胞遷移/細胞運動/細胞運動類別中下降的基因的qRT-PCR分析。數據通過與GAPDH mRNA水平進行標準化。圖5C顯示了在MKN28中以IgG或抗PHF8抗體針對參與在細胞遷移類別基因的啟動子區域上PHF8佔有率的ChIP分析。數據表示為三個重複樣本的平均值±SD * P<0.05，** P<0.01(雙尾學生t檢驗)。 Figure 5. PHF8 regulates gene expression related to cell migration and cell motility. Figure 5A shows the results of microarray analysis of MKN28 attenuating PHF8 expression, wherein genes with two-fold or less alterations were analyzed with DAVID functional annotation. Figure 5B shows qRT-PCR analysis of genes that declined in cell migration/cell motility/cell motility categories. Data were normalized to GAPDH mRNA levels. Figure 5C shows ChIP analysis of PHF8 occupancy on promoter regions of genes involved in cell migration classes with IgG or anti-PHF8 antibodies in MKN28. Data are presented as mean±SD of three replicates *P<0.05, **P<0.01 (two-tailed Student's t-test).

圖6、PRKCA的啟動子區域中PHF8佔有率的分析結果。圖6A顯示了用所示的四組引子對PRRKA的啟動子區域中的PHF8富集的ChIP分析。IgG作為對照。圖6B-D顯示了在三個區域中pLKO和shPHF8(# 1和# 2)MKN28中PRKCA啟動子上的PHF8、H3K9me2和H4K20me1的增加倍數(圖6B：-1,100至-906，圖6C：-932至-797，以及圖6D：-925至-744)。圖6A-6D表示為三個重複樣本的平均值±SD * P<0.05，** P<0.01(雙尾學生t檢驗)。 Figure 6. Analysis results of PHF8 occupancy in the promoter region of PRKCA. Figure 6A shows ChIP analysis of PHF8 enrichment in the promoter region of PRRKA with the four sets of primers indicated. IgG served as a control. Figures 6B-D show the fold increase of PHF8, H3K9me2 and H4K20me1 on the PRKCA promoter in pLKO and shPHF8 (#1 and #2) MKN28 in three regions (Figure 6B: -1,100 to -906, Figure 6C: - 932 to -797, and Figure 6D: -925 to -744). Figures 6A-6D are expressed as mean ± SD of three replicates *P<0.05, **P<0.01 (two-tailed Student's t-test).

圖7、PKCα作為PHF8/c-JUN的下游標的以促進胃癌進展。圖7A顯示了在pLKO、shPHF8 # 1或shPHF8 # 2 MKN28中PKCα的外源性表達的分析。以對照(ctl)或PKCα(PRKCA)表現載體轉染細胞，然後進行西方墨點法分析。圖7B顯示PKCα的外源性表達分別顯著恢復shPHF8 # 1和shPHF8 # 2中的細胞增殖程度。圖7C顯示shPHF8 MKN28中PKCα的外源性表達顯著恢復了遷移活性。圖7B-7C的數據表示為3次重複±SD的平均值。** p<0.01(雙尾學生t檢驗)。 Figure 7. PKCα acts as a downstream target of PHF8/c-JUN to promote gastric cancer progression. Figure 7A shows analysis of exogenous expression of PKCa in pLKO, shPHF8 #1 or shPHF8 #2 MKN28. Cells were transfected with control (ctl) or PKCa (PRKCA) expression vectors and then subjected to Western blot analysis. Figure 7B shows that exogenous expression of PKCα significantly restored the degree of cell proliferation in shPHF8 #1 and shPHF8 #2, respectively. Figure 7C shows that exogenous expression of PKCα in shPHF8 MKN28 significantly restored migratory activity. The data in Figures 7B-7C are presented as the mean of 3 replicates ± SD. **p<0.01 (two-tailed Student's t-test).

圖8、PKCα作為PHF8/c-JUN的下游標的，以促進MKN45中的胃癌進展。圖8A顯示了在pLKO、shPHF8 # 1或shPHF8 # 2 MKN45中外源性表達PKCα。用對照(ctl)或PKCα(PRKCA)表 現載體轉染細胞，然後進行西方墨點法分析。圖8B顯示PKCα的外源性表達分別顯著恢復了shPHF8 # 1和shPHF8 # 2中的細胞增殖程度。圖8C顯示shPHF8 MKN45中PKCα的外源性表達顯著恢復了遷移活性。圖8D顯示了PHF8和c-Jun之間內源性的關聯。MKN45細胞裂解物以兔IgG、抗PHF8或抗c-Jun進行IP分析。圖8E顯示了在耗盡PHF8細胞的中PRKCA啟動子上c-Jun富集的ChIP分析。圖8F顯示了AP-1報導質體和內部β-半乳糖苷酶控制質體共同轉染的MKN45細胞(pLKO、shPHF8 # 1或shPHF8 # 2)中的AP-1報導活性。圖8B-8C和8E-8F為3次重複±SD的平均值。** p<0.01(雙尾學生t檢驗)。 Figure 8. PKCα as a downstream target of PHF8/c-JUN to promote gastric cancer progression in MKN45. Figure 8A shows exogenous expression of PKCa in pLKO, shPHF8 #1 or shPHF8 #2 MKN45. Tables with control (ctl) or PKCα (PRKCA) Cells were transfected with the current vector and then subjected to Western blot analysis. Figure 8B shows that exogenous expression of PKCα significantly restored the degree of cell proliferation in shPHF8 #1 and shPHF8 #2, respectively. Figure 8C shows that exogenous expression of PKCα in shPHF8 MKN45 significantly restored migratory activity. Figure 8D shows the endogenous association between PHF8 and c-Jun. MKN45 cell lysates were subjected to IP analysis with rabbit IgG, anti-PHF8 or anti-c-Jun. Figure 8E shows ChIP analysis of c-Jun enrichment on the PRKCA promoter in depleted PHF8 cells. Figure 8F shows AP-1 reporter activity in MKN45 cells (pLKO, shPHF8 #1 or shPHF8 #2) co-transfected with AP-1 reporter plastids and internal β-galactosidase control plastids. Figures 8B-8C and 8E-8F are the mean of 3 replicates ± SD. **p<0.01 (two-tailed Student's t-test).

圖9、ICAM-1是PHF8的下游標的，可促進遷移活性。圖9A顯示了pLKO、shPHF8 # 1或shPHF8 # 2 MKN45中ICAM-1的qRT-PCR分析。數據透過GAPDH mRNA水平進行標準化。圖9B顯示了分別對pLKO和shPHF8(# 1和# 2)MKN28和MKN45中ICAM-1啟動子上的PHF8富集的ChIP分析。圖9C顯示耗盡ICAM-1在MKN28和MKN45中分別表現出降低的遷移活性。 Figure 9. ICAM-1 is a downstream target of PHF8 and promotes migratory activity. Figure 9A shows qRT-PCR analysis of ICAM-1 in pLKO, shPHF8 #1 or shPHF8 #2 MKN45. Data were normalized by GAPDH mRNA levels. Figure 9B shows ChIP analysis of PHF8 enrichment on the ICAM-1 promoter in pLKO and shPHF8 (#1 and #2) MKN28 and MKN45, respectively. Figure 9C shows that depletion of ICAM-1 exhibited reduced migratory activity in MKN28 and MKN45, respectively.

圖10、PHF8與c-JUN相互作用並調節AP-1報告基因的活性。圖10A顯示了PHF8和c-Jun之間的內源性結合。MKN28細胞 裂解物用於IgG、抗PHF8或抗c-Jun的IP分析。圖10B顯示了在耗盡PHF8的細胞中PRKCA啟動子上c-Jun富集的ChIP分析。圖10C顯示了用AP-1報導質體和內部β-半乳糖苷酶控制質體共同轉染的細胞(pLKO、shPHF8 # 1或shPHF8 # 2)中的AP-1報導活性。圖10D顯示了如圖中所示以載體(Flag、野生型PHF8(WT)或無活性PHF8(H247A)、HA或HA-c-JUN以及β-半乳糖苷酶內部對照)共轉染的MKN28中的AP-1報導分子活性。圖10B-10D的數據表示為三個重複樣本的平均值±SD。* P<0.05，** P<0.01(雙尾Student t檢驗)。 Figure 10. PHF8 interacts with c-JUN and regulates the activity of the AP-1 reporter gene. Figure 10A shows endogenous binding between PHF8 and c-Jun. MKN28 cells Lysates were used for IP analysis of IgG, anti-PHF8 or anti-c-Jun. Figure 10B shows ChIP analysis of c-Jun enrichment on the PRKCA promoter in PHF8-depleted cells. Figure 1OC shows AP-1 reporter activity in cells (pLKO, shPHF8 #1 or shPHF8 #2) co-transfected with AP-1 reporter plastids and internal β-galactosidase control plastids. Figure 10D shows MKN28 co-transfected with vectors (Flag, wild-type PHF8 (WT) or inactive PHF8 (H247A), HA or HA-c-JUN, and β-galactosidase internal control) as indicated in the figure AP-1 reporter activity in . The data of Figures 10B-10D are presented as the mean ± SD of three replicates. *P<0.05, **P<0.01 (two-tailed Student's t-test).

圖11、PHF8和c-Jun之間的相互作用區域比對的結果。如圖中所示以Flag-PHF8和HA-c-Jun質體共同轉染的HEK293T細胞中，用抗-Flag和抗-HA進行Co-IP分析(圖11A：具有抗Flag抗體的IP；圖11B：具有抗HA抗體的IP)。 Figure 11. Results of the alignment of the interaction regions between PHF8 and c-Jun. Co-IP analysis was performed with anti-Flag and anti-HA in HEK293T cells co-transfected with Flag-PHF8 and HA-c-Jun plastids as indicated in the figure (FIG. 11A: IP with anti-Flag antibody; FIG. 11B: IP with anti-HA antibody).

圖12、西方點墨微陣列晶片分析結果。pLKO、shPHF8 # 1和shPRKCA # 2 MKN28之間訊號蛋白質倍數變化的熱圖。 Figure 12. Western blotting microarray wafer analysis results. Heat map of signaling protein fold changes between pLKO, shPHF8 #1, and shPRKCA #2 MKN28.

圖13、PHF8-PKCα軸通過SRC激活調節了PTEN的去穩定作用。圖13A顯示了處理或未處理MG132的shPHF8(# 1和# 2)和shPRKCA(# 1和# 2)MKN28中的PTEN蛋白質表現量。圖13B顯示了以對照(ctl)或表現PRCCA的載體轉染的pLKO或shPHF8(# 1或# 2)MKN28中Src和pSrc(Y419)的含量。圖13C顯示了用激酶活化Src(Y419D)或激酶死亡Src(Y419F)載體轉染的shPRKCA(# 1和# 2)細胞中的PTEN蛋白質表現量。 Figure 13. The PHF8-PKCα axis modulates PTEN destabilization through SRC activation. Figure 13A shows PTEN protein expression in shPHF8 (#1 and #2) and shPRKCA (#1 and #2) MKN28 treated or untreated with MG132. Figure 13B shows pLKO or shPHF8 (# 1 or #2) Content of Src and pSrc(Y419) in MKN28. Figure 13C shows PTEN protein expression in shPRKCA (#1 and #2) cells transfected with kinase-activated Src (Y419D) or kinase-dead Src (Y419F) vectors.

圖14、PHF8-PKCα軸透過活化SRC調節了PTEN的去穩定作用。圖14A顯示了處理或未處理MG132的shPHF8(# 1和# 2)和shPRKCA(# 1和# 2)MKN28中的PTEN蛋白質表現量。圖14B顯示了用對照(ctl)或表達PRCCA的載體轉染的pLKO或shPHF8(# 1或# 2)MKN28中Src和pSrc(Y419)的含量。圖14C顯示了用激酶活化的Src(Y419D)或死亡的激酶的Src(Y419F)載體轉染的shPRKCA(# 1和# 2)MKN28細胞中的PTEN蛋白質表現量。 Figure 14. The PHF8-PKCα axis modulates PTEN destabilization through activation of SRC. Figure 14A shows the amount of PTEN protein expression in shPHF8 (#1 and #2) and shPRKCA (#1 and #2) MKN28 treated or untreated with MG132. Figure 14B shows the content of Src and pSrc(Y419) in pLKO or shPHF8 (#1 or #2) MKN28 transfected with control (ctl) or a PRCCA-expressing vector. Figure 14C shows PTEN protein expression in shPRKCA (#1 and #2) MKN28 cells transfected with kinase-activated Src (Y419D) or dead kinase Src (Y419F) vectors.

圖15、PHF8-PKCα-Src-PTEN的藥理抑製作用可在體外和體內阻斷胃癌進程。圖15A顯示了用0、1、2或4μM的Midostaurin(Mido)處理的MKN28中的PTEN蛋白質表現量。圖15B顯示了通過跨孔細胞遷移測定法分析以Mido處理的MKN28的遷移活性。圖15C顯示了用0、1、2或4μM Bosutinib(Bosu)處理的MKN28中的PTEN蛋白質表現量。圖15D顯示了用Bosu處理的MKN28的遷移活性。圖15B和15D的數據表示為三個重複的平均值±SD，** P<0.01(雙尾學生t檢驗)。使用Mido或Bosu進行的斑馬魚異種移 植測定。圖15E顯示出了斑馬魚胚胎的代表性螢光圖像，在3dpi下呈現細胞散佈(上圖)或無遷移(下圖)。青色，血管(比例尺，100μm)。圖15F顯示了在溶媒組或藥物處理組中具有遷移行為的胚胎的定量。括號中顯示了溶媒、Mido和Bosu中使用的胚胎總數。數據來自三項獨立研究。** P<0.01(雙尾學生t檢驗)。圖15G顯示了PHF8-c-Jun複合物對胃癌進展的貢獻的示意圖。 Figure 15. Pharmacological inhibition of PHF8-PKCα-Src-PTEN blocks gastric cancer progression in vitro and in vivo. Figure 15A shows PTEN protein expression in MKN28 treated with 0, 1, 2 or 4 μM of Midostaurin (Mido). Figure 15B shows the migration activity of Mido-treated MKN28 analyzed by a transwell cell migration assay. Figure 15C shows PTEN protein expression in MKN28 treated with 0, 1, 2 or 4 μM Bosutinib (Bosu). Figure 15D shows the migratory activity of MKN28 treated with Bosu. Data in Figures 15B and 15D are presented as mean ± SD of three replicates, **P<0.01 (two-tailed Student's t-test). Zebrafish xenotransplantation using Mido or Bosu Plant assay. Figure 15E shows representative fluorescence images of zebrafish embryos at 3 dpi with spreading of cells (upper panel) or no migration (lower panel). Cyan, blood vessels (scale bar, 100 μm). Figure 15F shows quantification of embryos with migratory behavior in vehicle or drug treated groups. The total number of embryos used in vehicle, Mido and Bosu are shown in parentheses. Data are from three independent studies. **P<0.01 (two-tailed Student's t-test). Figure 15G shows a schematic representation of the contribution of the PHF8-c-Jun complex to gastric cancer progression.

圖16、PHF8-PKCα-Src-PTEN的藥理抑製作用可在體外和體內阻斷胃癌進程。圖16A顯示出了用0、1、2或4μM的Midostaurin(Mido)處理的MKN45中的PTEN蛋白質表現量的分析。圖16B顯示了以跨孔遷移測定法分析用Mido處理的MKN45的遷移活性。圖16C顯示了用0、1、2或4μM Bosutinib(Bosu)處理的MKN45中的PTEN蛋白質表現量。圖16D顯示了通過跨孔遷移測定法分析用Bosu處理的MKN45的遷移活性。圖16B和16D的結果數據表示為3次重複±SD的平均值。* p<0.05，** p<0.01(雙尾學生t檢驗)。使用Mido或Bosu進行的斑馬魚異種移植測定。在1dpi和3dpi下進行螢光顯微鏡分析。如圖16E顯示出了斑馬魚胚胎的代表性螢光圖像，其在3dpi下顯示細胞散佈(上圖)或無遷移(下圖)。青色，血管。比例尺，100μm。圖16F顯示了在溶媒組或藥物處理組中具有遷 移行為的胚胎的定量。括號中顯示了溶媒、Mido以及Bosu中使用的胚胎總數。數據來自3項獨立研究。* p<0.05(雙尾學生t檢驗)。 Figure 16. Pharmacological inhibition of PHF8-PKCα-Src-PTEN blocks gastric cancer progression in vitro and in vivo. Figure 16A shows the analysis of PTEN protein expression in MKN45 treated with 0, 1, 2 or 4 μM of Midostaurin (Mido). Figure 16B shows the migration activity of MKN45 treated with Mido analyzed in a transwell migration assay. Figure 16C shows PTEN protein expression in MKN45 treated with 0, 1, 2 or 4 μM Bosutinib (Bosu). Figure 16D shows the analysis of the migratory activity of MKN45 treated with Bosu by a transpore migration assay. The resulting data in Figures 16B and 16D are presented as the mean of 3 replicates ± SD. *p<0.05, **p<0.01 (two-tailed Student's t-test). Zebrafish xenograft assay using Mido or Bosu. Fluorescence microscopy analysis was performed at 1dpi and 3dpi. Figure 16E shows a representative fluorescence image of a zebrafish embryo showing cell spread (upper panel) or no migration (lower panel) at 3 dpi. Cyan, blood vessels. Scale bar, 100 μm. FIG. 16F shows that in the vehicle group or the drug-treated group with migration Quantification of migratory behavior of embryos. The total number of embryos used in vehicle, Mido, and Bosu are shown in parentheses. Data are from 3 independent studies. *p<0.05 (two-tailed Student's t-test).

圖17、PHF8-PKCα-Src-PTEN的藥理抑製作用阻斷了MKN28異種移植物在體內的腫瘤生長。將MKN28細胞皮下植入裸鼠中。當腫瘤分期至100±30mm3時，分別用溶媒、Midostaurin(50mg/kg)和Bosutinib(50mg/kg)治療小鼠。植入7週後拍攝MKN28-luc異種移植物的圖像，每1周至7週測量一次腫瘤體積。數據表示為平均值±SD，SD來自五隻小鼠。** p<0.01(單因子變異數分析)。 Figure 17. Pharmacological inhibition of PHF8-PKCa-Src-PTEN blocks tumor growth of MKN28 xenografts in vivo. MKN28 cells were implanted subcutaneously into nude mice. When tumors were staged to 100 ± 30 mm, mice were treated with vehicle, Midostaurin (50 mg/kg) and Bosutinib (50 mg/kg), respectively. Images of MKN28-luc xenografts were taken 7 weeks after implantation, and tumor volumes were measured every 1 to 7 weeks. Data are presented as mean ± SD, SD from five mice. **p<0.01 (one-way ANOVA).

圖18、Midostaurin與Bosutinib在斑馬魚胚胎中的亞致死劑量。187.5nM的Midostaurin與1.25μM的Bosutinib合併使用對從3dpf開始治療2天的斑馬魚胚胎沒有毒性。因此，選擇此劑量進行異種移植測定。 Figure 18. Sublethal doses of Midostaurin and Bosutinib in zebrafish embryos. Midostaurin at 187.5 nM in combination with Bosutinib at 1.25 μM was not toxic to zebrafish embryos treated for 2 days from 3 dpf. Therefore, this dose was chosen for the xenograft assay.

圖19、Midostaurin與Bosutinib對MKN28抑制體內細胞增殖和遷移具有協同作用。圖19A顯示了在斑馬魚異種移植模型中的MKN28增殖測定。通過公式(3dpi-1dpi)/1dpi計算面積變化百分比。圖19B示出了透過公式(3dpi-1dpi)/1dpi計算的強度變化百分比。圖19C顯示出了對於DMSO、Mido、Bosu或Mido+Bosu，斑馬魚胚胎在1dpi與3dpi時的代表性螢光圖像。圖19D顯示了具有遷移 行為的胚胎的百分比。在1dpi和3dpi下進行螢光顯微鏡分析。圖19E顯示出斑馬魚胚胎的代表性螢光圖像，其在3dpi下顯示細胞散佈(上圖)或無遷移(下圖)。 Figure 19. Midostaurin and Bosutinib have a synergistic effect on the inhibition of cell proliferation and migration in vivo by MKN28. Figure 19A shows MKN28 proliferation assay in a zebrafish xenograft model. The percent change in area is calculated by the formula (3dpi-1dpi)/1dpi. Figure 19B shows the percent change in intensity calculated through the formula (3dpi-1dpi)/1dpi. Figure 19C shows representative fluorescence images of zebrafish embryos at 1 dpi and 3 dpi for DMSO, Mido, Bosu or Mido+Bosu. Figure 19D shows migration with Percentage of embryos that behave. Fluorescence microscopy analysis was performed at 1dpi and 3dpi. Figure 19E shows representative fluorescence images of zebrafish embryos showing cell spread (upper panel) or no migration (lower panel) at 3 dpi.

圖20、從CGMH獲得的42位胃癌受試者中PHF8、PKCα和PTEN的臨床相關性。圖20A顯示免疫組織化學圖譜的代表性圖像。對每個胃組織標本進行了PHF8、PKCα和PTEN免疫染色(n=42)(比例尺，100μm)。圖20B顯示出胃癌樣品的IRS得分。IHC訊號的相關性用於兩組間的比較。圖20C表示PHF8和PKCα的相關性，圖20D表示PHF8和PTEN的相關性，圖20E表示PKCα和PTEN的相關性(E)。使用χ2檢驗評估統計學顯著性。圖20F顯示在患有胃癌的患者中，PHF8表達與腫瘤分期顯著相關。使用單因子變異數分析進行統計計算。圖20G示出了根據胃癌患者(n=42)中PHF8和PKCα表達水平的五年OS和5-y DFS分析。高：IRS

Figure 110104309-A0202-12-0013-136
8；低：IRS
Figure 110104309-A0202-12-0013-137
6。通過對數秩檢驗確定統計學顯著性(PHF8highPKCαhigh與PHF8lowPKCαlow)。 Figure 20. Clinical correlation of PHF8, PKCa and PTEN in 42 gastric cancer subjects obtained from CGMH. Figure 20A shows representative images of immunohistochemical profiles. Immunostaining for PHF8, PKCα, and PTEN was performed on each gastric tissue specimen (n=42) (scale bar, 100 μm). Figure 20B shows IRS scores for gastric cancer samples. Correlation of IHC signals was used for comparison between the two groups. Figure 20C shows the correlation between PHF8 and PKCa, Figure 20D shows the correlation between PHF8 and PTEN, and Figure 20E shows the correlation between PKCa and PTEN (E). Statistical significance was assessed using the χ test. Figure 20F shows that in patients with gastric cancer, PHF8 expression is significantly correlated with tumor stage. Statistical calculations were performed using one-way analysis of variance. Figure 20G shows five-year OS and 5-y DFS analysis according to the expression levels of PHF8 and PKCa in gastric cancer patients (n=42). High: IRS
Figure 110104309-A0202-12-0013-136
8; low: IRS
Figure 110104309-A0202-12-0013-137
6. Statistical significance was determined by the log-rank test (PHF8 high PKCα high vs. PHF8 low PKCα low ).

圖21、在從Biomax組織陣列ST1505購買的42位胃癌受試者中，PHF8、PKCα和PTEN的臨床相關性。圖21A示出了免疫組織化學圖譜的代表性圖像。對每個胃組織標本進行了PHF8、PKCα和 PTEN免疫染色(n=50)。比例尺，100μm。兩組之間IHC訊號的相關性。圖21B示出了PHF8和PKCα的相關性(B)。圖21C示出了PHF8和PTEN的相關性(C)。圖21D示出了PKCα和PTEN的相關性。使用卡方檢驗評估統計學顯著性。圖21E顯示了在患有胃癌的患者中，PHF8表達與腫瘤階段顯著相關。使用單因子變異數分析分析進行統計計算。 Figure 21. Clinical correlation of PHF8, PKCa and PTEN in 42 gastric cancer subjects purchased from Biomax tissue array ST1505. Figure 21A shows representative images of immunohistochemical profiles. PHF8, PKCα and PTEN immunostaining (n=50). Scale bar, 100 μm. Correlation of IHC signals between the two groups. Figure 21B shows the correlation of PHF8 and PKCa (B). Figure 21C shows the correlation of PHF8 and PTEN (C). Figure 21D shows the correlation of PKCa and PTEN. Statistical significance was assessed using the chi-square test. Figure 21E shows that in patients with gastric cancer, PHF8 expression is significantly correlated with tumor stage. Statistical calculations were performed using one-way ANOVA analysis.

以下實施例是非限制性的，並且僅代表本發明的各個方面和特徵。 The following examples are non-limiting and represent only various aspects and features of the present invention.

材料和方法 Materials and methods

細胞培養 cell culture

腺癌細胞株MKN28(JCRB編號JCRB0253)和MKN45(JCRB編號JCRB0254)在提供有10%胎牛血清(Hyclone,Logan UT,USA)的RPMI 1640培養基(Thermo,Waltham,Massachusetts,USA)中，在37℃下於5%CO2中培養。人胚胎腎細胞株293T(ATCC編號CRL-3216)則在提供有10%胎牛血清的DMEM培養基(Thermo，Waltham，Massachusetts，USA)中培養。 Adenocarcinoma cell lines MKN28 (JCRB No. JCRB0253) and MKN45 (JCRB No. JCRB0254) were grown in RPMI 1640 medium (Thermo, Waltham, Massachusetts, USA) supplied with 10% fetal bovine serum (Hyclone, Logan UT, USA) at 37 Incubate in 5% CO2 at ℃. Human embryonic kidney cell line 293T (ATCC number CRL-3216) was cultured in DMEM medium (Thermo, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum.

抗體、試劑和質體 Antibodies, Reagents and Plasmids

兔抗PHF8抗體購自Bethyl Laboratories(Montgomery,TX,USA)。兔抗c-Jun、抗PTEN、抗Src和抗HA抗體購自Cell Signaling Technology(Danvers,MA,USA)。小鼠抗β-肌動蛋白和抗flag購自Sigma-Aldirich((St.Louis,Missouri,USA)。兔抗IgG購自Santa Cruz Biotechnology(Dallas,TX,USA)。兔抗H3K9me2和抗H4K20me1購自Active Motif(Carlsbad,CA,USA)。兔抗p-Src(Y419)購自Thermo。兔抗PKCα購自Abcam(Cambridge，MA，USA)。慢病毒載體pLKO-control(pLKO)、pLKO-shPHF8(shPHF8 # 1：TRCN0000118319；shPHF8 # 2：TRCN0000118320)、shPRKCA(shPRKCA # 1：TRCN0000001692；shPRKCA # 2：TRCN0000001693)質體購自The RNAi Consortium(TRC)。抑製劑Midostaurin和Bosutinib購自Sigma-Aldirich。pHACE-PKCα(Addgene質體# 21232)，pCDNA3-SRC(Addgene質體# 44652)p6600-c-Jun(Addgene質體# 34898)。 Rabbit anti-PHF8 antibody was purchased from Bethyl Laboratories (Montgomery, TX, USA). Rabbit anti-c-Jun, anti-PTEN, anti-Src and anti-HA antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-β-actin and anti-flag were purchased from Sigma-Aldirich ((St. Louis, Missouri, USA). Rabbit anti-IgG was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-H3K9me2 and anti-H4K20me1 were purchased from Active Motif (Carlsbad, CA, USA). Rabbit anti-p-Src (Y419) was purchased from Thermo. Rabbit anti-PKCa was purchased from Abcam (Cambridge, MA, USA). Lentiviral vectors pLKO-control (pLKO), pLKO-shPHF8 (shPHF8 #1: TRCN0000118319; shPHF8 #2: TRCN0000118320), shPRKCA (shPRKCA #1: TRCN0000001692; shPRKCA #2: TRCN0000001693) plastids were purchased from The RNAi Consortium (TRC) ). Inhibitors Midostaurin and Bosutinib were purchased from Sigma-Aldirich. pHACE-PKCa (Addgene plastid #21232), pCDNA3-SRC (Addgene plastid #44652) p6600-c-Jun (Addgene plastid #34898).

減弱細胞(knockdown cells)的建立 Establishment of knockdown cells

根據使用者操作手冊，使用pLP1、pLP2和pLP/VSVG套組系統(Thermo)在293T細胞中產生了慢病毒顆粒。用攜帶pLKO、shPHF8或shPRKCA的慢病毒感染MKN28和MKN45細胞，然後以嘌呤黴素(2μg/ml)進行篩選。通過免疫墨點法分析評估減弱的效率。 Lentiviral particles were generated in 293T cells using the pLP1, pLP2 and pLP/VSVG kit systems (Thermo) according to the user's manual. MKN28 and MKN45 cells were infected with lentiviruses carrying pLKO, shPHF8 or shPRKCA and then selected with puromycin (2 μg/ml). The efficiency of attenuation was assessed by immunoblotting analysis.

斑馬魚中的遷移測定和化學反應測定 Migration assays and chemical reaction assays in zebrafish

Tg(fli1：EGFP)斑馬魚轉基因菌株購自ZIRC(Oregon,USA)。斑馬魚的胚胎、幼蟲和成年魚都保存在NHRI的台灣斑馬魚核心設施 中。受精後兩天，將胚胎去絨毛膜，然後用三卡因(0.04mg/ml)麻醉。用CFSE或CM-DiI標記MKN28或MKN45細胞。使用Nanoject II自動納升注射器(Drummond Science,Broomall,PA,USA)，將兩百個標記細胞(4.6nl)注射到兩天大的卵黃中。之後，通過螢光顯微鏡檢查(Leica)，在注射後第1天和第3天檢查單個受體的螢光細胞。斑馬魚實驗的動物實驗方案已由NHRI的IACUC批准(IACUC編號：IACUC-107057-AC1)。 The Tg(fli1:EGFP) zebrafish transgenic strain was purchased from ZIRC (Oregon, USA). Zebrafish embryos, larvae and adults are kept at NHRI's Taiwan Zebrafish Core Facility middle. Two days after fertilization, the embryos were dechorionated and then anesthetized with tricaine (0.04 mg/ml). MKN28 or MKN45 cells were labeled with CFSE or CM-DiI. Two hundred labeled cells (4.6 nl) were injected into two-day-old yolks using a Nanoject II automated nanoliter injector (Drummond Science, Broomall, PA, USA). Afterwards, individual recipients were examined for fluorescent cells on days 1 and 3 post-injection by fluorescence microscopy (Leica). The animal experimental protocol for the zebrafish experiments has been approved by the IACUC of NHRI (IACUC number: IACUC-107057-AC1).

細胞增殖測定和跨孔細胞遷移測定法 Cell Proliferation Assays and Transwell Cell Migration Assays

通過在六孔板的每孔中接種1×105個細胞，然後在第0、24、48、72和96小時計數細胞數量來測量細胞增殖。遷移試驗是在帶有8μm濾膜的BD的跨孔24孔板中進行的(BD,Franklin Lakes,NJ,USA)。將7×104細胞(在200μl 0.5%培養基中稀釋)接種到上腔室中，然後將500μl完全培養基添加到下腔室中。24小時作用後，以棉籤除去上側的非遷移細胞，並通過結晶紫染色檢查和檢測下側的細胞。 Cell proliferation was measured by seeding 1 x 105 cells in each well of a six-well plate and then counting the number of cells at 0, 24, 48, 72 and 96 hours. Migration assays were performed in BD transwell 24-well plates with 8 μm filters (BD, Franklin Lakes, NJ, USA). 7×10 4 cells (diluted in 200 μl of 0.5% medium) were seeded into the upper chamber, then 500 μl of complete medium was added to the lower chamber. After 24 hours of action, non-migrating cells on the upper side were removed with a cotton swab, and cells on the lower side were examined and detected by crystal violet staining.

小鼠異種移植和抑制實驗 Mouse Xenograft and Suppression Experiments

在每組五隻裸鼠的兩個側面皮下注射五百萬個MKN28細胞 (pLKO、shPHF8 # 1或shPHF8 # 2)。植入後兩週，每週測量一次腫瘤體積，連續測六週。NHRI的機構動物護理使用委員會(IACUC)批准了用於小鼠實驗的動物實驗方案。 Five million MKN28 cells were subcutaneously injected into both flanks of five nude mice in each group (pLKO, shPHF8 #1 or shPHF8 #2). Two weeks after implantation, tumor volumes were measured weekly for six consecutive weeks. The NHRI's Institutional Animal Care and Use Committee (IACUC) approved animal experimental protocols for mouse experiments.

免疫墨點法和免疫沉澱測定 Immunoblotting and immunoprecipitation assays

為了進行免疫墨點測定，將通過刮擦收集的PBS洗滌的細胞直接溶解在補充有蛋白酶抑製劑(Santa Cruz)和PhosSTOP(Sigma)的RIPA緩衝液中。在SDS-PAGE上分離出適量的蛋白質，然後電轉移到PVDF膜上(PALL,Protein Washington,NY,USA)。轉移後，將每個膜與適當的一抗在4℃作用過夜，然後用與螢光偶聯的二抗進行探測。利用奧德賽紅外成像系統(LI-COR Biosciences,Lincoln,NE,USA)來檢測螢光水平。為了進行免疫沉澱測定，將細胞沉澱物在含有蛋白酶抑製劑(Santa Cruz)的裂解緩衝液(50mM Tris-HCl(pH 7.4)、150mM NaCl、0.5%NP40)中裂解。然後將裂解物與相應的相應抗體(1μg)和所示的10μl PureProteome蛋白A/G磁珠(Millipore)在4℃下作用過夜。將小珠用IP洗滌緩衝液(137mM NaCl，2.7mM KCL，10mM Na2HPO4、1.8mM K2HPO4、0.1%Tween 20，pH 7.4)洗滌，並用IP裂解緩衝液洗脫，然後如上所述進行免疫墨點法測定。 For immunoblotting assays, PBS-washed cells collected by scraping were directly lysed in RIPA buffer supplemented with protease inhibitors (Santa Cruz) and PhosSTOP (Sigma). Appropriate amounts of protein were separated on SDS-PAGE and then electrotransferred to PVDF membranes (PALL, Protein Washington, NY, USA). After transfer, each membrane was incubated with the appropriate primary antibody overnight at 4°C and then probed with a fluorescently conjugated secondary antibody. Fluorescence levels were detected using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). For immunoprecipitation assays, cell pellets were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40) containing protease inhibitors (Santa Cruz). Lysates were then reacted with the corresponding corresponding antibodies (1 μg) and 10 μl PureProteome protein A/G magnetic beads (Millipore) as indicated overnight at 4°C. Beads were washed with IP wash buffer (137 mM NaCl, 2.7 mM KCL, 10 mM Na2HPO4, 1.8 mM K2HPO4, 0.1% Tween 20, pH 7.4) and eluted with IP lysis buffer, followed by immunoblotting as described above Determination.

ChIP測定 ChIP assay

MKN28細胞與甲醛(1%)交聯10分鐘，然後在0.125M甘氨酸中淬減。通過超音波處理(100-500bps)裂解細胞裂解液，然後IP分析則使用所示的相應抗體(兔IgG、抗PHF8、抗c-Jun、抗H3K9me2和抗H4K20me1)和Magna ChIP G磁珠(Millipore)在4°C過夜。沉澱的ChIP複合物通過qRT-PCR進行分析和定量。表1顯示了所示基因的引子序列和靶位點。根據閾值循環(Ct)進行倍數富集計算：2-△(△Ct)，其中△Ct=Ct(IP)-Ct(輸入)，△(△Ct)=△Ct(抗體)-△Ct(IgG)。 MKN28 cells were cross-linked with formaldehyde (1%) for 10 minutes and then quenched in 0.125M glycine. Cell lysates were lysed by sonication (100-500bps) followed by IP analysis using the corresponding antibodies (rabbit IgG, anti-PHF8, anti-c-Jun, anti-H3K9me2 and anti-H4K20me1) and Magna ChIP G magnetic beads (Millipore) as indicated ) overnight at 4°C. Precipitated ChIP complexes were analyzed and quantified by qRT-PCR. Table 1 shows the primer sequences and target sites of the indicated genes. The fold enrichment calculation was performed according to the threshold cycle (Ct): 2-△(△Ct), where △Ct=Ct(IP)-Ct(input), △(△Ct)=△Ct(antibody)-△Ct(IgG) ).

表1.用於ChIP測定的所示基因的引子序列和靶位點

Figure 110104309-A0202-12-0023-1
Table 1. Primer sequences and target sites of the indicated genes used for ChIP assays
Figure 110104309-A0202-12-0023-1

Figure 110104309-A0202-12-0024-2
Figure 110104309-A0202-12-0024-2

微陣列 microarray

整體的的表達分析是在MKN28 pLKO與shPHF8 # 1(GEO：GSE117980)中進行的。美國國家衛生研究院(NHRI)使用Affymertix GeneChip人類基因2.0 ST陣列(Affymetrix,Santa Clara,California,USA)進行了微陣列分析。隨後的分析由DAVID(The Database for Annotation,Visualization and Integrated Discovery)生物信息數據庫和USCS基因體瀏覽器工具完成。 Overall expression analysis was performed in MKN28 pLKO and shPHF8 #1 (GEO: GSE117980). Microarray analysis was performed by the National Institutes of Health (NHRI) using the Affymertix GeneChip Human Gene 2.0 ST Array (Affymetrix, Santa Clara, California, USA). Subsequent analysis was performed by the DAVID (The Database for Annotation, Visualization and Integrated Discovery) bioinformatics database and the USCS Genome Browser tool.

西方點墨微陣列晶片 Western Ink Microarray Wafers

訊息傳遞路徑分析是由NHRI的Micro-western Core設施按照指示使用適當的抗體，並依照了詳細描述的內容和流程進行分析(Ciaccio，Wagner等，2010)。 Messaging pathway analysis was performed by NHRI's Micro-western Core facility using appropriate antibodies as instructed and according to the content and protocols described in detail (Ciaccio, Wagner et al., 2010).

收集和即時定量PCR(qRT-PCR) Collection and real-time quantitative PCR (qRT-PCR)

用TRizol試劑(Thermo)提取總RNA，使用SuperScript III逆轉錄酶(Thermo)、dNTP(Genedirex,Las Vegas,Nevada,USA)和隨機引物(Thermo)製備cDNA。使用SensiMixTM Hi-ROX試劑盒(Bioline，Taunton，MA，USA)和ABI StepOnePlus即時定量PCR系統(Thermo)檢測cDNA樣品的量。GAPDH作為內部控制。引子列表在表2中提供。 Total RNA was extracted with TRizol reagent (Thermo) and cDNA was prepared using SuperScript III reverse transcriptase (Thermo), dNTPs (Genedirex, Las Vegas, Nevada, USA) and random primers (Thermo). The amount of cDNA samples was detected using the SensiMix™ Hi-ROX kit (Bioline, Taunton, MA, USA) and the ABI StepOnePlus real-time quantitative PCR system (Thermo). GAPDH as an internal control. A list of primers is provided in Table 2.

表2. 即時定量PCR的引子序列

Figure 110104309-A0202-12-0025-3
Table 2. Primer sequences for real-time quantitative PCR
Figure 110104309-A0202-12-0025-3

Figure 110104309-A0202-12-0026-4
Figure 110104309-A0202-12-0026-4

IHC和IHC評分 IHC and IHC scoring

從台灣桃園的長庚紀念醫院(CGMH)獲得了三個連續的石蠟包埋的胃癌活體組織切片(n=42)。組織陣列ST1505(n=50)購自US Biomax。表3顯示了包埋的胃癌活體組織切片的資訊。使用抗PHF8、抗PKCα和抗PTEN進行IHC染色，並由合格的病理學家進行評分。根據強度等級(分數：1-3)和陽性腫瘤細胞的比例(分數：1-4)對IHC結果進行評分。然後通過將染色強度乘以陽性細胞的比例來計算免疫反應評分(IRS)。本發明由長庚紀念醫院的機構審查委員會批准(IRB號：201800374B0C501)。 Three serial paraffin-embedded gastric cancer biopsies (n=42) were obtained from Chang Gung Memorial Hospital (CGMH) in Taoyuan, Taiwan. Tissue array ST1505 (n=50) was purchased from US Biomax. Table 3 shows information on embedded gastric cancer biopsies. IHC staining was performed using anti-PHF8, anti-PKCα and anti-PTEN and scored by a qualified pathologist. The IHC results were scored according to the intensity scale (score: 1-3) and the proportion of positive tumor cells (score: 1-4). The immune response score (IRS) was then calculated by multiplying the staining intensity by the proportion of positive cells. This invention was approved by the Institutional Review Board of Chang Gung Memorial Hospital (IRB number: 201800374B0C501).

表3. 包埋的胃癌活體組織切片的資訊

Figure 110104309-A0202-12-0026-5
Table 3. Information on embedded gastric cancer biopsies
Figure 110104309-A0202-12-0026-5

Figure 110104309-A0202-12-0027-6
Figure 110104309-A0202-12-0027-6

統計分析 Statistical Analysis

學生的t檢驗用於計算兩組之間實驗結果的統計顯著性(顯著性p<0.05)。通過對數秩檢驗分析總體生存率和無病進展。卡方檢驗用於比較IHC分析中具有分類變量的組。通過單因子變異數分析計算腫瘤與PHF8表現量之間的比較計算p值。 Student's t-test was used to calculate the statistical significance of the experimental results between the two groups (p<0.05 for significance). Overall survival and disease-free progression were analyzed by the log-rank test. Chi-square test was used to compare groups with categorical variables in IHC analysis. Comparisons between tumor and PHF8 expression levels were calculated by one-way analysis of variance to calculate p-values.

實施例1、在HER2陰性胃癌中，PHF8的過量表達與較差的臨床結果的相關性Example 1. Correlation of PHF8 overexpression with poor clinical outcome in HER2-negative gastric cancer

本發明首先評估了從Oncomine數據庫(www.oncomine.org/)選定來作為研究的正常胃粘膜和腫瘤組織中21種KDMs的表現量(表4)。 The present inventors first evaluated the expression levels of 21 KDMs in normal gastric mucosa and tumor tissues selected for study from the Oncomine database (www.oncomine.org/) (Table 4).

表4.基於Oncomine數據庫的KDMs表現量的統計分析

Figure 110104309-A0202-12-0028-7
Table 4. Statistical analysis of KDMs performance based on Oncomine database
Figure 110104309-A0202-12-0028-7

Figure 110104309-A0202-12-0029-8
Figure 110104309-A0202-12-0029-8

Figure 110104309-A0202-12-0030-9
Figure 110104309-A0202-12-0030-9

如表4結果所示，與正常組織相比，腫瘤標本中的KDM1B、KDM2A和PHF8/KDM7B顯著上升(p<0.05)。接著，使用從Kaplan-Meier Plotter(KM Plotter)中所取得的數據，針對HER2陰性的胃癌患者的5年總生存期(OS)和首次進展(FP)終點進行了臨床相關性評估(表5)。 As shown in the results in Table 4, KDM1B, KDM2A and PHF8/KDM7B were significantly increased in tumor specimens compared with normal tissues (p<0.05). Next, clinical relevance was assessed for the 5-year overall survival (OS) and first progression (FP) endpoints of HER2-negative gastric cancer patients using data from the Kaplan-Meier Plotter (KM Plotter) (Table 5). .

表5. Kaplan-Meier Plotter分析中選出KDMs的臨床結果

Figure 110104309-A0202-12-0030-10
Table 5. Clinical outcomes of selected KDMs in Kaplan-Meier Plotter analysis
Figure 110104309-A0202-12-0030-10

Figure 110104309-A0202-12-0031-11
Figure 110104309-A0202-12-0031-11

對於HER2陰性病例，較高的PHF8/KDM7B表達與較差的OS顯著相關。然而，針對KDM1B和KDM2A則會根據不同的探針而顯示出不一致的結果(圖1A和1B)。此外，在轉移部位比在原發腫瘤部位中觀察到甚至具有更高的PHF8/KDM7B表現量(圖2A)。另外，腫瘤組織中的PHF8/KDM7B表現量高於相鄰的正常組織中的表現量(圖2B)。這些結果共同指出PHF8在HER2陰性胃癌中是一個預後的表觀遺傳調控因子。 For HER2-negative cases, higher PHF8/KDM7B expression was significantly associated with worse OS. However, KDM1B and KDM2A showed inconsistent results depending on the probes (Figures 1A and 1B). Furthermore, an even higher amount of PHF8/KDM7B expression was observed in metastatic sites than in primary tumor sites (Fig. 2A). In addition, the expression level of PHF8/KDM7B in tumor tissues was higher than that in adjacent normal tissues ( FIG. 2B ). These results collectively point to PHF8 as a prognostic epigenetic regulator in HER2-negative gastric cancer.

實施例2、PHF8在體外和體內對轉移性胃癌細胞的作用Example 2. Effects of PHF8 on metastatic gastric cancer cells in vitro and in vivo

在Oncomine分析中，發現到PHF8在胃癌轉移位點的表現量甚至更高。為了釐清PHF8在胃癌進展中的扮演的生物上的功能，使用了兩個類似於染色體不穩定腫瘤(CIN)亞型的HER2陰性、轉 移性MKN28和MKN45細胞株。MKN45從一名彌散性組織學低分化原發性胃癌的患者的肝轉移中獲得，其特徵為微小衛星體的不穩定性(MSI)低和Epstein-Barr病毒(EBV)陰性。MKN28源自具有腸道分化原發性胃癌的淋巴結轉移，並具有中等基因體拷貝數改變和中等數量的單核苷酸變異基因。 In the Oncomine analysis, the expression of PHF8 was found to be even higher at the metastatic sites of gastric cancer. To clarify the biological function of PHF8 in gastric cancer progression, two HER2-negative, transduced chromosomally unstable tumor (CIN)-like subtypes were used. Transgenic MKN28 and MKN45 cell lines. MKN45 was obtained from liver metastases from a patient with diffuse histologically poorly differentiated primary gastric cancer characterized by low microsatellite instability (MSI) and Epstein-Barr virus (EBV) negativity. MKN28 is derived from lymph node metastases with intestinal differentiated primary gastric cancer and has moderate gene somatic copy number alterations and moderate numbers of single-nucleotide variant genes.

通過使用慢病毒方法，為MKN28建立了對照(pLKO)或耗盡PHF8的細胞株(shPHF8 # 1和shPHF8 # 2)(圖3A)。與對照細胞相比，耗盡PHF8的細胞顯著降低了細胞增殖水平(圖3B)和遷移水平(圖3E)。重要的是，對於PHF8-KD MKN28細胞，MKN28異種移植物的腫瘤生長顯著受損(圖3D)。與對照細胞相比，PHF8耗盡的MKN45細胞(shPHF8 # 1和shPHF8 # 2)也發現了相似的結果(圖4A-4C)。 Using a lentiviral approach, control (pLKO) or PHF8-depleted cell lines (shPHF8 #1 and shPHF8 #2) were established for MKN28 (Figure 3A). Compared with control cells, cells depleted of PHF8 significantly reduced the level of cell proliferation (FIG. 3B) and migration (FIG. 3E). Importantly, tumor growth of MKN28 xenografts was significantly impaired for PHF8-KD MKN28 cells (Fig. 3D). Similar results were also found in PHF8-depleted MKN45 cells (shPHF8 #1 and shPHF8 #2) compared to control cells (Figures 4A-4C).

接下來，要使用斑馬魚異種移植測定法評估遷移行為，這是一種有效的體內系統，該系統利用少量細胞(100至200個細胞)在兩天內準確地監測細胞遷移活動。細胞(pLKO與shPHF8)用羧基熒光素琥珀酰亞胺酯(CFSE)(一種胺反應性綠色螢光染料)標記，並注入斑馬魚胚胎中。在注射後1天(1dpi和3dpi)通過螢光顯微鏡監測遷移活性。如圖3F和4D所示，與保留在胚胎中的細胞相比， 在3dpi中(上圖)，對照細胞已擴散到遠端。具有遠端腫瘤灶的胚胎的定量結果顯示出，與shPFH8組相比，pLKO組的轉移活性(36.1%)明顯更高於shPFH8組(shPHF8 # 1，17.1%；shPHF8 # 2，16.7%)(圖3G)。同樣，與對照組相比，降低PHF8表現的MKN45組別的遷移程度明顯降低(pLKO：31.3%；shPHF8 # 1：8.9%；和shPHF8 # 2：11.8%)(圖4E)。本發明發現PHF8對於腫瘤生長和遷移至關重要。 Next, migration behavior was assessed using the zebrafish xenograft assay, an efficient in vivo system that utilizes a small number of cells (100 to 200 cells) to accurately monitor cell migration activity over two days. Cells (pLKO and shPHF8) were labeled with carboxyfluorescein succinimidyl ester (CFSE), an amine-reactive green fluorescent dye, and injected into zebrafish embryos. Migration activity was monitored by fluorescence microscopy 1 day after injection (1 dpi and 3 dpi). As shown in Figures 3F and 4D, compared to cells retained in the embryo, At 3dpi (top panel), control cells have spread to the distal end. Quantification of embryos with distal tumor foci revealed significantly higher metastatic activity in the pLKO group (36.1%) compared to the shPFH8 group (shPHF8 #1, 17.1%; shPHF8 #2, 16.7%) ( Figure 3G). Likewise, the MKN45 group with reduced PHF8 expression had significantly less migration compared to the control group (pLKO: 31.3%; shPHF8 #1: 8.9%; and shPHF8 #2: 11.8%) (Figure 4E). The present inventors found that PHF8 is essential for tumor growth and migration.

實施例3、PHF8透過調節PKCα和ICAM-1促進胃癌進展Example 3. PHF8 promotes gastric cancer progression by regulating PKCα and ICAM-1

為了表徵PHF8有助於胃癌進程的分子機制，對pLKO和shPHF8 MKN28細胞(GSE117980)進行了比較微陣列分析。DAVID功能註釋表明，shPHF8細胞中被下調(小於或等於兩倍；n=150)的基因主要參與在細胞遷移(n=8，P=0.00041)和細胞運動性(n=8，P=0.00079)(https：//david.ncifcrf.gov/)(圖5A)。qRT-PCR證實與細胞遷移和運動相關的八個基因(UGT8、HBEGF、ROBO1、STATB2、PRKCA、AXL、ICAM1和TUBE1)在兩個獨立的shPHF8細胞系中的表現量明顯低於在pLKO細胞中的表現量(圖5B)。 To characterize the molecular mechanism by which PHF8 contributes to gastric cancer progression, comparative microarray analysis was performed on pLKO and shPHF8 MKN28 cells (GSE117980). DAVID functional annotation indicated that genes that were down-regulated (less than or equal to two-fold; n=150) in shPHF8 cells were mainly involved in cell migration (n=8, P=0.00041) and cell motility (n=8, P=0.00079) (https://david.ncifcrf.gov/) (Figure 5A). qRT-PCR confirmed that eight genes associated with cell migration and motility (UGT8, HBEGF, ROBO1, STATB2, PRKCA, AXL, ICAM1, and TUBE1) were significantly less expressed in two independent shPHF8 cell lines than in pLKO cells The amount of expression (Figure 5B).

接下來，評估PHF8是否直接參與調節微陣列分析中鑑定的基因的表達。在染色質免疫沉澱(ChIP)分析中，在MKN28的PRCCA 和ICAM-1的啟動子區域上，PHF8的信號明顯高於IgG的信號(圖5C)。使用橫跨MKN28的PRKCA啟動子區域設計的四組引子進行的另一項ChIP分析顯示出在-1,100bp和-744bp區域之間，尤其是在-925到-744bp的位點之間有陽性PHF8結合信號(圖6A)。比較在沒有或缺乏PHF8的細胞中PRKCA基因座上的H3K9me2和H4K20me1的ChIP信號。如圖所示，在圖6B-6D中，在兩個獨立的shPHF8系中H3K9me2信號，但未在H4K20me1中觀察到顯著增加，顯示出PHF8通過消除PRRKA基因座上的抑制性H3K9me2標記來調節PRCKA的表達。 Next, it was assessed whether PHF8 is directly involved in regulating the expression of genes identified in the microarray analysis. In chromatin immunoprecipitation (ChIP) analysis, PRCCA at MKN28 and ICAM-1 promoter region, the signal of PHF8 was significantly higher than that of IgG (Fig. 5C). Another ChIP analysis using four sets of primers designed across the PRKCA promoter region of MKN28 showed positive PHF8 between the -1,100bp and -744bp regions, especially between the -925 and -744bp sites Binding signal (FIG. 6A). Comparison of ChIP signals for H3K9me2 and H4K20me1 at the PRKCA locus in cells without or lacking PHF8. As shown in Figures 6B-6D, H3K9me2 signaling was observed in two independent shPHF8 lines, but no significant increase was observed in H4K20me1, showing that PHF8 regulates PRCKA by eliminating the repressive H3K9me2 mark at the PRRKA locus expression.

為了證實PRRKA作為PHF8的下游靶標，在兩條shPHF8系(MKN28，圖7A和MKN45，圖8A)的每條中異位表達PKCα。有趣的是，與pLKO系列相比，shPHF8中PKCα的異位表達顯著恢復了細胞增殖(MKN28：圖7B和MKN45：圖8B)和遷移(MKN28：圖7C和MKN45：圖8C)的水平。總體而言，本發明表明PHF8呈現出H3K9me2去甲基化活性進而提高了參與細胞增殖和遷移的PRCKA的表現量。 To demonstrate PRRKA as a downstream target of PHF8, PKCa was ectopically expressed in each of two shPHF8 lines (MKN28, Figure 7A and MKN45, Figure 8A). Interestingly, ectopic expression of PKCα in shPHF8 significantly restored the levels of cell proliferation (MKN28: Figure 7B and MKN45: Figure 8B) and migration (MKN28: Figure 7C and MKN45: Figure 8C) compared to the pLKO series. Overall, the present invention demonstrates that PHF8 exhibits H3K9me2 demethylation activity and thereby increases the expression of PRCKA involved in cell proliferation and migration.

另外，評估PHF8是否直接調節ICAM-1的表現。圖9A顯示在MKN45中耗盡PHF8表現量降低了ICAM-1 mRNA的表現量， 與在MKN28中觀察到的一致(圖5B)。此外，使用ChIP分析已經檢測到ICAM-1的啟動子區域中的PHF8信號(圖9B)。與對照相比，耗盡ICAM-1的表現量導致遷移作用程度降低(圖9C)，顯示出PHF8調節的ICAM-1表現也有助於胃癌進展。 Additionally, it was assessed whether PHF8 directly regulates ICAM-1 expression. Figure 9A shows that depletion of PHF8 expression in MKN45 reduced ICAM-1 mRNA expression, Consistent with that observed in MKN28 (Fig. 5B). In addition, PHF8 signaling in the promoter region of ICAM-1 has been detected using ChIP analysis (Figure 9B). Depletion of the expressed amount of ICAM-1 resulted in a reduced degree of migratory effects compared to controls (Fig. 9C), showing that PHF8-regulated ICAM-1 expression also contributes to gastric cancer progression.

實施例4、PHF8與c-Jun相互作用，並且它們被共同招募到PRCCA基因座Example 4. PHF8 interacts with c-Jun and they are co-recruited to the PRCCA locus

接下來，使用加利福尼亞大學聖克魯斯大學的基因組瀏覽器來識別與PHF8相互作用的潛在轉錄因子。c-Jun，為一AP-1轉錄因子的組成部分，在兩個ChIP-Seq數據集(來自A549的ChIP-Seq(ENCLB202COI)(Ab：PHF8)：GSM2700325；來自A549的ChIP-Seq(ENCLB403GIO)(Ab：c-Jun)：GSM2437720)中顯示出與PRKCA啟動子區域中具有結合峰。使用抗PHF8或抗c-Jun抗體對MKN28或MKN45的裂解物進行免疫沉澱(IP)，顯示出內源性PHF8與c-Jun相關(MKN28，圖10A和MKN45，圖8D)。進一步確定與c-Jun相互作用的PHF8的關鍵區域。製備了全長PHF8和各種帶有Flag標籤的截短變體(帶有標記的全長[FL]，N端[△N440]和C端[△C589]截短變體)。IP分析顯示出PHF8的C末端區域(441至1,024殘基)對於其與c-Jun的相互作用最為關鍵(圖11A)。透過製備與HA標籤融 合的全長和截短的c-Jun變體(HA標籤FL，N端[△N223]和C端[△C108]截短形式)，對c-Jun進行了對等實驗。IP分析顯示c-Jun△N223而非△C108保留了與PHF8的結合，表明c-Jun的C端區域(殘基224至331)對於PHF8-c-Jun相互作用至關重要(圖11B)。 Next, the University of California, Santa Cruz genome browser was used to identify potential transcription factors that interact with PHF8. c-Jun, a component of an AP-1 transcription factor, was tested in two ChIP-Seq datasets (ChIP-Seq from A549 (ENCLB202COI) (Ab: PHF8): GSM2700325; ChIP-Seq from A549 (ENCLB403GIO) (Ab: c-Jun): GSM2437720) showed a binding peak to the PRKCA promoter region. Immunoprecipitation (IP) of lysates of MKN28 or MKN45 using anti-PHF8 or anti-c-Jun antibodies showed that endogenous PHF8 was associated with c-Jun (MKN28, Figure 10A and MKN45, Figure 8D). The key regions of PHF8 that interact with c-Jun were further identified. Full-length PHF8 and various Flag-tagged truncated variants (full-length tagged [FL], N-terminal [ΔN440] and C-terminal [ΔC589] truncated variants) were prepared. IP analysis revealed that the C-terminal region of PHF8 (residues 441 to 1,024) was most critical for its interaction with c-Jun (FIG. 11A). Fusion with HA tag by preparation Peer experiments were performed on c-Jun using the combined full-length and truncated c-Jun variants (HA-tag FL, N-terminal [ΔN223] and C-terminal [ΔC108] truncated forms). IP analysis showed that c-Jun ΔN223, but not ΔC108, retained binding to PHF8, indicating that the C-terminal region of c-Jun (residues 224 to 331) is critical for the PHF8-c-Jun interaction (Fig. 11B).

【00100】為了支持PHF8作為c-Jun的共激活子並調節PRKCA的表現的觀點，因此使用抗c-Jun和IgG進行了pLKO和shPHF8細胞的ChIP分析以進行比較。在PRKCA位點的中檢測到在pLKO細胞中c-Jun的統計學顯著富集(MKN28：圖10B和MKN45：圖8E)。值得注意的是，根據ChIP-Seq數據(基因表達綜合[GEO]登錄號GSM2437720)，在PRCAA的啟動子區域中發現了c-Jun/AP-1結合位點。使用AP-1報導分子活性測定法，顯示出耗盡PHF8的表現顯著降低了反式活化AP-1報導分子活性(MKN28：圖10C和MKN45：圖8F)。接下來檢測大量表達PHF8和/或c-Jun是否能刺激AP-1轉錄活性。圖10D顯示單獨大量表達PHF8或c-Jun顯著增強了AP-1反式活化的表達。值得注意的是，與c-Jun一起過量表達PHF8時，甚至顯著增加。有趣的是，當過表達無活性的突變體PHF8(H247A)時，則看不到這種增強(圖10D)。本發明顯示出PHF8透過去甲基酶的活性與c-Jun結合來調節PRKCA的表達。 [00100] To support the notion that PHF8 acts as a co-activator of c-Jun and regulates PRKCA expression, ChIP analysis of pLKO and shPHF8 cells was therefore performed using anti-c-Jun and IgG for comparison. Statistically significant enrichment of c-Jun in pLKO cells was detected in PRKCA loci (MKN28: Figure 10B and MKN45: Figure 8E). Notably, a c-Jun/AP-1 binding site was found in the promoter region of PRCAA based on ChIP-Seq data (Gene Expression Omnibus [GEO] accession number GSM2437720). Using the AP-1 reporter activity assay, it was shown that depletion of PHF8 significantly reduced transactivated AP-1 reporter activity (MKN28: Figure 10C and MKN45: Figure 8F). We next tested whether high expression of PHF8 and/or c-Jun could stimulate AP-1 transcriptional activity. Figure 10D shows that high expression of PHF8 or c-Jun alone significantly enhanced the expression of AP-1 transactivation. Notably, when PHF8 was overexpressed together with c-Jun, there was even a significant increase. Interestingly, this enhancement was not seen when the inactive mutant PHF8 (H247A) was overexpressed (Fig. 10D). The present invention shows that PHF8 regulates the expression of PRKCA through the combination of demethylase activity and c-Jun.

【00101】實施例5、PHF8-PKCα軸與Src調節PTEN去穩定 Embodiment 5, PHF8-PKCα axis and Src regulate PTEN destabilization

【00102】RKCA編碼出PKCα，為一種絲胺酸/蘇胺酸蛋白激酶，作為被Ca2+和磷脂激活的信號分子。因此，進一步探索由PHF8-PKCα軸介導的訊息傳遞路徑。西方點墨微陣列晶片分析用於同時評估pLKO、shPHF8和shPRKCA細胞中96種抗體的模式。圖12顯示兩個途徑發生了顯著變化：PI3K和MAPK。特別是，抑癌基因PTEN/pPTEN在shPHF8和shPRKCA細胞中均具有最高的信號強度。 [00102] RKCA encodes PKCα, a serine/threonine protein kinase that acts as a signaling molecule activated by Ca 2+ and phospholipids. Therefore, the signaling pathway mediated by the PHF8-PKCα axis was further explored. Western blotting microarray wafer analysis was used to simultaneously assess the pattern of 96 antibodies in pLKO, shPHF8 and shPRKCA cells. Figure 12 shows significant changes in two pathways: PI3K and MAPK. In particular, the tumor suppressor gene PTEN/pPTEN had the highest signal intensity in both shPHF8 and shPRKCA cells.

【00103】對pLKO和shPHF8系的西方墨點法分析證實耗盡PHF8表現提高了PTEN表現(MKN28：圖13A和MKN45：圖14A)。此外，在兩個shPRKCA系中的每一個中，PTEN的表現量均顯著高於pLKO中的表現，顯示出PHF8-PKCα信號傳導導致PTEN去穩定化(MKN28：圖13A和MKN45：圖14A)。因此，試問在PHF8-PKCα軸的範圍下，PTEN是否透過轉錄沉默或轉譯修飾表現來被調控。首先，評估蛋白酶體抑製劑MG132的治療是否可以恢復PTEN信號，由於PTEN對蛋白酶體降解非常敏感，特別是轉譯後修飾。MG132處理的pLKO細胞中確實能回復PTEN的蛋白表現(MKN28：圖13A和MKN45：圖14A)。有趣的是，分析pLKO和兩個shPHF8系中Src和活化的Src(pY419)的含量，結果顯示出在耗盡了PHF8的細胞 (MKN28：圖13B和MKN45：圖14B)中活化Src(pY419)的表現量降低。此外，在兩條shPHF8系的每條中使用表達PRKα的載體來互補PKCα能恢復了活化的Src的表現量(MKN28：圖13B和MKN45：圖14B)，因此，支持了Src作為PKCα下游效應子的觀點。引入持續活化型的Src變體(Y419D)，而非激酶死亡的Src變體(Y419F)，能極大地減少了PTEN的含量(MKN28：圖13C和MKN45：圖14C)。因此，本發明顯示了PHF8透過PKCα-Src引起的訊號傳遞路徑負向調控PTEN去穩定作用。 [00103] Western blot analysis of pLKO and shPHF8 lines confirmed that depletion of PHF8 expression enhanced PTEN expression (MKN28: Figure 13A and MKN45: Figure 14A). Furthermore, in each of the two shPRKCA lines, the amount of PTEN expression was significantly higher than in pLKO, showing that PHF8-PKCα signaling leads to PTEN destabilization (MKN28: Figure 13A and MKN45: Figure 14A). Therefore, it was asked whether PTEN is regulated through transcriptional silencing or expression of translational modification in the context of the PHF8-PKCα axis. First, it was assessed whether treatment with the proteasome inhibitor MG132 could restore PTEN signaling, since PTEN is very sensitive to proteasomal degradation, especially post-translational modifications. The protein expression of PTEN was indeed restored in MG132-treated pLKO cells (MKN28: Figure 13A and MKN45: Figure 14A). Interestingly, analysis of the content of Src and activated Src(pY419) in pLKO and two shPHF8 lines showed that in PHF8-depleted cells (MKN28: Fig. 13B and MKN45: Fig. 14B) The expression of activated Src (pY419) was decreased. Furthermore, complementation of PKCα with a PRKα-expressing vector in each of the two shPHF8 lines restored the expression of activated Src (MKN28: Figure 13B and MKN45: Figure 14B), thus supporting Src as a downstream effector of PKCα the opinion of. Introduction of a persistently activated Src variant (Y419D), but not a kinase-dead Src variant (Y419F), greatly reduced PTEN content (MKN28: Figure 13C and MKN45: Figure 14C). Therefore, the present invention shows that PHF8 negatively regulates PTEN destabilization through the signaling pathway induced by PKCα-Src.

【00104】實施例6、在轉移胃癌中靶向PKCα-Src途徑 [00104] Example 6. Targeting the PKC α -Src pathway in metastatic gastric cancer

【00105】使用藥理抑製劑測試是否可透過靶向PHF8-PKCα-Src-PTEN軸抑制胃癌轉移。在MKN28(圖15A)和MKN45(圖16A)中，PKCα抑製劑Midostaurin的治療確實導致PTEN表現量升高，並呈現劑量依賴性。重要的是，細胞遷移的程度也被顯著抑制(MKN28：圖15B和MKN45：圖16B)。同樣，Bosutinib對Src的抑制導致MKN28(圖15C)和MKN45(圖16C)中PTEN表現量提高。因此，細胞遷移的程度也降低了(MKN28：圖15D和MKN45：圖16D)。 [00105] Pharmacological inhibitors were used to test whether gastric cancer metastasis could be inhibited by targeting the PHF8-PKCα-Src-PTEN axis. In MKN28 (FIG. 15A) and MKN45 (FIG. 16A), treatment with the PKC alpha inhibitor Midostaurin did result in increased PTEN expression in a dose-dependent manner. Importantly, the extent of cell migration was also significantly inhibited (MKN28: Figure 15B and MKN45: Figure 16B). Likewise, inhibition of Src by bosutinib resulted in increased PTEN expression in MKN28 (FIG. 15C) and MKN45 (FIG. 16C). Consequently, the extent of cell migration was also reduced (MKN28: Figure 15D and MKN45: Figure 16D).

【00106】接下來，使用斑馬魚異種移植模型來證實此發現。將標記 有Vybrant CM-DiI(CM-Dil)(紅色螢光染料)的細胞注射到Tg(fli1：EGFP)(血管中帶有綠色螢光的魚)的胚胎中，然後在1dpi條件下，浸入含1μM Midostaurin或Bosutinib(亞致死劑量)。在胚胎中植入MKN28或MKN45細胞通常會導致細胞擴散。細胞明顯轉移到身體的遠端部分(MKN28：圖15E和MKN45：圖16E)。大部分以Midostaurin或Bosutinib治療的MKN28注射的胚胎具有降低的散佈和侵襲現象(溶媒組為39.3%、Midostaurin組為11%以及Bosutinib組為11.95%)(圖15F)。與兩個抑製劑組(Midostaurin組為13.88%，Bosutinib組為18.08%)相比，MKN45注射的胚胎在溶媒組(34.25%)中轉移活性顯著更高(圖16F)。接下來，透過使用MKN28異種移植模型來測試Midostaurin或Bosutinib是否會在體內阻礙腫瘤生長。圖17顯示出與溶媒組相比，兩種藥物均顯著損害腫瘤的生長。總體而言，本發明揭示抑制PKCα或Src是抑制體內腫瘤進展的有效策略。 [00106] Next, a zebrafish xenograft model was used to confirm this finding. will mark Cells with Vybrant CM-DiI(CM-Dil) (red fluorescent dye) were injected into embryos of Tg(fli1:EGFP) (fish with green fluorescence in blood vessels) and then submerged in 1 μM at 1 dpi Midostaurin or Bosutinib (sublethal dose). Implantation of MKN28 or MKN45 cells in embryos usually results in cell spreading. Cells clearly metastasized to distal parts of the body (MKN28: Figure 15E and MKN45: Figure 16E). The majority of MKN28-injected embryos treated with Midostaurin or Bosutinib had reduced spreading and invasion (39.3% in the vehicle group, 11% in the Midostaurin group, and 11.95% in the Bosutinib group) (Figure 15F). MKN45-injected embryos had significantly higher transfer activity in the vehicle group (34.25%) compared to the two inhibitor groups (13.88% in the Midostaurin group and 18.08% in the Bosutinib group) (Fig. 16F). Next, we tested whether Midostaurin or Bosutinib would block tumor growth in vivo by using an MKN28 xenograft model. Figure 17 shows that both drugs significantly impair tumor growth compared to the vehicle group. Overall, the present invention reveals that inhibition of PKCa or Src is an effective strategy to inhibit tumor progression in vivo.

【00107】為了測試斑馬魚胚胎體內模型中Midostaurin和Bosutinib用於靶向PKCα-Src途徑的作用的亞致死劑量。本發明顯示187.5nM的Midostaurin與1.25μM的Bosutinib組合對於從3dpf開始處理2天的斑馬魚胚胎沒有毒性。因此，選擇該劑量作為用於異種移植測定(圖 18)。此外，結果顯示出Midostaurin與Bosutinib對於MKN28去抑制體內細胞增殖和遷移具有協同作用(圖19)。187.5nM的Midostaurin降低了MKN28的細胞增殖，1250nM的Bosutinib對細胞增殖影響很小。然而，Midostaurin(187.5nM)結合Bosutinib(1250nM)在斑馬魚胚胎中顯示出MKN28的細胞增殖具有統計學意義的降低(圖19A-19C)。在MKN28中187.5nM的Midostaurin在MKN28中表現出具有遷移行為的胚胎比例降低，1250nM的Bosutinib對細胞遷移的影響很小，而Midostaurin(187.5nM)結合Bosutinib(1250nM)在比例上表現出顯著降低的胚胎遷移率(圖19D-19E)。 [00107] To test sublethal doses of Midostaurin and Bosutinib for targeting the PKCα-Src pathway in an in vivo model of zebrafish embryos. The present invention shows that Midostaurin at 187.5 nM in combination with Bosutinib at 1.25 μM is not toxic to zebrafish embryos treated for 2 days from 3dpf. Therefore, this dose was chosen as used in the xenograft assay (Fig. 18). Furthermore, the results showed that Midostaurin and Bosutinib had a synergistic effect on MKN28 de-inhibition of cell proliferation and migration in vivo (Figure 19). Midostaurin at 187.5nM reduced cell proliferation of MKN28, and Bosutinib at 1250nM had little effect on cell proliferation. However, Midostaurin (187.5 nM) combined with Bosutinib (1250 nM) showed a statistically significant reduction in MKN28 cell proliferation in zebrafish embryos (Figures 19A-19C). Midostaurin at 187.5nM in MKN28 showed a reduced proportion of embryos with migratory behavior in MKN28, Bosutinib at 1250nM had little effect on cell migration, while Midostaurin (187.5nM) combined with Bosutinib (1250nM) showed a significantly reduced proportion of Embryo migration rates (Figures 19D-19E).

【00108】實施例7、胃癌受試者中PHF8、PKCα和PTEN的免疫組織化學分析 [00108] Embodiment 7. Immunohistochemical analysis of PHF8, PKCα and PTEN in gastric cancer subjects

【00109】鑑於PHF8-PKCα-PTEN軸在體外和體內中對於HER2陰性胃癌進程中的作用，透過對大量胃癌患者以IHC分析PHF8、PKCα和PTEN評估這些標誌物的臨床相關性(受試者來自CGMH：圖20和Biomax組織陣列ST1505：圖21)。根據兩個參數對IHC結果進行評分：強度等級(分數：1-3)和陽性腫瘤細胞的比例(分數：1-4)。透過將強度等級乘以陽性比例得分獲得免疫反應得分(IRS)(圖20A-20B和圖21A)。值得注意的是，這些標記之間存在顯著的相關性(圖 20C-20E和圖21B-21D)，如：在PHF8和PKCα之間呈正相關，而在PHF8和PTEN之間以及與PKCα和PTEN之間呈負相關。值得注意的是，PHF8含量與腫瘤分期顯著正相關(圖20F和圖21E)。PHF8顯著預測了較差的OS和無病生存期。此外，PHF8highPKCαhigh組(IRS評分

Figure 110104309-A0202-12-0041-138
6)與OS差和無病生存顯著相關(圖20G)。 [00109] Given the role of the PHF8-PKCα-PTEN axis in HER2-negative gastric cancer progression in vitro and in vivo, the clinical relevance of these markers was assessed by IHC analysis of PHF8, PKCα, and PTEN in a large number of gastric cancer patients (subjects from CGMH: Figure 20 and Biomax Tissue Array ST1505: Figure 21). IHC results were scored according to two parameters: intensity grade (score: 1-3) and proportion of positive tumor cells (score: 1-4). The Immune Response Score (IRS) was obtained by multiplying the intensity scale by the positive proportion score (FIGS. 20A-20B and 21A). Notably, there were significant correlations between these markers (Figures 20C-20E and 21B-21D), such as a positive correlation between PHF8 and PKCα, and a positive correlation between PHF8 and PTEN and with PKCα and PTEN were negatively correlated. Notably, PHF8 content was significantly positively correlated with tumor stage (Figures 20F and 21E). PHF8 significantly predicted poorer OS and disease-free survival. In addition, the PHF8 high PKCα high group (IRS score
Figure 110104309-A0202-12-0041-138
6) was significantly associated with poor OS and disease-free survival (Fig. 20G).

<110> 國立清華大學和財團法人國家衛生研究院 <110> National Tsing Hua University and National Institutes of Health

<120> 抑制胃癌腫瘤生長或判斷胃癌腫瘤生長階段之方法 <120> Method for inhibiting the growth of gastric cancer tumor or judging the growth stage of gastric cancer tumor

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<210> 10 <210> 10

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於ChIP的ROBO1 R引子 <223> ROBO1 R primer for ChIP

<400> 10 <400> 10

Figure 110104309-A0202-12-0046-21
Figure 110104309-A0202-12-0046-21

<210> 11 <210> 11

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於ChIP的SATB2 F引子 <223> SATB2 F primer for ChIP

<400> 11 <400> 11

Figure 110104309-A0202-12-0046-22
Figure 110104309-A0202-12-0046-22

<210> 12 <210> 12

<211> 19 <211> 19

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於ChIP的SATB2 R引子 <223> SATB2 R primer for ChIP

<400> 12 <400> 12

Figure 110104309-A0202-12-0047-23
Figure 110104309-A0202-12-0047-23

<210> 13 <210> 13

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於ChIP的TUBE1 F引子 <223> TUBE1 F primer for ChIP

<400> 13 <400> 13

Figure 110104309-A0202-12-0047-24
Figure 110104309-A0202-12-0047-24

<210> 14 <210> 14

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於ChIP的TUBE1 R引子 <223> TUBE1 R primer for ChIP

<400> 14 <400> 14

Figure 110104309-A0202-12-0047-25
Figure 110104309-A0202-12-0047-25

<210> 15 <210> 15

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於ChIP的UGT8 F引子 <223> UGT8 F primer for ChIP

<400> 15 <400> 15

Figure 110104309-A0202-12-0048-26
Figure 110104309-A0202-12-0048-26

<210> 16 <210> 16

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於ChIP的UGT8 R引子 <223> UGT8 R primer for ChIP

<400> 16 <400> 16

Figure 110104309-A0202-12-0048-27
Figure 110104309-A0202-12-0048-27

<210> 17 <210> 17

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的AXL F引子 <223> AXL F primer for PCR

<400> 17 <400> 17

Figure 110104309-A0202-12-0049-28
Figure 110104309-A0202-12-0049-28

<210> 18 <210> 18

<211> 18 <211> 18

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的AXL R引子 <223> AXL R primer for PCR

<400> 18 <400> 18

Figure 110104309-A0202-12-0049-29
Figure 110104309-A0202-12-0049-29

<210> 19 <210> 19

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列e <213> Artificial sequence e

<220> <220>

<223> 用於PCR的HBEGF F引子 <223> HBEGF F primer for PCR

<400> 19 <400> 19

Figure 110104309-A0202-12-0050-30
Figure 110104309-A0202-12-0050-30

<210> 20 <210> 20

<211> 21 <211> 21

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的HBEGF R引子 <223> HBEGF R primer for PCR

<400> 20 <400> 20

Figure 110104309-A0202-12-0050-31
Figure 110104309-A0202-12-0050-31

<210> 21 <210> 21

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的ICAM-1 F引子 <223> ICAM-1 F primer for PCR

<400> 21 <400> 21

Figure 110104309-A0202-12-0050-135
Figure 110104309-A0202-12-0050-135

<210> 22 <210> 22

<211> 21 <211> 21

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的ICAM-1 R引子 <223> ICAM-1 R primer for PCR

<400> 22 <400> 22

Figure 110104309-A0202-12-0051-33
Figure 110104309-A0202-12-0051-33

<210> 23 <210> 23

<211> 21 <211> 21

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的PTEN F引子 <223> PTEN F primer for PCR

<400> 23 <400> 23

Figure 110104309-A0202-12-0051-34
Figure 110104309-A0202-12-0051-34

<210> 24 <210> 24

<211> 21 <211> 21

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的PTEN R引子 <223> PTEN R primer for PCR

<400> 24 <400> 24

Figure 110104309-A0202-12-0052-35
Figure 110104309-A0202-12-0052-35

<210> 25 <210> 25

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的PRKCA F引子 <223> PRKCA F primer for PCR

<400> 25 <400> 25

Figure 110104309-A0202-12-0052-36
Figure 110104309-A0202-12-0052-36

<210> 26 <210> 26

<211> 19 <211> 19

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的PRKCA R引子 <223> PRKCA R primer for PCR

<400> 26 <400> 26

Figure 110104309-A0202-12-0052-37
Figure 110104309-A0202-12-0052-37

<210> 27 <210> 27

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的ROBO1 F引子 <223> ROBO1 F primer for PCR

<400> 27 <400> 27

Figure 110104309-A0202-12-0053-38
Figure 110104309-A0202-12-0053-38

<210> 28 <210> 28

<211> 23 <211> 23

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的ROBO1 R引子 <223> ROBO1 R primer for PCR

<400> 28 <400> 28

Figure 110104309-A0202-12-0053-39
Figure 110104309-A0202-12-0053-39

<210> 29 <210> 29

<211> 19 <211> 19

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的SATB2 F引子 <223> SATB2 F primer for PCR

<400> 29 <400> 29

Figure 110104309-A0202-12-0054-40
Figure 110104309-A0202-12-0054-40

<210> 30 <210> 30

<211> 19 <211> 19

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的SATB2 R引子 <223> SATB2 R primer for PCR

<400> 30 <400> 30

Figure 110104309-A0202-12-0054-41
Figure 110104309-A0202-12-0054-41

<210> 31 <210> 31

<211> 24 <211> 24

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的TUBE1 F引子 <223> TUBE1 F primer for PCR

<400> 31 <400> 31

Figure 110104309-A0202-12-0054-42
Figure 110104309-A0202-12-0054-42

<210> 32 <210> 32

<211> 25 <211> 25

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的TUBE1 R引子 <223> TUBE1 R primer for PCR

<400> 32 <400> 32

Figure 110104309-A0202-12-0055-43
Figure 110104309-A0202-12-0055-43

<210> 33 <210> 33

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的UGT8 F引子 <223> UGT8 F primer for PCR

<400> 33 <400> 33

Figure 110104309-A0202-12-0055-44
Figure 110104309-A0202-12-0055-44

<210> 34 <210> 34

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於PCR的UGT8 R引子 <223> UGT8 R primer for PCR

<400> 34 <400> 34

Figure 110104309-A0202-12-0056-45
Figure 110104309-A0202-12-0056-45

Claims (16)

一種組合物用於製備抑制患有一胃癌的受試者的一腫瘤進展的藥物組合物的用途，其中該組合物包含靶向PHF8-c-Jun-PKCα-Src-PTEN軸的一抑製劑或其可藥用鹽。 Use of a composition for the manufacture of a pharmaceutical composition for inhibiting tumor progression in a subject with a gastric cancer, wherein the composition comprises an inhibitor targeting the PHF8-c-Jun-PKC α -Src-PTEN axis or Its pharmaceutically acceptable salt. 根據請求項1所述之用途，其中該腫瘤進展包括腫瘤生長、癌症擴散以及轉移。 The use according to claim 1, wherein the tumor progression includes tumor growth, cancer spread and metastasis. 根據請求項1所述之用途，其中該胃癌係為一HER2陰性的胃癌。 The use according to claim 1, wherein the gastric cancer is a HER2-negative gastric cancer. 根據請求項1所述之用途，其中該抑製劑係透過破壞PHF8和c-Jun之間的相互作用，進而抑制活化PRKCA表達。 The use according to claim 1, wherein the inhibitor inhibits activated PRKCA expression by disrupting the interaction between PHF8 and c-Jun. 根據請求項1所述之用途，其中該抑製劑係為一PKCα的抑制劑。 The use according to claim 1, wherein the inhibitor is a PKCα inhibitor. 根據請求項1所述之用途，其中該抑製劑係為一Src的抑制劑。 The use according to claim 1, wherein the inhibitor is a Src inhibitor. 根據請求項1所述之用途，其中該抑製劑係為PKCα抑製劑和Src抑製劑的組合。 The use according to claim 1, wherein the inhibitor is a combination of a PKCα inhibitor and a Src inhibitor. 根據請求項5所述之用途，其中該PKCα抑製劑係為 Midostaurin。 The use according to claim 5, wherein the PKCα inhibitor is Midostaurin. 根據請求項6所述之用途，其中該Src抑製劑係為Bosutinib。 The use according to claim 6, wherein the Src inhibitor is Bosutinib. 一種確定一患有胃癌的受試者中的腫瘤進展狀態的方法，其包括： A method of determining tumor progression status in a subject with gastric cancer, comprising: (a)從該患有胃癌的受試者提供一樣品； (a) providing a sample from the subject with gastric cancer; (b)檢測該樣品中PHF8的表達水平； (b) detecting the expression level of PHF8 in the sample; (c)將該樣品的PHF8表達水平與一預定閾值進行比較；以及 (c) comparing the PHF8 expression level of the sample to a predetermined threshold; and (d)確定該患有胃癌的受試者的腫瘤進展狀態，其中該樣品的PHF8表達水平高於該預定閾值，表明該患有胃癌的受試者為一胃癌晚期。 (d) determining the tumor progression status of the subject with gastric cancer, wherein the PHF8 expression level of the sample is higher than the predetermined threshold, indicating that the subject with gastric cancer is an advanced gastric cancer. 根據請求項1所述之方法，其中該PHF8表達係為一PHF8基因表達或是一PHF8蛋白質表達。 The method according to claim 1, wherein the PHF8 expression is a PHF8 gene expression or a PHF8 protein expression. 根據請求項11所述之方法，該PHF8基因表達為透過定量即時聚合酶連鎖反應或原位雜交法來確定。 According to the method of claim 11, the PHF8 gene expression is determined by quantitative real-time polymerase chain reaction or in situ hybridization. 根據請求項11所述之方法，該PHF8蛋白質表達為透 過免疫墨點法、免疫組織化學法或免疫磁減量分析來確定。 According to the method of claim 11, the expression of the PHF8 protein is transparent Determined by immunoblotting, immunohistochemistry, or immunomagnetic decrement analysis. 根據請求項10所述之方法，其中進一步包括檢測一PKCα表達水平，其中該PKCα表達水平高於一預定閾值，則預測該受試者具有不良預後。 The method of claim 10, further comprising detecting a PKCα expression level, wherein the PKCα expression level is above a predetermined threshold, predicting that the subject has a poor prognosis. 根據請求項10所述之方法，其中該胃癌晚期是從第II期到IV期。 The method according to claim 10, wherein the advanced gastric cancer is from stage II to stage IV. 根據請求項10所述之方法，其中該胃癌晚期是具有淋巴結轉移或遠處轉移的腫瘤。 The method according to claim 10, wherein the advanced gastric cancer is a tumor with lymph node metastasis or distant metastasis.
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