WO2017104912A1 - Composition for diagnosis of radio-resistance, and use thereof - Google Patents

Composition for diagnosis of radio-resistance, and use thereof Download PDF

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WO2017104912A1
WO2017104912A1 PCT/KR2016/004706 KR2016004706W WO2017104912A1 WO 2017104912 A1 WO2017104912 A1 WO 2017104912A1 KR 2016004706 W KR2016004706 W KR 2016004706W WO 2017104912 A1 WO2017104912 A1 WO 2017104912A1
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cancer cells
her2
stat3
breast cancer
survivin
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PCT/KR2016/004706
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French (fr)
Korean (ko)
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김재성
김현아
노우철
성민기
황상구
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한국원자력의학원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a biomarker for measuring radiation resistance and its medical use discovered from breast cancer cells showing radiation resistance.
  • breast cancer is the most common cancer among women around the world, and the incidence is also steadily increasing.
  • the treatment methods for breast cancer include surgical surgery, radiation therapy, hormonal therapy or targeted therapy targeting estrogen receptors or HER2. .
  • breast cancers are mainly hormone-positive breast cancers, which are treated with surgical methods or drugs such as tamoxifen, but the chemotherapy methods for breast cancers are easily tolerated and metastatic.
  • Radiation therapy is recommended for effective local site management in patients with high risk of recurrence, as well as breast conserving surgery, but some patients have decreased survival due to radiation therapy resistance and local site management failure.
  • HER2 is a gene that is overexpressed in about 20-30% of breast cancer patients and plays an important role in the progression and metastasis of breast cancer. Overexpression of HER2 has been reported to be associated with poor prognosis and decreased survival of patients. It can be an effective strategy for reducing aggression, and when HER2 is inhibited, it has been found that the radiation sensitivity of breast cancer cells is increased, so HER2 is a target molecule for radiotherapy in breast cancer patients and a biomarker for predicting the results of radiation therapy. Although it has been suggested that it can be used as a test, it has been reported that it is difficult to predict the survival rate of radiotherapy after mastectomy alone.
  • the present invention provides one or more genes or proteins thereof selected from STAT3 or survivin as a biomarker for predicting the outcome of radiation therapy for effective radiotherapy of a breast cancer patient, and using the biomarker for radiation sensitivity It is intended to provide a way to increase.
  • the present invention provides a composition for diagnosing radiation resistance of cancer cells, comprising an agent for measuring mRNA of one or more genes selected from STAT3 or survivin, or protein levels thereof.
  • the present invention provides a kit for diagnosing radiation resistance of cancer cells comprising the composition.
  • the present invention comprises the steps of measuring at least one gene mRNA or protein level thereof selected from STAT3 or survivin from a biological sample; And comparing the gene mRNA or protein level with at least one gene mRNA or protein level thereof selected from STAT3 or survivin measured from a normal control group. To provide.
  • the present invention provides a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, which contains an agent that inhibits the expression or activity of one or more genes selected from STAT3 or survivin as an active ingredient.
  • the present invention also provides a pharmaceutical composition for enhancing radiosensitivity to cancer cells, which contains an agent that inhibits the expression or activity of at least one protein selected from STAT3 or survivin.
  • the STAT3 or survivin gene or proteins thereof may be expressed in HR- / HER2 breast cancer. It can be provided as a biomarker for predicting patient radiation resistance, and through the control of their expression can enhance the radiation treatment effect of breast cancer patients.
  • FIG. 1 shows the correlation between HR- / HER2 + subtype and radiation resistance in breast cancer patients and breast cancer cell lines.
  • FIG. 1A shows Kaplan confirming the survival rate of patients who received breast conservative treatment with assisted radiotherapy. -Meier results (log-rank test, P ⁇ 0.001)
  • FIG. 1B shows cell viability by directly irradiating MCF7, MDA-MB231, SKBR3, T47D and BT-474 cells with various doses of radiation and performing colony formation assays.
  • 1C shows the results of immunoblot analysis using anti-HER2 and anti-ER antibodies in MCF7, MDA-MB231, SKBR3, T47D and BT-474 cells, and ⁇ -actin was used as a loading control. The data represents the mean ⁇ standard deviation of the results of the experiment repeated three times.
  • FIG. 2 is a result of confirming the radiation resistance of HR- / HER2 + breast cancer cells deficient in HER2,
  • Figure 2A is a 100 nM control siRNA or 100 nM HER2 siRNA 48 hours after transfection into SKBR3 cells treated with various doses of radiation
  • 2B shows 10 Gy radiation after transfection of 100 nM control siRNA or HER2 siRNA to SKBR3 cells or MDA-MB453 cells for 48 hours, and irradiated 48 hours after the irradiation (IR) )
  • IR irradiation
  • Ctrl cell viability of the irradiated cell group
  • 2C shows anti-irradiation of SKBR3 cells or MDA-MB453 cells after 100 nM control siRNA or HER2 siRNA transfection.
  • ⁇ -actin was used as a loading control, and each experimental data was repeated three times with the mean ⁇ standard deviation.
  • Were tanae the value * P ⁇ 0.05 or ** P ⁇ 0.01 compared with irradiated siRNA control cells (A and B).
  • FIG. 3 is a result of confirming the radiation sensitivity according to STAT3 regulation in HR- / HER2 + breast cancer cells deficient in HER2,
  • Figures 3A and 3B shows a 100 nM control siRNA or HER2 siRNA in SKBR3 cells or MDA-MB453 cells for 48 hours Immunoblot was performed using the loading control ⁇ -actin and each antibody 48 hours after transfection treatment (IR) with or without 10 Gy radiation (Ctrl), and FIG. 3C was treated with the above method.
  • IR transfection treatment
  • Ctrl 10 Gy radiation
  • FIG. 4 shows the radiosensitivity of HR- / HER2 + breast cancer cells according to HER2, STAT3 and survivin inhibition.
  • FIG. 4A shows 10 Gy radiation to SKBR3 cells in the presence of 1 ⁇ M lapatinib or 100 ⁇ M S3I-201. After culturing for 24 hours without treatment, the expression levels of HER2, STAT3 and survivin proteins were confirmed.
  • FIG. 4B shows the cell viability of SKBR3 cells treated by the above procedure by flow cytometry and the percentage of PI-positive cells.
  • 4C shows the survival rate of SKBR3 cells treated with or without 3 Gy radiation in the presence of 1 ⁇ M lapatinib, 100 ⁇ M S3I-201 or 1 ⁇ M lapatinib + 100 ⁇ M S3I-201, and FIG. 4D
  • This is the result of immunoblotting of SKBR3 cells transfected with 100 nM control siRNA or survivin siRNA for 48 hours and subjected to 10 Gy radiation and confirming the amount of protein expression using each antibody.
  • 4E was treated with 10 Gy radiation to SKBR3 cells transfected with 100 nM control siRNA or survivin siRNA for 48 hours, and colony formation was confirmed by colony formation analysis.
  • a and D) each experimental data is the average ⁇ standard deviation of the results of the experiment repeated three times, ** P ⁇ 0.01 compared with irradiated siRNA control cells (B, C and E) value.
  • FIG. 5 shows the positive correlation between recurrent HER2-positive breast cancer and phosphorylated STAT3, STAT3 and survivin expression after radiation treatment.
  • FIG. 5A shows anti-phosphorylated STAT3 ( Y705), micrographs confirming anti-STAT3 and anti-survivin staining, FIG.
  • FIG. 5B shows anti-phosphorylated STAT3 (Y705), anti-STAT3 and anti-survivin staining in non-recurrent HER2-positive breast cancer tissues
  • the above results are representative of the box-and-whisker plot.
  • Staining intensity was evaluated by the following scores: 0, not stained; +1, weak staining; +2, moderate; And +3, strong staining; * P ⁇ 0.05 compared
  • the present invention may provide a composition for diagnosing radiation resistance of cancer cells, including an agent for measuring mRNA of one or more genes selected from STAT3 or survivin, or protein levels thereof.
  • the cancer cells may be breast cancer cells, more preferably HR- / HER2 + subtype breast cancer cells.
  • the agent for measuring the level of the gene mRNA may comprise a primer or probe that specifically binds to the gene.
  • Agents for measuring the protein level may include antibodies or aptamers specific for the protein.
  • the survival rate analysis was performed by confirming colony formation rate after 3Gy irradiation, and as shown in FIG. 4C, the survival rate of HER2-positive SKBR3 breast cancer cell group treated with lapatinib or S3I-201 and radiation was significantly decreased. It could be confirmed.
  • the STAT3 and survivin genes or proteins thereof can be used as biomarkers for predicting radiation sensitivity of HR- / HER2 breast cancer patients, and their expression can be controlled to enhance the radiation treatment effect of breast cancer patients.
  • the present invention can provide a kit for diagnosing radiation resistance of cancer cells comprising the composition.
  • the kit may be, but is not limited to, RT-PCR kit, DNA chip kit, microarray, or protein chip kit.
  • the present invention comprises the steps of measuring at least one gene mRNA or protein level thereof selected from STAT3 or survivin from a biological sample; And comparing the gene mRNA or protein level with at least one gene mRNA or protein level thereof selected from STAT3 or survivin measured from a normal control group. Can be provided.
  • the biological sample may be selected from the group consisting of tissue and blood, and the gene mRNA or protein level thereof may be reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunoassay. It can be measured by any one selected from the group consisting of immunohistochemistry, Western blotting, and flow cytometry (FACS).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • ELISA enzyme immunoassay
  • FACS flow cytometry
  • the present invention can provide a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, containing as an active ingredient an agent that inhibits the expression or activity of one or more genes selected from STAT3 or survivin.
  • the cancer cells may be breast cancer cells, more preferably HR- / HER2 + subtype breast cancer cells.
  • the agent may be selected from the group consisting of antisense nucleotides, siRNAs and shRNAs that complementarily bind to mRNA of the STAT3 or survivin gene, but is not limited thereto.
  • the present invention can provide a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, containing as an active ingredient an agent that inhibits the expression or activity of one or more proteins selected from STAT3 or survivin.
  • the cancer cells may be breast cancer cells, more preferably HR- / HER2 + subtype breast cancer cells.
  • the agent may be selected from the group consisting of compounds, peptides, aptamers, antibodies and natural products that specifically bind to STAT3 or survivin proteins.
  • the term "enhancement of radiation sensitivity” refers to enhancing the sensitivity of cells to radiation in treating diseases using radiation. Through this, the radiation treatment efficiency can be increased. In particular, when the cancer treatment is performed in parallel, the radiation sensitivity of the cancer cells can be enhanced, and the killing effect and the proliferation inhibitory effect of the cancer cells can be exhibited.
  • composition for enhancing radiation sensitivity of the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier.
  • pharmaceutically acceptable carrier is meant a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • composition for enhancing radiation sensitivity of the present invention can be applied in any dosage form, can be prepared in oral or parenteral dosage form, and can be formulated in unit dosage form for ease of administration and uniformity of dosage.
  • Pharmaceutical formulations of the invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.
  • the oral dosage form may be formulated, for example, in tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs.
  • lactose saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate
  • binders such as cellulose or gelatin
  • excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate
  • It may include a lubricating oil, such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • parenteral formulation may be formulated for injection such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection method, or aerosol for inhalation through the respiratory system.
  • the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials.
  • solutions or suspensions For infusion into suppositories, it may be formulated as a rectal composition such as suppositories or body enema including conventional suppository bases such as cocoa butter or other glycerides.
  • a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.
  • Human breast cancer cells MCF7, MDA-MB231, SKBR3, T47D and BT474 were purchased from the American Type Culture Collection (ATCC) Manassas, VA.
  • the purchased cells were incubated at 37 ° C. and 5% CO 2 in DMEM medium containing 10% fetal bovine serum (FBS; HyClone, South Logan, UT) and penicillin / streptomycin.
  • FBS fetal bovine serum
  • Cs cesium
  • S3I-201 100 ⁇ M (EMD Millipore, Billerica, Mass.)
  • lapatinib 1 ⁇ M lapatinib; Selleckchem , Houston, TX
  • RNA interference was synthesized in (Seoul, Korea) and were as follows: HER2, 5'-CUGGUGUAUGCAGAUUGCC-3 'and survivin, 5'-AAGGAGAUCAACAUUUUCA-3'.
  • siRNA transfection was performed using G-Fectin (Genolution Pharmaceuticals Inc.) according to the manufacturer's instructions.
  • the proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and the proteins were detected using specific antibodies.
  • rabbit monoclonal anti-survivin rabbit polyclonal anti-phospho-STAT3 (Tyr705) and anti-cleaved-PARP (Asp214) were purchased from Cell Signaling Technology (Beverly, MA) and used as mice.
  • Monoclonal anti-STAT3, rabbit polyclonal anti-ER ⁇ and anti-HER2 are listed in Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.)
  • mouse monoclonal anti- ⁇ -actin was purchased from Sigma (St. Louis, MO).
  • Blots were detected using HRP-binding secondary antibodies and an enhanced chemiluminescent detection system (Amersham Life Science, Piscataway, NJ).
  • Cells were transfected with 21pSTAT3-TA-Luc and control siRNA or HER2 siRNA for 48 hours using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) And treated with or without radiation 10 Gy.
  • Apoptosis analysis was performed by a previously reported method (Kim JS et al., Proteomics. 2010; 10 (14): 2589-2604.).
  • the cells were incubated with propidium iodide (5 ⁇ M / mL) for 10 minutes and analyzed for apoptosis using a FACScan flow cytometer (Becton Dickson, Franklin Lakes, NJ).
  • IHC immunohistochemistry
  • the patients were classified according to the following four subtypes based on the degree of tumor expression of ER, PR and HER2.
  • Specimens for IHC were obtained from HER2 overexpressed primary breast cancer tissue paraffin blocks removed through radical surgery.
  • anti-STAT3 mouse monoclonal antibody (1: 200 dilution; Santa Cruz), anti-phospho-STAT3 rabbit polyclonal antibody (1:50 dilution; GeneTex, Irvine, CA), or anti-survivin lash single Immunohistochemical staining was performed using clone antibody (1: 100 dilution; Cell Signaling Technology).
  • Immunostaining was performed using the avidin-biotin-peroxide as instructed by the manufacturer (Invitrogen).
  • Dye intensity was evaluated based on the following criteria: 0 (no staining observed), 1+ (faint staining), 2+ (normal staining intensity), and 3+ (strong staining confirmed).
  • radiation therapy sensitivity is related to molecular subtype differences in breast cancer.
  • HER2 expression was associated with radiation resistance in breast cancer
  • HER2 siRNA is a major mediator of radiation resistance through gene inhibition induced by HER2 siRNA in HER2-positive breast cancer cells.
  • the SKBR3 cell group whose HER2 expression was suppressed by siRNA was sensitive to radiation of various doses and showed low survival rate, and also in HER2-positive SKBR3 and MDA-MB453 breast cancer cells as shown in FIGS. 2B and 2C.
  • HER2 is deficient, it was confirmed that cell death by radiation is increased.
  • HER2 is a major factor regulating radiation resistance in HER2-positive breast cancer cells.
  • HER2 promotes the activity of STAT3 induced by radiation, and this signaling process was identified as it is suggested to be one of the major signaling processes of complex radiation resistant cancer.
  • the luciferase assay was performed using a STAT3 reporter plasmid with a STAT3 binding element to confirm direct transcriptional activity of STAT3.
  • STAT3 reporter plasmid with a STAT3 binding element to confirm direct transcriptional activity of STAT3.
  • HER2 was deficient in irradiated cells, radiation-induced STAT3 activity was inhibited. there was.
  • Lapatinib and S3I-201, inhibitors of HER2 and STAT3, were treated with HER2-positive SKBR3 breast cancer cells, respectively, and irradiated with 10 Gy dose.
  • the survival rate analysis was performed by confirming colony formation rate after 3Gy irradiation, and as shown in FIG. 4C, the survival rate of HER2-positive SKBR3 breast cancer cell group treated with lapatinib or S3I-201 and radiation was significantly decreased. It could be confirmed.
  • HER2-STAT3-survivin signaling process was found to be a major factor in radiation resistance in HER2-positive SKBR3 breast cancer cells, so HER2-STAT3-survivin signal is a radiation support for the treatment of HER2-positive breast cancer. It can be suggested as the main target of treatment.
  • FIGS. 5A and 5B a strong nucleic acid staining pattern of phosphorylated STAT3 and survivin was observed in the relapsed HER2-positive breast cancer patient group than in the relapsed HER2-positive breast cancer patient group.
  • the increased expression of phosphorylated STAT3, STAT3 and survivin through FIGS. 5C to 5E showed a positive correlation with the group not showing sensitivity to radiation therapy.

Abstract

The present invention relates to: a biomarker composition for diagnosis of radio-resistance in breast cancer cells, containing, as active ingredients, gene mRNAs comprising STAT3 and survivin or protein groups thereof; and a use thereof. More specifically, since it has been identified that radio-resistance, exhibited in HR-/HER2+ breast cancer patients, is due to an increase in the activity or the expression of STAT3 and survivin, STAT3 and survivin genes or proteins thereof can be provided as biomarkers for predicting radio-resistance in an HR-/HER2 breast cancer patient, and an effect of radiotherapy in a breast cancer patient can be enhanced by regulating the expression of STAT3 and survivin.

Description

방사선 저항성 진단용 조성물 및 이의 용도Radiation resistance diagnostic composition and use thereof
본 발명은 방사선 저항성을 나타내는 유방암세포로부터 발굴된 방사선 저항성 측정용 바이오마커 및 이의 의학적 용도에 관한 것이다.The present invention relates to a biomarker for measuring radiation resistance and its medical use discovered from breast cancer cells showing radiation resistance.
유방암은 전세계 여성에게 가장 흔히 발병되는 암으로, 발병률 또한 꾸준히 증가하고 있으며, 이러한 유방암의 치료방법으로는 외과적 수술이나 방사선 치료, 에스트로겐 수용체나 HER2를 타겟팅하는 호르몬 치료 또는 표적치료 방법이 이용되고 있다.Breast cancer is the most common cancer among women around the world, and the incidence is also steadily increasing. The treatment methods for breast cancer include surgical surgery, radiation therapy, hormonal therapy or targeted therapy targeting estrogen receptors or HER2. .
대부분의 유방암은 주로 호르몬 양성 유방암으로 외과적 수술 방법이나, 타목시펜과 같은 약물요법으로 치료되고 있으나, 유방암의 화학적 치료방법은 내성이 잘 생기며, 전이능을 가진 형태로 쉽게 변화하게 한다.Most breast cancers are mainly hormone-positive breast cancers, which are treated with surgical methods or drugs such as tamoxifen, but the chemotherapy methods for breast cancers are easily tolerated and metastatic.
방사선 치료는 유방 보존수술뿐만 아니라 재발 위험이 높은 유방 절제술을 받은 환자들의 효과적인 국소 부위 관리를 위해 추천되는 치료방법이나, 일부 환자들에게는 방사선 치료 저항성과 국소 부위 관리 실패에 따른 생존율 감소가 나타난다.Radiation therapy is recommended for effective local site management in patients with high risk of recurrence, as well as breast conserving surgery, but some patients have decreased survival due to radiation therapy resistance and local site management failure.
HER2는 약 20-30%의 유방암 환자에서 과발현되어 유방암의 진행 및 전이에 중요한 역할을 하는 유전자로, HER2의 과발현은 나쁜 예후 및 환자의 생존율 감소와 관련 있다고 보고되어짐에 따라, HER2 발현 억제는 종양 공격성 감소를 위한 효과적인 전략이 될 수 있으며, HER2가 억제될 경우, 유방암세포의 방사선 민감성이 증가되는 것이 확인됨에 따라, HER2는 유방암 환자의 방사선 치료를 위한 타겟 분자 및 방사선 치료 결과 예측을 위한 바이오마커로 사용할 수 있을 것으로 제안되었으나, 아직까지는 HER2 상태만으로는 유방절제 후 방사선체료에 대한 생존율 예측이 어렵다는 연구 결과가 보고되었다.HER2 is a gene that is overexpressed in about 20-30% of breast cancer patients and plays an important role in the progression and metastasis of breast cancer. Overexpression of HER2 has been reported to be associated with poor prognosis and decreased survival of patients. It can be an effective strategy for reducing aggression, and when HER2 is inhibited, it has been found that the radiation sensitivity of breast cancer cells is increased, so HER2 is a target molecule for radiotherapy in breast cancer patients and a biomarker for predicting the results of radiation therapy. Although it has been suggested that it can be used as a test, it has been reported that it is difficult to predict the survival rate of radiotherapy after mastectomy alone.
이러한 방사선 치료의 문제점을 해결하기 위해, 방사선 치료 결과를 예측할 수 있을 뿐만 아니라 방사선 저항성을 갖는 세포에 민감성을 증가시키기 위한 분자 신호에 대한 연구가 필요한 실정이다. In order to solve the problems of radiation therapy, research on molecular signals for increasing the sensitivity to radiation resistant cells as well as predicting the results of radiation therapy is required.
본 발명은 유방암 환자의 효과적인 방사선 치료를 위해 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자 또는 이의 단백질을 방사선 치료 결과 예측을 위한 바이오마커로 제공하며, 상기 바이오마커를 이용하여 방사선 민감성을 증가시킬 수 있는 방법을 제공하고자 한다.The present invention provides one or more genes or proteins thereof selected from STAT3 or survivin as a biomarker for predicting the outcome of radiation therapy for effective radiotherapy of a breast cancer patient, and using the biomarker for radiation sensitivity It is intended to provide a way to increase.
본 발명은 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자의 mRNA, 또는 이의 단백질 수준을 측정하는 제제를 포함하는, 암세포의 방사선 저항성 진단용 조성물을 제공한다.The present invention provides a composition for diagnosing radiation resistance of cancer cells, comprising an agent for measuring mRNA of one or more genes selected from STAT3 or survivin, or protein levels thereof.
본 발명은 상기 조성물을 포함하는 암세포의 방사선 저항성 진단용 키트를 제공한다.The present invention provides a kit for diagnosing radiation resistance of cancer cells comprising the composition.
본 발명은 생물학적 시료로부터 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자 mRNA 또는 이의 단백질 수준을 측정하는 단계; 및 상기 유전자 mRNA 또는 단백질 수준을 정상 대조군으로부터 측정한 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자 mRNA 또는 이의 단백질 수준과 비교하는 단계를 포함하는 방사선 저항성 예후 진단에 필요한 정보를 제공하는 방법을 제공한다.The present invention comprises the steps of measuring at least one gene mRNA or protein level thereof selected from STAT3 or survivin from a biological sample; And comparing the gene mRNA or protein level with at least one gene mRNA or protein level thereof selected from STAT3 or survivin measured from a normal control group. To provide.
본 발명은 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자의 발현 또는 활성을 억제하는 제제를 유효성분으로 함유하는, 암세포에 대한 방사선 민감성 증진용 약학조성물을 제공한다.The present invention provides a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, which contains an agent that inhibits the expression or activity of one or more genes selected from STAT3 or survivin as an active ingredient.
또한, 본 발명은 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 단백질의 발현 또는 활성을 억제하는 제제를 유효성분으로 함유하는, 암세포에 대한 방사선 민감성 증진용 약학조성물을 제공한다.The present invention also provides a pharmaceutical composition for enhancing radiosensitivity to cancer cells, which contains an agent that inhibits the expression or activity of at least one protein selected from STAT3 or survivin.
본 발명에 따르면, HR-/HER2 유방암 환자에게서 나타나는 방사선 저항성은 STAT3 또는 서바이빈의 활성 또는 발현 증가에 의한 것으로 확인됨에 따라, 상기 STAT3 또는 서바이빈 유전자 또는 이들의 단백질들은 HR-/HER2 유방암 환자 방사선 저항성 예측용 바이오마커로 제공될 수 있으며, 이들의 발현 조절을 통하여 유방암 환자의 방사선 치료 효과를 증진시킬 수 있다.According to the present invention, as radiation resistance in HR- / HER2 breast cancer patients is found to be due to increased activity or expression of STAT3 or survivin, the STAT3 or survivin gene or proteins thereof may be expressed in HR- / HER2 breast cancer. It can be provided as a biomarker for predicting patient radiation resistance, and through the control of their expression can enhance the radiation treatment effect of breast cancer patients.
도 1은 유방암 환자 및 유방암 세포주에서 HR-/HER2+ 서브타입(subtype)과 방사선 저항성의 상관관계를 확인한 결과로, 도 1A는 보조적인 방사선치료를 수행한 유방 보존 치료를 받은 환자의 생존율을 확인한 Kaplan-Meier 결과이며(log-rank test, P < 0.001), 도 1B는 MCF7, MDA-MB231, SKBR3, T47D 및 BT-474 세포에 다양한 선량의 방사선을 직접 조사하고 집락형성 분석을 수행하여 세포 생존율을 확인한 결과이며, 도 1C는 MCF7, MDA-MB231, SKBR3, T47D 및 BT-474 세포에서 항-HER2 및 항-ER 항체를 이용한 면역블롯 분석결과로, β-액틴을 로딩 대조군으로 이용하였으며, 각 실험 데이터는 3번씩 반복 수행된 실험 결과를 평균±표준편차로 나타내었다.FIG. 1 shows the correlation between HR- / HER2 + subtype and radiation resistance in breast cancer patients and breast cancer cell lines. FIG. 1A shows Kaplan confirming the survival rate of patients who received breast conservative treatment with assisted radiotherapy. -Meier results (log-rank test, P <0.001), FIG. 1B shows cell viability by directly irradiating MCF7, MDA-MB231, SKBR3, T47D and BT-474 cells with various doses of radiation and performing colony formation assays. 1C shows the results of immunoblot analysis using anti-HER2 and anti-ER antibodies in MCF7, MDA-MB231, SKBR3, T47D and BT-474 cells, and β-actin was used as a loading control. The data represents the mean ± standard deviation of the results of the experiment repeated three times.
도 2는 HER2가 결핍된 HR-/HER2+ 유방암세포의 방사선 저항성을 확인한 결과로, 도 2A는 SKBR3 세포에 100 nM 대조군 siRNA 또는 100 nM HER2 siRNA를 48시간 동안 형질주입한 후 다양한 선량의 방사선을 처리하고 세포 생존율을 확인한 결과이며, 도 2B는 48시간 동안 SKBR3 세포 또는 MDA-MB453 세포에 100 nM 대조군 siRNA 또는 HER2 siRNA가 형질주입 후 10 Gy 방사선을 조사하고, 48시간 후 방사선이 조사된 세포군(IR)과 조사되지 않은 세포군(Ctrl)의 세포 생존율을 유세포분석기(FACScan flow cytometer)로 확인한 결과이며, 도 2C는 100 nM 대조군 siRNA 또는 HER2 siRNA 형질주입 후 방사선 조사된 SKBR3 세포 또는 MDA-MB453 세포에 항-cleaved-PARP 및 항-HER2 항체를 이용한 면역블롯 분석결과로, β-액틴을 로딩 대조군으로 이용하였으며, 각 실험 데이터는 3번씩 반복 수행된 실험 결과를 평균±표준편차로 나타내었으며, *P < 0.05 또는 **P < 0.01 compared with irradiated siRNA control cells (A 및 B) 값이다.2 is a result of confirming the radiation resistance of HR- / HER2 + breast cancer cells deficient in HER2, Figure 2A is a 100 nM control siRNA or 100 nM HER2 siRNA 48 hours after transfection into SKBR3 cells treated with various doses of radiation 2B shows 10 Gy radiation after transfection of 100 nM control siRNA or HER2 siRNA to SKBR3 cells or MDA-MB453 cells for 48 hours, and irradiated 48 hours after the irradiation (IR) ) And the cell viability of the irradiated cell group (Ctrl) was confirmed by a FACScan flow cytometer. FIG. 2C shows anti-irradiation of SKBR3 cells or MDA-MB453 cells after 100 nM control siRNA or HER2 siRNA transfection. As a result of immunoblot analysis using -cleaved-PARP and anti-HER2 antibody, β-actin was used as a loading control, and each experimental data was repeated three times with the mean ± standard deviation. Were tanae, the value * P <0.05 or ** P <0.01 compared with irradiated siRNA control cells (A and B).
도 3은 HER2가 결핍된 HR-/HER2+ 유방암세포에서 STAT3 조절에 따른 방사선 민감성을 확인한 결과로, 도 3A 및 도 3B는 SKBR3 세포 또는 MDA-MB453 세포에 100 nM 대조군 siRNA 또는 HER2 siRNA를 48시간 동안 형질주입하여 10 Gy 방사선을 처리(IR)하거나 처리하지 않고(Ctrl) 48시간 후 로딩 대조군인 β-액틴 및 각각의 항체를 이용하여 면역블롯을 수행한 결과이며, 도 3C는 상기 방법으로 처리된 각 세포에서 STAT3 활성을 확인한 결과로, 각 실험 데이터는 3번씩 반복 수행된 실험 결과를 평균±표준편차로 나타내었으며, **P < 0.01 compared with irradiated siRNA control cells (C) 값이다.3 is a result of confirming the radiation sensitivity according to STAT3 regulation in HR- / HER2 + breast cancer cells deficient in HER2, Figures 3A and 3B shows a 100 nM control siRNA or HER2 siRNA in SKBR3 cells or MDA-MB453 cells for 48 hours Immunoblot was performed using the loading control β-actin and each antibody 48 hours after transfection treatment (IR) with or without 10 Gy radiation (Ctrl), and FIG. 3C was treated with the above method. As a result of confirming the STAT3 activity in each cell, each experimental data represents the mean ± standard deviation of the results of the experiment repeated three times, ** P <0.01 compared with irradiated siRNA control cells (C) value.
도 4는 HER2, STAT3 및 서바이빈 억제에 따른 HR-/HER2+ 유방암세포의 방사선 민감성을 확인한 결과로, 도 4A는 1 μM 라파티닙 또는 100 μM S3I-201 존재하에서 SKBR3 세포에 10 Gy 방사선을 처리 또는 처리하지 않고 24시간 동안 배양한 후 HER2, STAT3 및 서바이빈 단백질 발현량을 확인한 결과이며, 도 4B는 상기 과정으로 처리된 SKBR3 세포의 세포 생존율을 유세포 분석기로 측정하고 PI-양성 세포에 대한 백분율로 나타낸 결과이며, 도 4C는 1 μM 라파티닙, 100 μM S3I-201 또는 1 μM 라파티닙 + 100 μM S3I-201 존재하에서 3 Gy 방사선이 처리 또는 처리하지 않은 SKBR3 세포의 생존율을 확인한 결과이며, 도 4D는 48시간 동안 100 nM 대조군 siRNA 또는 서바이빈 siRNA가 형질주입된 SKBR3 세포에 10 Gy 방사선을 처리하고 각각의 항체를 이용하여 단백질 발현량을 확인한 면역블롯팅 결과이며, 도 4E는 48시간 동안 100 nM 대조군 siRNA 또는 서바이빈 siRNA가 형질주입된 SKBR3 세포에 10 Gy 방사선을 처리하고 콜로니 형성 분석을 통하여 집락형성 생존율을 확인한 결과로, β-액틴을 로딩 대조군을 사용하였으며(A 및 D), 각 실험 데이터는 3번씩 반복 수행된 실험 결과를 평균±표준편차로 나타내었으며, **P < 0.01 compared with irradiated siRNA control cells (B, C 및 E) 값이다. 4 shows the radiosensitivity of HR- / HER2 + breast cancer cells according to HER2, STAT3 and survivin inhibition. FIG. 4A shows 10 Gy radiation to SKBR3 cells in the presence of 1 μM lapatinib or 100 μM S3I-201. After culturing for 24 hours without treatment, the expression levels of HER2, STAT3 and survivin proteins were confirmed. FIG. 4B shows the cell viability of SKBR3 cells treated by the above procedure by flow cytometry and the percentage of PI-positive cells. 4C shows the survival rate of SKBR3 cells treated with or without 3 Gy radiation in the presence of 1 μM lapatinib, 100 μM S3I-201 or 1 μM lapatinib + 100 μM S3I-201, and FIG. 4D This is the result of immunoblotting of SKBR3 cells transfected with 100 nM control siRNA or survivin siRNA for 48 hours and subjected to 10 Gy radiation and confirming the amount of protein expression using each antibody. 4E was treated with 10 Gy radiation to SKBR3 cells transfected with 100 nM control siRNA or survivin siRNA for 48 hours, and colony formation was confirmed by colony formation analysis. A and D), each experimental data is the average ± standard deviation of the results of the experiment repeated three times, ** P <0.01 compared with irradiated siRNA control cells (B, C and E) value.
도 5는 방사선 치료 후 재발된 HER2-양성 유방암과 인산화된 STAT3, STAT3 및 서바이빈 발현 사이의 정적 상관관계를 확인한 결과로, 도 5A는 재발된 HER2-양성 유방암 조직에서 항-인산화된 STAT3 (Y705), 항-STAT3 및 항-서바이빈 염색을 확인한 현미경 사진이며, 도 5B는 재발되지 않은 HER2-양성 유방암 조직에서 항-인산화된 STAT3 (Y705), 항-STAT3 및 항-서바이빈 염색하고 각 단백질의 핵산 염색 패턴을 확인한 고배율 현미경 사진이며(Scale bar, 50 μm), 도 3C는 재발군 조직(n=7)과 비재발군 조직(n=7)의 인산화된 STAT3의 염색 강도를 각각 정량한 결과이며, 도 3D는 재발군 조직(n=7)과 비재발군 조직(n=7)의 STAT3 염색 강도를 정량한 결과이며, 도 3E는 재발군 조직(n=7)과 비재발군 조직(n=7)의 서바이빈 염색 강도를 정량한 결과로, 상기 결과들은 box-and-whisker plot의 대표적인 결과이며, 염색강도는 다음과 같은 점수로 평가하였다: 0, 염색되지 않음; +1, 약한 염색; +2, 보통; 및 +3, 강한 염색; *P < 0.05 compared with responder group.FIG. 5 shows the positive correlation between recurrent HER2-positive breast cancer and phosphorylated STAT3, STAT3 and survivin expression after radiation treatment. FIG. 5A shows anti-phosphorylated STAT3 ( Y705), micrographs confirming anti-STAT3 and anti-survivin staining, FIG. 5B shows anti-phosphorylated STAT3 (Y705), anti-STAT3 and anti-survivin staining in non-recurrent HER2-positive breast cancer tissues High magnification micrograph (Scale bar, 50 μm) confirming the nucleic acid staining pattern of each protein, Figure 3C shows the staining intensity of phosphorylated STAT3 of recurrent tissue (n = 7) and non-recurrent tissue (n = 7) Figure 3D is a result of quantifying the STAT3 staining intensity of the relapsed tissue (n = 7) and non-recurrent tissue (n = 7), Figure 3E is a recurrent tissue (n = 7) and non-recurrent tissue As a result of quantifying survivin staining intensity of (n = 7), the above results are representative of the box-and-whisker plot. , Staining intensity was evaluated by the following scores: 0, not stained; +1, weak staining; +2, moderate; And +3, strong staining; * P <0.05 compared with responder group.
본 발명은 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자의 mRNA, 또는 이의 단백질 수준을 측정하는 제제를 포함하는, 암세포의 방사선 저항성 진단용 조성물을 제공할 수 있다.The present invention may provide a composition for diagnosing radiation resistance of cancer cells, including an agent for measuring mRNA of one or more genes selected from STAT3 or survivin, or protein levels thereof.
상기 암세포는 유방암세포일 수 있으며, 보다 바람직하게는 HR-/HER2+ 서브타입 유방암세포일 수 있다.The cancer cells may be breast cancer cells, more preferably HR- / HER2 + subtype breast cancer cells.
상기 유전자 mRNA의 수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 프라이머 또는 프로브를 포함할 수 있다.The agent for measuring the level of the gene mRNA may comprise a primer or probe that specifically binds to the gene.
상기 단백질 수준을 측정하는 제제는 상기 단백질에 특이적인 항체 또는 앱타머를 포함할 수 있다.Agents for measuring the protein level may include antibodies or aptamers specific for the protein.
본 발명의 일실시예에 따르면, 유방암 환자의 분자 서브타입에 따라 국소부위 무재발 생존율이 유의한 차이를 나타내는 것으로 확인되었는데, 도 1A를 참고하면, HR+/HER2- 유방암 환자들은 매우 높은 국소 부위 무재발 생존율을 나타낸 반면, HR-/HER2+ 환자들은 재발할 경우 매우 낮은 생존율이 나타나는 것으로 확인되었다. 상기 결과를 재확인하기 위해, MCF7 및 T47D (HR+/HER2-), MDA-MB231 (HR-/HER2-), BT474 (HR+/HER2+), 및 SKBR3 (HR-/HER2+)을 포함한 유방암 세포주에 다양한 선량의 방사선을 조사하여 반응성을 확인한 결과, 도 1B 및 1C와 같이 다른 유방암 세포와 비교하여 HER2-양성(HR-/HER2+) 유방암 세포주인 SKBR3에서 매우 높은 방사선 저항성이 나타나는 것을 확인할 수 있었다.According to one embodiment of the present invention, it was confirmed that there is a significant difference in the local recurrence-free survival rate according to the molecular subtype of the breast cancer patient, referring to Figure 1A, HR + / HER2- breast cancer patients are very high While relapse survival was observed, HR- / HER2 + patients were found to have very low survival rates when relapsed. To reconfirm the above results, various doses to breast cancer cell lines including MCF7 and T47D (HR + / HER2-), MDA-MB231 (HR- / HER2-), BT474 (HR + / HER2 +), and SKBR3 (HR- / HER2 +) As a result of irradiating with radiation, it was confirmed that very high radiation resistance was observed in SKBR3, a HER2-positive (HR- / HER2 +) breast cancer cell line, as compared with other breast cancer cells as shown in FIGS. 1B and 1C.
상기 결과로부터 방사선 치료 민감성은 유방암의 분자 서브타입 차이와 관련이 있으며, 특히 HR-/HER2 유방암 환자의 방사선 치료 예후가 매우 나쁜 것으로 확인되었다.The results indicate that radiation therapy sensitivity is related to the difference in molecular subtypes of breast cancer, especially the prognosis of radiotherapy in HR- / HER2 breast cancer patients.
상기 결과에서 확인된 HR-/HER2 유방암 환자에게서 나타나는 방사선 저항성의 신호과정을 확인한 결과, 도 3A와 같이 HER2가 결핍된 HER2-양성 SKBR3 및 MDA-MB453 유방암 세포에서 인산화된 STAT3와 서바이빈의 감소가 확인되었으며, 실제로 STAT3의 억제가 HER2-양성 SKBR3 유방암세포의 방사선 민감성을 증가시킬 수 있는 지를 확인하기 위해 HER2 및 STAT3의 억제자인 라파티닙(lapatinib) 및 S3I-201를 HER2-양성 SKBR3 유방암세포에 각각 처리하고, 10 Gy 선량의 방사선을 조사하였다.As a result of confirming the radiation resistance signaling process in HR- / HER2 breast cancer patients identified in the above results, reduction of phosphorylated STAT3 and survivin in HER2-deficient HER2-positive SKBR3 and MDA-MB453 breast cancer cells as shown in FIG. 3A In order to confirm that inhibition of STAT3 could actually increase the radiation sensitivity of HER2-positive SKBR3 breast cancer cells, lapatinib and S3I-201, inhibitors of HER2 and STAT3, were applied to HER2-positive SKBR3 breast cancer cells, respectively. The treatments were irradiated with 10 Gy dose of radiation.
그 결과, 도 4A와 같이 라파티닙(lapatinib) 및 S3I-201이 처리된 HER2-양성 SKBR3 유방암세포에서 방사선 조사에 의해 유도된 세포사멸이 매우 효과적으로 증가된 것이 확인되었을 뿐만 아니라, 도 4B와 같이 방사선에 의해 유도되는 STAT3 인산화 및 서바이빈 발현이 매우 효과적으로 감소된 것을 확인할 수 있었다.As a result, it was confirmed that the apoptosis induced by irradiation in HER2-positive SKBR3 breast cancer cells treated with lapatinib and S3I-201 as shown in FIG. It was confirmed that the STAT3 phosphorylation and survivin expression induced by HCl were very effectively reduced.
또한, 3Gy 방사선 조사 후 콜로니 형성율을 확인하여 생존율 분석을 수행한 결과, 도 4C와 같이 라파티닙(lapatinib) 또는 S3I-201과 방사선이 함께 처리된 HER2-양성 SKBR3 유방암세포군의 생존율이 매우 유의하게 감소된 것을 확인할 수 있었다.In addition, the survival rate analysis was performed by confirming colony formation rate after 3Gy irradiation, and as shown in FIG. 4C, the survival rate of HER2-positive SKBR3 breast cancer cell group treated with lapatinib or S3I-201 and radiation was significantly decreased. It could be confirmed.
상기 결과로부터 HR-/HER2 유방암 환자에게서 나타나는 방사선 저항성은 STAT3 및 서바이빈의 활성 또는 발현에 의한 것으로 확인되었다.From these results, radiation resistance in HR- / HER2 breast cancer patients was confirmed by the activity or expression of STAT3 and survivin.
따라서 상기 STAT3 및 서바이빈 유전자 또는 이들의 단백질들은 HR-/HER2 유방암 환자 방사선 민감성 예측용 바이오마커로 사용할 수 있으며, 이들의 발현 조절을 통하여 유방암 환자의 방사선 치료 효과를 증진시킬 수 있다.Therefore, the STAT3 and survivin genes or proteins thereof can be used as biomarkers for predicting radiation sensitivity of HR- / HER2 breast cancer patients, and their expression can be controlled to enhance the radiation treatment effect of breast cancer patients.
본 발명은 상기 조성물을 포함하는 암세포의 방사선 저항성 진단용 키트를 제공할 수 있다.The present invention can provide a kit for diagnosing radiation resistance of cancer cells comprising the composition.
상기 키트는 RT-PCR 키트, DNA 칩 키트, 마이크로어레이 또는 단백질 칩 키트일 수 있으나, 이에 한정되지 않는다.The kit may be, but is not limited to, RT-PCR kit, DNA chip kit, microarray, or protein chip kit.
본 발명은 생물학적 시료로부터 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자 mRNA 또는 이의 단백질 수준을 측정하는 단계; 및 상기 유전자 mRNA 또는 단백질 수준을 정상 대조군으로부터 측정한 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자 mRNA 또는 이의 단백질 수준과 비교하는 단계를 포함하는 방사선 저항성 예후 진단에 필요한 정보를 제공하는 방법을 제공할 수 있다.The present invention comprises the steps of measuring at least one gene mRNA or protein level thereof selected from STAT3 or survivin from a biological sample; And comparing the gene mRNA or protein level with at least one gene mRNA or protein level thereof selected from STAT3 or survivin measured from a normal control group. Can be provided.
상기 생물학적 시료는 조직 및 혈액으로 이루어진 군에서 선택될 수 있으며, 상기 유전자 mRNA 또는 이의 단백질 수준은 역전사 중합효소 연쇄반응(Reverse Transcription-Polymerase chain Reaction, RT-PCR), 효소면역분석법(ELISA), 면역조직화학(Immunohistochemistry), 웨스턴 블랏(Western Blotting) 및 유세포 분석법(FACS)으로 구성된 군으로부터 선택된 어느 하나로 측정될 수 있다.The biological sample may be selected from the group consisting of tissue and blood, and the gene mRNA or protein level thereof may be reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunoassay. It can be measured by any one selected from the group consisting of immunohistochemistry, Western blotting, and flow cytometry (FACS).
또한, 본 발명은 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자의 발현 또는 활성을 억제하는 제제를 유효성분으로 함유하는, 암세포에 대한 방사선 민감성 증진용 약학조성물을 제공할 수 있다.In addition, the present invention can provide a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, containing as an active ingredient an agent that inhibits the expression or activity of one or more genes selected from STAT3 or survivin.
상기 암세포는 유방암세포일 수 있으며, 보다 바람직하게는 HR-/HER2+ 서브타입 유방암세포일 수 있다.The cancer cells may be breast cancer cells, more preferably HR- / HER2 + subtype breast cancer cells.
상기 제제는 STAT3 또는 서바이빈(survivin) 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오타이드, siRNA 및 shRNA로 이루어진 군에서 선택될 수 있으나, 이에 한정되는 것을 아니다.The agent may be selected from the group consisting of antisense nucleotides, siRNAs and shRNAs that complementarily bind to mRNA of the STAT3 or survivin gene, but is not limited thereto.
또한, 본 발명은 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 단백질의 발현 또는 활성을 억제하는 제제를 유효성분으로 함유하는, 암세포에 대한 방사선 민감성 증진용 약학조성물을 제공할 수 있다.In addition, the present invention can provide a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, containing as an active ingredient an agent that inhibits the expression or activity of one or more proteins selected from STAT3 or survivin.
상기 암세포는 유방암세포일 수 있으며, 보다 바람직하게는 HR-/HER2+ 서브타입 유방암세포일 수 있다.The cancer cells may be breast cancer cells, more preferably HR- / HER2 + subtype breast cancer cells.
상기 제제는 STAT3 또는 서바이빈(survivin) 단백질에 특이적으로 결합하는 화합물, 펩티드, 앱타머, 항체 및 천연물로 이루어진 군에서 선택될 수 있다.The agent may be selected from the group consisting of compounds, peptides, aptamers, antibodies and natural products that specifically bind to STAT3 or survivin proteins.
상기 “방사선 민감성 증진”이란 방사선을 이용한 질병 치료에 있어서, 방사선에 대한 세포의 민감성을 증진시키는 것을 의미한다. 이를 통해 방사선 치료 효율을 상승시킬 수 있는데, 특히, 암치료시 병행처리되면 암세포의 방사선 민감성이 증진되어 암세포의 살상 효과 및 증식 억제효과를 나타낼 수 있다.The term "enhancement of radiation sensitivity" refers to enhancing the sensitivity of cells to radiation in treating diseases using radiation. Through this, the radiation treatment efficiency can be increased. In particular, when the cancer treatment is performed in parallel, the radiation sensitivity of the cancer cells can be enhanced, and the killing effect and the proliferation inhibitory effect of the cancer cells can be exhibited.
본 발명의 방사선 민감성 증진용 조성물은 약학적으로 허용가능한 담체를 추가로 포함할 수 있으며, 담체와 함께 제제화될 수 있다.The composition for enhancing radiation sensitivity of the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier.
본 발명에서 "약학적으로 허용가능한 담체"란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 의미한다.By "pharmaceutically acceptable carrier" is meant a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
액상 용액으로 제제화되는 조성물에 있어서 허용되는 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
본 발명의 방사선 민감성 증진용 조성물은 어떠한 제형으로도 적용가능하며, 경구용 또는 비경구용 제형으로 제조할 수 있으며, 투여의 용이성과 투여량의 균일화를 위해 단위 투여 형태로 제형화할 수 있다. 본 발명의 약학적 제형은 구강(oral), 직장(rectal), 비강(nasal), 국소(topical; 볼 및 혀 밑을 포함), 피하, 질(vaginal) 또는 비경구(parenteral; 근육내, 피하 및 정맥 내를 포함) 투여에 적당한 것 또는 흡입(inhalation) 또는 주입(insufflation)에 의한 투여에 적당한 형태를 포함한다.The composition for enhancing radiation sensitivity of the present invention can be applied in any dosage form, can be prepared in oral or parenteral dosage form, and can be formulated in unit dosage form for ease of administration and uniformity of dosage. Pharmaceutical formulations of the invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.
상기 경구 투여용 제형으로는, 예를 들어 정제, 트로키제, 로렌지, 수용성 또는 유성현탁액, 조제분말 또는 과립, 에멀젼, 하드 또는 소프트 캡슐, 시럽 또는 엘릭시르제로 제제화할 수 있다. 정제 및 캡슐 등의 제형으로 제제화하기 위해, 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴과 같은 결합제, 디칼슘 포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕괴제, 스테아르산 마그네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활유를 포함할 수 있으며, 캡슐제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 더 함유할 수 있다.The oral dosage form may be formulated, for example, in tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate It may include a lubricating oil, such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
또한, 상기 비경구 투여용 제형으로는, 피하주사, 정맥주사 또는 근육내 주사 등의 주사용 형태, 좌제 주입방식 또는 호흡기를 통하여 흡입이 가능하도록 하는 에어로졸제 등 스프레이용으로 제제화할 수 있다. 주사용 제형으로 제제화하기 위해서는 본 발명의 조성물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알의 단위 투여용으로 제제화할 수 있다. 좌제로 주입하기 위해서는, 코코아버터 또는 다른 글리세라이드 등 통상의 좌약 베이스를 포함하는 좌약 또는 체료 관장제와 같은 직장투여용 조성물로 제제화할 수 있다. 에어로졸제 등의 스프레이용으로 제형화하는 경우, 수분산된 농축물 또는 습윤 분말이 분산되도록 추진제 등이 첨가제와 함께 배합될 수 있다.In addition, the parenteral formulation may be formulated for injection such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection method, or aerosol for inhalation through the respiratory system. To formulate injectable formulations, the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials. For infusion into suppositories, it may be formulated as a rectal composition such as suppositories or body enema including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
<실험예 1> 세포배양 및 방사선 조사Experimental Example 1 Cell Culture and Irradiation
사람 유방암 세포 MCF7, MDA-MB231, SKBR3, T47D 및 BT474를 ATCC(American Type Culture Collection; Manassas, VA)에서 구입하였다.Human breast cancer cells MCF7, MDA-MB231, SKBR3, T47D and BT474 were purchased from the American Type Culture Collection (ATCC) Manassas, VA.
상기 구입한 세포를 10% 소태아혈청(fetal bovine serum; FBS; HyClone, South Logan, UT) 및 페니실린/스트렙토마이신이 포함된 DMEM 배지에서 37℃, 5% CO2 조건으로 배양하였다.The purchased cells were incubated at 37 ° C. and 5% CO 2 in DMEM medium containing 10% fetal bovine serum (FBS; HyClone, South Logan, UT) and penicillin / streptomycin.
137세슘(Cs) 광원(Atomic Energy of Canada Ltd., Mississauga, Canada)을 분당 3.81Gy 선량으로 세포에 조사하였으며, S3I-201 100 μM (EMD Millipore, Billerica, MA) 및 라파티닙 1 μM(lapatinib; Selleckchem., Houston, TX)을 세포에 처리하여 각각 STAT3 및 HER2 활성을 억제하였다. A 137 cesium (Cs) light source (Atomic Energy of Canada Ltd., Mississauga, Canada) was irradiated with cells at a dose of 3.81 Gy per minute, and S3I-201 100 μM (EMD Millipore, Billerica, Mass.) And lapatinib 1 μM (lapatinib; Selleckchem , Houston, TX) cells were treated to inhibit STAT3 and HER2 activity, respectively.
<실험예 2> 집락형성 분석법(Clonogenic assay)Experimental Example 2 Colonogenic Assay
방사선 조사 후 세포 생존율을 확인하기 위해 이전에 보고된 방법(Kim JS et al., Proteomics. 2010; 10(14):2589-2604.)으로 집락형성 분석을 수행하였다.Colony formation analysis was performed by the previously reported method (Kim JS et al., Proteomics. 2010; 10 (14): 2589-2604.) To confirm cell viability after irradiation.
먼저, 각각 다른 선량의 방사선 처리된 세포를 다양한 밀도로 60 mm 배양 디쉬에 접종하였다. 10-14일 후 콜로니를 메탄올로 고정하고 트립토판 블루로 염색하였다.First, different doses of radiation treated cells were seeded in 60 mm culture dishes at various densities. After 10-14 days colonies were fixed with methanol and stained with tryptophan blue.
균락수측정기(Image Products, Chantilly, VA)를 이용하여 50 세포 이상을 포함하는 콜로니만을 생존 콜로니로 측정하였다.Only colonies containing 50 cells or more were measured as viable colonies by using a colony counter (Image Products, Chantilly, VA).
<실험예 3> RNA 간섭Experimental Example 3 RNA Interference
siRNA를 Genolution　Pharmaceuticals Inc. (Seoul, Korea)에서 합성하여 사용하였으며, RNA 간섭에 사용된 서열은 다음과 같다: HER2, 5'-CUGGUGUAUGCAGAUUGCC-3' 및 서바이빈(survivin), 5'-AAGGAGAUCAACAUUUUCA-3'. siRNA Genolution Pharmaceuticals Inc. The sequences used for RNA interference were synthesized in (Seoul, Korea) and were as follows: HER2, 5'-CUGGUGUAUGCAGAUUGCC-3 'and survivin, 5'-AAGGAGAUCAACAUUUUCA-3'.
non-targeting siRNA(Genolution　Pharmaceuticals Inc.)를 음성 대조군으로 사용하였으며, G-Fectin (Genolution Pharmaceuticals Inc.)을 이용하여 제조사의 설명서대로 siRNA 형질주입을 수행하였다.A non-targeting siRNA (Genolution® Pharmaceuticals Inc.) was used as a negative control, and siRNA transfection was performed using G-Fectin (Genolution Pharmaceuticals Inc.) according to the manufacturer's instructions.
<실험예 4> 웨스턴 블롯 분석Experimental Example 4 Western Blot Analysis
이전에 보고된 방법(Kim JS et al., Proteomics. 2010; 10(14):2589-2604.)으로 웨스턴 블롯 분석을 수행하였다. Western blot analysis was performed by the previously reported method (Kim JS et al., Proteomics. 2010; 10 (14): 2589-2604.).
먼저, SDS-폴리아크릴아마이드 겔 전기영동을 통하여 단백질을 분리하고 니트로셀룰로스 막으로 옮긴 후 특이적인 항체를 이용하여 단백질을 검출하였다. First, the proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and the proteins were detected using specific antibodies.
사용된 항체로는 래빗 단일클론 항-서바이빈, 래빗 다중클론 항-phospho-STAT3(Tyr705) 및 항-cleaved-PARP (Asp214)를 Cell Signaling Technology (Beverly, MA)에서 구입하여 사용하였으며, 마우스 단일클론 항-STAT3, 래빗 다중클론 항-ERα 및 항-HER2는 Santa Cruz Biotechnology Inc. (Santa Cruz, CA)에서 구입하여 사용하였으며, 마우스 단일클론 항-β-액틴은 Sigma (St. Louis, MO)에서 구입하여 사용하였다.As the antibodies used, rabbit monoclonal anti-survivin, rabbit polyclonal anti-phospho-STAT3 (Tyr705) and anti-cleaved-PARP (Asp214) were purchased from Cell Signaling Technology (Beverly, MA) and used as mice. Monoclonal anti-STAT3, rabbit polyclonal anti-ERα and anti-HER2 are listed in Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.) And used, mouse monoclonal anti-β-actin was purchased from Sigma (St. Louis, MO).
HRP-결합 2차 항체와 향상된 화학발광 검출 시스템(Amersham Life Science, Piscataway, NJ)을 이용하여 블롯을 검출하였다.Blots were detected using HRP-binding secondary antibodies and an enhanced chemiluminescent detection system (Amersham Life Science, Piscataway, NJ).
<실험예 5> STAT3 활성 분석Experimental Example 5 Analysis of STAT3 Activity
이미 보고된 방법(Shin DS et al., Cancer Res. 2009; 69(1):193-202.)으로 STAT3 활성을 확인하였다.STAT3 activity was confirmed by previously reported methods (Shin DS et al., Cancer Res. 2009; 69 (1): 193-202.).
Lipofectamine 2000 (Invitrogen, Carlsbad, CA)을 이용하여 세포에 21pSTAT3-TA-Luc와 대조군 siRNA 또는 HER2 siRNA를 48시간 동안 함께 형질주입한 후 방사선 10 Gy를 처리 또는 비처리하였다.Cells were transfected with 21pSTAT3-TA-Luc and control siRNA or HER2 siRNA for 48 hours using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) And treated with or without radiation 10 Gy.
24시간 후 패시브 용해 버퍼를 이용하여 세포를 수집하고 Dual Luciferase Reporter Assay Kit (Promega, Madison, WI)와 Wallac Victor2 plate reader (Perkin Elmer Corp., Norwalk, CT)를 이용하여 루시퍼레이즈 활성을 확인하였다. After 24 hours, cells were collected using passive lysis buffer and luciferase activity was confirmed using Dual Luciferase Reporter Assay Kit (Promega, Madison, WI) and Wallac Victor2 plate reader (Perkin Elmer Corp., Norwalk, CT).
<실험예 6> 세포사멸 분석Experimental Example 6 Apoptosis Analysis
이전에 보고된 방법(Kim JS et al., Proteomics. 2010; 10(14):2589-2604.)으로 세포사멸 분석을 수행하였다.Apoptosis analysis was performed by a previously reported method (Kim JS et al., Proteomics. 2010; 10 (14): 2589-2604.).
트립신 처리된 세포를 세척한 후 10분간 프로피디움 아이오다이드(5 μM/mL)와 인큐배이션하고, FACScan flow cytometer (Becton Dickson, Franklin Lakes, NJ)을 이용하여 세포사멸을 분석하였다.After washing the trypsin-treated cells, the cells were incubated with propidium iodide (5 μM / mL) for 10 minutes and analyzed for apoptosis using a FACScan flow cytometer (Becton Dickson, Franklin Lakes, NJ).
<실험예 7> 국소부위 무병생존율 분석을 위한 환자군Experimental Example 7 Patient Group for Analysis of Disease-Free Survival
1980년 1월부터 2010년 9월 사이의 근치 수술과 보조적인 방사선 치료를 받은 일차 유방암 환자 총 1,693명을 대상으로 향후 분석을 수행하였다. A total of 1,693 primary breast cancer patients undergoing radical surgery and assisted radiotherapy between January 1980 and September 2010 were analyzed.
모든 임상 및 병리학적 데이터는 한국 암센터 병원 유방암 센터에서 제공받았다.All clinical and pathological data were obtained from the Korean Cancer Center Hospital Breast Cancer Center.
<< 실험예Experimental Example 8> 종양의 분자 서브타입(molecular subtypes)에 따른 유방암 환자 분류 8> Classification of breast cancer patients based on molecular subtypes of tumor
수술 직후 immunohistochemistry (IHC)를 수행하여 종양 조직에서 ER, PR 및 HER2 발현 정도를 평가하였다. ER 또는 PR 양성 반응은 10 고배율 시야관찰에서 핵내 최소 10% 염색이 확인된 경우로 분류하였으며, IHC 염색반응 또는 fluorescence in situ hybridization을 통한 HER2 유전자 증폭에서 3+일 경우 HER2 양성 반응으로 분류하였다.Immediately after surgery, immunohistochemistry (IHC) was performed to evaluate the expression of ER, PR and HER2 in tumor tissues. ER or PR positive reactions were classified as cases where at least 10% staining in the nucleus was confirmed by 10 high magnification field observations, and HER2 positive reactions when 3+ in HER2 gene amplification by IHC staining or fluorescence in situ hybridization.
상기 환자들은 ER, PR 및 HER2의 종양 발현 정도를 기본으로 한 하기 4종류의 서브타입에 따라 분류되었다.The patients were classified according to the following four subtypes based on the degree of tumor expression of ER, PR and HER2.
(a) 호르몬 수용체 HR+/HER2- : ER- 및/또는 PR-양성 및 HER2-음성(a) Hormone receptors HR + / HER2-: ER- and / or PR-positive and HER2-negative
(b) HR+/HER2+ : ER- 및/또는 PR-양성 및 HER2-양성(b) HR + / HER2 +: ER- and / or PR-positive and HER2-positive
(c) HR-/HER2+ : ER-음성, PR-음성 및 HER2-양성(c) HR- / HER2 +: ER-negative, PR-negative and HER2-positive
(d) HR-/HER2- : ER-음성, PR-음성 및 HER2-음성(d) HR- / HER2-: ER-negative, PR-negative and HER2-negative
<실험예 9> 면역조직화학(Immunohistochemistry)Experimental Example 9 Immunohistochemistry
근치 수술을 통하여 제거된 HER2 과발현된 1차 유방암 조직 파라핀 블록으로부터 IHC를 위한 표본을 얻었다.Specimens for IHC were obtained from HER2 overexpressed primary breast cancer tissue paraffin blocks removed through radical surgery.
방사선 치료 후 1년 이내 국소 재발이 나타난 환자들을 민감 반응군으로 분류하였으며, 방사선 치료 후 최소 2년간의 추적 치료기간 동안 질환의 증후가 나타나지 않은 환자들을 비민감 반응군으로 분류하고, 각 그룹의 환자들의 병리학적 TNM 염색 및 HR 상태를 매치시켰다.Patients who showed local recurrence within 1 year after radiation therapy were classified as sensitive response group. Patients who did not show symptoms of disease during follow-up period of at least 2 years after radiation therapy were classified as non-sensitive response group. Their pathological TNM staining and HR status were matched.
이전에 보고된 방법(Kim JS et al., Cancer Res. 2013; 73(22):6667-6678.; Min JW et al., Biochem Biophys Res Commun. 2013; 440(1):137-142.)에 따라, IHC 실험을 수행하였다.Previously reported method (Kim JS et al., Cancer Res. 2013; 73 (22): 6667-6678 .; Min JW et al., Biochem Biophys Res Commun. 2013; 440 (1): 137-142.) According to the IHC experiment was performed.
간략하게, 항-STAT3 마우스 단일클론 항체(1:200 dilution; Santa Cruz), 항-phospho-STAT3 래빗 다중클론 항체(1:50 dilution; GeneTex, Irvine, CA), 또는 항-서바이빈 래시 단일클론 항체(1:100 dilution; Cell Signaling Technology)를 이용하여 면역조직화학 염색을 수행하였다.Briefly, anti-STAT3 mouse monoclonal antibody (1: 200 dilution; Santa Cruz), anti-phospho-STAT3 rabbit polyclonal antibody (1:50 dilution; GeneTex, Irvine, CA), or anti-survivin lash single Immunohistochemical staining was performed using clone antibody (1: 100 dilution; Cell Signaling Technology).
면역염색은 아비딘-바이오틴-퍼옥사이드를 이용하여 제조사(Invitrogen)의 설명서대로 수행하였다.Immunostaining was performed using the avidin-biotin-peroxide as instructed by the manufacturer (Invitrogen).
염색 강도는 다음과 같은 기준으로 평가하였다: 0 (염색이 관찰되지 않음), 1+ (희미한 염색정도), 2+ (보통강도의 염색정도), and 3+ (강한 염색이 확인됨).Dye intensity was evaluated based on the following criteria: 0 (no staining observed), 1+ (faint staining), 2+ (normal staining intensity), and 3+ (strong staining confirmed).
<실시예 1> 방사선 저항성과 HER2-양성 유방암 관련성 확인<Example 1> Confirmation of the relationship between radiation resistance and HER2-positive breast cancer
근치 수술 후 적절한 보조적 방사선 치료를 받은 1693명의 1차 유방암 환자들을 대상으로 방사선 저항성과 HER2-양성 유방암 관련성을 확인하였다.We investigated the association between radiation resistance and HER2-positive breast cancer in 1693 primary breast cancer patients who received appropriate adjuvant radiotherapy after curative surgery.
상기 환자들은 그들의 종양조직에서 HER2 및 HR (Estrogen receptor [ER] and/or progesterone receptor [PR]) 분자 발현을 기반으로 한 네 범주(HR+/HER2-, HR+/HER2+, HR-/HER2+, 및 HR-/HER2-)로 분류하였으며, 임상 병리학적 특징은 하기 표 1과 같이 나타났다.These patients have four categories (HR + / HER2-, HR + / HER2 +, HR- / HER2 +, and HR) based on HER2 and HR (Estrogen receptor [ER] and / or progesterone receptor [PR]) molecule expression in their tumor tissues. -/ HER2-), and the clinical pathological characteristics are shown in Table 1 below.
그 결과, 표 1과 같이 HR+/HER2- (54.9%, 929 of 1,693 patients) 환자가 가장 많은 것으로 확인되었으며, 그 다음으로 HR-/HER2- (20.5%, 1,693 환자 중 347 명), HR+/HER2+ (13.6%, 1,693 환자 중 231 명) 및 HR-/HER2+ (11.0%, 1,693 환자 중 183 명)순인 것으로 확인되었다.As a result, the number of patients with HR + / HER2- (54.9%, 929 of 1,693 patients) was the highest as shown in Table 1, followed by HR- / HER2- (20.5%, 347 of 1,693 patients), HR + / HER2 +. (13.6%, 231 of 1,693 patients) and HR- / HER2 + (11.0%, 183 of 1,693 patients).
또한, 도 1A를 참고하면, 유방암 환자의 분자 서브타입을 기반으로 그룹을 분리한 결과, 서브타입에 따라 국소부위 무재발 생존율의 유의한 차이를 나타내는 것으로 확인되었는데, HR+/HER2- 유방암 환자들은 매우 높은 국소 부위 무재발 생존율을 나타낸 반면, HR-/HER2+ 환자들은 재발할 경우 매우 낮은 생존율을 나타내는 것을 확인할 수 있었다.In addition, referring to FIG. 1A, as a result of separating the groups based on molecular subtypes of breast cancer patients, it was confirmed that the subtypes showed a significant difference in local recurrence-free survival rates. HR + / HER2- breast cancer patients were very different. While there was a high local recurrence-free survival rate, HR- / HER2 + patients showed very low survival rates when relapsed.
상기 결과로부터 방사선 치료 민감성은 유방암의 분자 서브타입 차이와 관련있을 것으로 제안될 수 있다.From these results it may be suggested that radiation therapy sensitivity is related to molecular subtype differences in breast cancer.
이러한 HR-/HER2+ 서브타입과 높은 방사선 치료 저항성의 관련성을 확인하기 위해, 유방암의 다른 서브타입들과 방사선 치료 효과를 비교하였다.To determine the association of these HR- / HER2 + subtypes with high radiotherapy resistance, we compared the radiotherapy effects with other subtypes of breast cancer.
클론성 생존 분석을 위해, MCF7 및 T47D (HR+/HER2-), MDA-MB231 (HR-/HER2-), BT474 (HR+/HER2+), 및 SKBR3 (HR-/HER2+)을 포함한 유방암 세포주에 다양한 선량의 방사선을 조사하여 반응성을 확인하였다.For clonal survival analysis, various doses to breast cancer cell lines, including MCF7 and T47D (HR + / HER2-), MDA-MB231 (HR- / HER2-), BT474 (HR + / HER2 +), and SKBR3 (HR- / HER2 +) The radiation was checked for reactivity.
그 결과, 도 1B 및 1C와 같이 다른 유방암 세포와 비교하여 HER2-양성(HR-/HER2+) 유방암 세포주인 SKBR3에서 매우 높은 방사선 저항성 표현형이 나타나는 것으로 확인되었다.As a result, it was confirmed that a very high radiation resistance phenotype appears in SKBR3, a HER2-positive (HR- / HER2 +) breast cancer cell line, compared to other breast cancer cells as shown in FIGS. 1B and 1C.
상기 결과들로부터 HER2-양성 유방암에서 방사선 저항성이 나타나는 것으로 확인되었다.From these results, it was confirmed that radiation resistance appeared in HER2-positive breast cancer.
Figure PCTKR2016004706-appb-T000001
Figure PCTKR2016004706-appb-T000001
<실시예 2> HER2-양성 유방암 세포의 방사선 저항성 신호과정 확인Example 2 Confirmation of Radiation Resistance Signaling Process of HER2-positive Breast Cancer Cells
앞선 실험을 통하여 HER2 발현이 유방암의 방사선 저항성과 관련있음이 확인됨에 따라, HER2 양성 유방암세포에서 HER2의 siRNA에 의해 유도되는 유전자 억제를 통하여 HER2가 방사선 저항성의 주요 매개자인지를 확인하였다.As previous studies confirmed that HER2 expression was associated with radiation resistance in breast cancer, it was confirmed that HER2 is a major mediator of radiation resistance through gene inhibition induced by HER2 siRNA in HER2-positive breast cancer cells.
그 결과, 도 2A와 같이 siRNA에 의해 HER2 발현이 억제된 SKBR3 세포군은 다양한 선량의 방사선에 민감하게 반응하여 낮은 생존율을 나타내었으며, 도 2B 및 2C와 같이 HER2-양성 SKBR3 및 MDA-MB453 유방암세포에서도 HER2가 결핍될 경우, 방사선에 의한 세포 죽음이 증가되는 것을 확인할 수 있었다.As a result, as shown in FIG. 2A, the SKBR3 cell group whose HER2 expression was suppressed by siRNA was sensitive to radiation of various doses and showed low survival rate, and also in HER2-positive SKBR3 and MDA-MB453 breast cancer cells as shown in FIGS. 2B and 2C. When HER2 is deficient, it was confirmed that cell death by radiation is increased.
상기 결과로부터 HER2는 HER2-양성 유방암 세포에서 방사선 저항성을 조절하는 주요 인자인 것으로 확인되었다.From these results, it was confirmed that HER2 is a major factor regulating radiation resistance in HER2-positive breast cancer cells.
다음으로, 다양한 발암경로를 통하여, HER2는 방사선에 의해 유도되는 STAT3의 활성을 촉진시키고, 이러한 신호과정은 복합 방사선 저항성 암의 주요 신호과정 중 하나일 것으로 제안됨에 따라, 이를 확인하였다.Next, through various carcinogenic pathways, HER2 promotes the activity of STAT3 induced by radiation, and this signaling process was identified as it is suggested to be one of the major signaling processes of complex radiation resistant cancer.
그 결과, 도 3A와 같이 HER2가 결핍된 HER2-양성 SKBR3 및 MDA-MB453 유방암 세포에서 인산화된 STAT3와 서바이빈의 감소가 확인되었으며, 이를 통하여 방사선에 의해 유도되는 STAT3 활성이 억제된 것을 확인할 수 있었다.As a result, as shown in Fig. 3A, the reduction of phosphorylated STAT3 and survivin in HER2-deficient HER2-positive SKBR3 and MDA-MB453 breast cancer cells was confirmed, and it was confirmed that STAT3 activity induced by radiation was suppressed. there was.
추가적으로 STAT3 결합 요소를 가진 STAT3 리포터 플라스미드를 이용하여 루시퍼레이즈 분석을 수행하여 STAT3의 직접적인 전사활동을 확인한 결과, 방사선이 조사된 세포에서 HER2를 결핍시킨 경우, 방사선 유도성 STAT3 활성이 억제되는 것을 확인할 수 있었다.In addition, the luciferase assay was performed using a STAT3 reporter plasmid with a STAT3 binding element to confirm direct transcriptional activity of STAT3. When HER2 was deficient in irradiated cells, radiation-induced STAT3 activity was inhibited. there was.
상기 결과로부터 HER2 양성 유방암 세포에서 HER2가 STAT3 신호 활성화를 통하여 방사선 저항성을 향상시키는 것으로 확인되었다.From these results, it was confirmed that HER2 enhances radiation resistance through STAT3 signal activation in HER2-positive breast cancer cells.
<< 실시예Example 3> 3> HER2HER2 -양성 유방암 세포에서 In benign breast cancer cells HER2HER2 -- STAT3STAT3 -- SurvivinSurvivin 신호과정 억제를 통한 방사선 민감성 증가 확인 Confirmation of radiation sensitivity increase through signal process inhibition
HER2 및 STAT3의 억제가 HER2-양성 SKBR3 유방암세포의 방사선 민감성을 증가시킬 수 있는 지를 확인하였다.It was confirmed that inhibition of HER2 and STAT3 can increase the radiation sensitivity of HER2-positive SKBR3 breast cancer cells.
HER2 및 STAT3의 억제자인 라파티닙(lapatinib) 및 S3I-201를 HER2-양성 SKBR3 유방암세포에 각각 처리하고, 10 Gy 선량의 방사선을 조사하였다.Lapatinib and S3I-201, inhibitors of HER2 and STAT3, were treated with HER2-positive SKBR3 breast cancer cells, respectively, and irradiated with 10 Gy dose.
그 결과, 도 4A와 같이 라파티닙(lapatinib) 및 S3I-201이 처리된 HER2-양성 SKBR3 유방암세포에서 방사선 조사에 의해 유도된 세포사멸이 매우 효과적으로 증가된 것이 확인되었을 뿐만 아니라, 도 4B와 같이 방사선에 의해 유도되는 STAT3 인산화 및 서바이빈 발현이 역시 효과적으로 감소되는 것을 확인할 수 있었다.As a result, it was confirmed that the apoptosis induced by irradiation in HER2-positive SKBR3 breast cancer cells treated with lapatinib and S3I-201 as shown in FIG. It was confirmed that the STAT3 phosphorylation and survivin expression induced by the efflux were also effectively reduced.
또한, 3Gy 방사선 조사 후 콜로니 형성율을 확인하여 생존율 분석을 수행한 결과, 도 4C와 같이 라파티닙(lapatinib) 또는 S3I-201과 방사선이 함께 처리된 HER2-양성 SKBR3 유방암세포군의 생존율이 매우 유의하게 감소된 것을 확인할 수 있었다.In addition, the survival rate analysis was performed by confirming colony formation rate after 3Gy irradiation, and as shown in FIG. 4C, the survival rate of HER2-positive SKBR3 breast cancer cell group treated with lapatinib or S3I-201 and radiation was significantly decreased. It could be confirmed.
다음으로, HER2-양성 SKBR3 유방암세포에서 siRNA를 통하여 서바이빈이 억제된 경우, 방사선 민감성이 향상되는지 확인하였다.Next, when survivin is inhibited through siRNA in HER2-positive SKBR3 breast cancer cells, it was confirmed whether radiation sensitivity was improved.
그 결과, 도 4D 및 4E를 참고하면 앞서 확인된 HER2 및 STAT3 억제 효과와 유사하게, 서바이빈이 결핍된 세포에서는 HER2 및 STAT3 인산화와 관계없이 방사선에 의해 유도되는 세포 사멸이 증가가 확인되었다.As a result, with reference to FIGS. 4D and 4E, similar to the HER2 and STAT3 inhibitory effects previously confirmed, the increase in cell death induced by radiation was confirmed in survivin-deficient cells regardless of HER2 and STAT3 phosphorylation.
상기 결과로부터 HER2-STAT3-서바이빈 신호과정은 HER2-양성 SKBR3 유방암세포에서 나타나는 방사선 저항성의 주요 인자인 것으로 확인됨에 따라, HER2-STAT3-서바이빈 신호는 HER2-양성 유방암 치료를 위한 방사선 보조치료의 주요 타겟으로 제안될 수 있다.From the above results, the HER2-STAT3-survivin signaling process was found to be a major factor in radiation resistance in HER2-positive SKBR3 breast cancer cells, so HER2-STAT3-survivin signal is a radiation support for the treatment of HER2-positive breast cancer. It can be suggested as the main target of treatment.
<< 실시예Example 4>  4> HER2HER2 -양성 유방암 조직에서 In benign breast cancer tissues HER2HER2 -- STAT3STAT3 -- 서바이빈Survival 신호와 방사선 치료 저항성 사이의 정적 상관관계(positive correlation) 확인  Identify positive correlation between signal and radiation therapy resistance
HER2-양성 유방암에서 HER2-STAT3-서바이빈 조절과 방사선 치료 저항성 사이의 생리학적 관련성을 확인하기 위해, 방사선 치료 후 재발된 HER2-양성 유방암 환자(비민감성 군; n = 7) 또는 재발되지 않은 HER2-양성 유방암 환자군(민감성 군; n = 8)에서 인산화된 STAT3 (Tyr705), STAT3 및 서바이빈의 발현 수준을 확인하였다.To confirm the physiological relationship between HER2-STAT3-survivin control and radiation therapy resistance in HER2-positive breast cancer, patients with HER2-positive breast cancer (insensitive group; n = 7) or not relapsed after radiation treatment The expression levels of phosphorylated STAT3 (Tyr705), STAT3 and survivin in HER2-positive breast cancer patients group (sensitive group; n = 8) were identified.
그 결과, 도 5A 및 도 5B와 같이 재발된 HER2-양성 유방암 환자군의 연속 절편에서는 인산화된 STAT3, STAT3 및 서바이빈의 염색 패턴이 유사하게 나타났으나, 재발되지 않은 환자군에서는 상이하게 나타났다.As a result, staining patterns of phosphorylated STAT3, STAT3 and survivin were similar in the continuous sections of the recurrent HER2-positive breast cancer patient group as shown in FIGS. 5A and 5B, but different in the non-relapsed patient group.
추가적으로 STAT3 활성을 나타내는 인산화된 STAT3 및 서바이빈의 강한 핵산 염색 패턴을 확인하였다.Additionally, strong nucleic acid staining patterns of phosphorylated STAT3 and survivin exhibiting STAT3 activity were identified.
그 결과, 도 5A 및 도 5B의 오른쪽 상측 이미지를 참고하면 재발되지 않은 HER2-양성 유방암 환자군보다 재발된 HER2-양성 유방암 환자군에서 인산화된 STAT3 및 서바이빈의 강한 핵산 염색 패턴이 확인되었다. 또한, 도 5C 내지 도 5E를 통하여 인산화된 STAT3, STAT3 및 서바이빈의 발현 증가는 방사선치료에 민감성을 나타내지 않은 군과 정적 상관관계를 나타내는 것을 확인할 수 있었다. As a result, referring to the upper right image of FIGS. 5A and 5B, a strong nucleic acid staining pattern of phosphorylated STAT3 and survivin was observed in the relapsed HER2-positive breast cancer patient group than in the relapsed HER2-positive breast cancer patient group. In addition, it was confirmed that the increased expression of phosphorylated STAT3, STAT3 and survivin through FIGS. 5C to 5E showed a positive correlation with the group not showing sensitivity to radiation therapy.
상기 결과들로부터 HER2-양성 유방암에서 나타나는 방사선치료 저항성은 HER2-STAT3-서바이빈 신호과정에 의한 것임이 확인되었다.From these results, it was confirmed that radiotherapy resistance in HER2-positive breast cancer was due to HER2-STAT3-survivin signaling.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (18)

  1. STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자의 mRNA, 또는 이의 단백질 수준을 측정하는 제제를 포함하는, 암세포의 방사선 저항성 진단용 조성물.At least one gene selected from STAT3 or survivin (survivin), comprising a preparation for measuring the mRNA, or protein level thereof, the composition for diagnosing radiation resistance of cancer cells.
  2. 청구항 1에 있어서, 상기 암세포는 유방암세포인 것을 특징으로 하는, 암세포의 방사선 저항성 진단용 조성물.The composition for diagnosing radiation resistance of cancer cells according to claim 1, wherein the cancer cells are breast cancer cells.
  3. 청구항 1에 있어서, 상기 유방암세포는 HR-/HER2+ 서브타입의 유방암세포인 것윽 특징으로 하는, 암세포의 방사선 저항성 진단용 조성물.The composition for diagnosing radiation resistance of cancer cells according to claim 1, wherein the breast cancer cells are breast cancer cells of the HR- / HER2 + subtype.
  4. 청구항 1에 있어서, 상기 유전자 mRNA의 수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 프라이머 또는 프로브를 포함하는 것을 특징으로 하는, 암세포의 방사선 저항성 진단용 조성물.The composition for diagnosing radiation resistance of cancer cells according to claim 1, wherein the agent for measuring the level of the gene mRNA comprises a primer or a probe specifically binding to the gene.
  5. 청구항 1에 있어서, 상기 단백질 수준을 측정하는 제제는 상기 단백질에 특이적인 항체 또는 앱타머를 포함하는 것을 특징으로 하는, 암세포의 방사선 저항성 진단용 조성물.The composition for diagnosing radiation resistance of cancer cells according to claim 1, wherein the agent for measuring the protein level comprises an antibody or aptamer specific for the protein.
  6. 청구항 1의 조성물을 포함하는 암세포의 방사선 저항성 진단용 키트.Radiation resistance diagnostic kit for cancer cells comprising the composition of claim 1.
  7. 청구항 6에 있어서, 상기 키트는 RT-PCR 키트, DNA 칩 키트, 마이크로어레이 또는 단백질 칩 키트인 것을 특징으로 하는 암세포의 방사선 저항성 진단용 키트.The kit for diagnosing radiation resistance of cancer cells according to claim 6, wherein the kit is an RT-PCR kit, a DNA chip kit, a microarray, or a protein chip kit.
  8. 생물학적 시료에서 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자 mRNA 또는 이의 단백질 수준을 측정하는 단계;Measuring at least one gene mRNA or protein level thereof selected from STAT3 or survivin in the biological sample;
    상기 유전자 mRNA 또는 이의 단백질 수준을 정상 대조군으로부터 측정한 STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자 mRNA 또는 이의 단백질 수준과 비교하는 단계를 포함하는 방사선 저항성 예후 진단에 필요한 정보를 제공하는 방법.Comparing said gene mRNA or protein level thereof with one or more gene mRNA or protein levels selected from STAT3 or survivin measured from a normal control group to provide information necessary for diagnosing a radiation resistance prognosis .
  9. 청구항 8에 있어서, 상기 생물학적 시료는 조직 및 혈액으로 이루어진 군에서 선택되는 것을 특징으로 하는 방사선 저항성 예후 진단에 필요한 정보를 제공하는 방법.The method of claim 8, wherein the biological sample is selected from the group consisting of tissue and blood.
  10. 청구항 8에 있어서, 상기 유전자 mRNA 또는 이의 단백질 수준은 역전사 중합효소 연쇄반응(Reverse Transcription-Polymerase chain Reaction, RT-PCR), 효소면역분석법(ELISA), 면역조직화학(Immunohistochemistry), 웨스턴 블랏(Western Blotting) 및 유세포 분석법(FACS)으로 구성된 군으로부터 선택된 어느 하나로 측정하는 것을 특징으로 하는 방사선 저항성 예후 진단에 필요한 정보를 제공하는 방법.The method according to claim 8, wherein the gene mRNA or its protein level is Reverse Transcription-Polymerase chain Reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemistry, Western Blotting ) And flow cytometry (FACS). The method for providing information necessary for diagnosing a radiation-resistant prognosis, characterized in that it is measured by any one selected from the group consisting of.
  11. STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 유전자의 발현 또는 활성을 억제하는 제제를 유효성분으로 함유하는, 암세포에 대한 방사선 민감성 증진용 약학조성물.A pharmaceutical composition for enhancing radiation sensitivity to cancer cells, comprising as an active ingredient an agent that inhibits the expression or activity of one or more genes selected from STAT3 or survivin.
  12. 청구항 11에 있어서, 상기 암세포는 유방암세포인 것을 특징으로 하는, 암세포에 대한 방사선 민감성 증진용 약학조성물.The pharmaceutical composition for enhancing radiation sensitivity to cancer cells according to claim 11, wherein the cancer cells are breast cancer cells.
  13. 청구항 12에 있어서, 상기 유방암세포는 HR-/HER2+ 서브타입인 것을 특징으로 하는, 암세포에 대한 방사선 민감성 증진용 약학조성물.The pharmaceutical composition for enhancing radiation sensitivity to cancer cells according to claim 12, wherein the breast cancer cells are HR- / HER2 + subtypes.
  14. 청구항 11에 있어서, 상기 제제는 STAT3 또는 서바이빈(survivin) 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오타이드, siRNA 및 shRNA로 이루어진 군에서 선택되는 것을 특징으로 하는, 암세포에 대한 방사선 민감성 증진용 약학조성물.The pharmaceutical composition for enhancing radiation sensitivity of cancer cells according to claim 11, wherein the agent is selected from the group consisting of antisense nucleotides, siRNAs and shRNAs that complementarily bind to mRNA of STAT3 or survivin genes. Composition.
  15. STAT3 또는 서바이빈(survivin)에서 선택되는 하나 이상의 발현 단백질의 발현 또는 활성을 억제하는 제제를 유효성분으로 함유하는, 암세포에 대한 방사선 민감성 증진용 약학조성물.A pharmaceutical composition for enhancing radiation sensitivity to cancer cells, comprising as an active ingredient an agent that inhibits the expression or activity of one or more expression proteins selected from STAT3 or survivin.
  16. 청구항 15에 있어서, 상기 암세포는 유방암세포인 것을 특징으로 하는, 암세포에 대한 방사선 민감성 증진용 약학조성물.The pharmaceutical composition for improving radiation sensitivity to cancer cells according to claim 15, wherein the cancer cells are breast cancer cells.
  17. 청구항 16에 있어서, 상기 유방암세포는 HR-/HER2+ 서브타입인 것을 특징으로 하는, 암세포에 대한 방사선 민감성 증진용 약학조성물.The pharmaceutical composition for improving radiation sensitivity to cancer cells according to claim 16, wherein the breast cancer cells are HR- / HER2 + subtypes.
  18. 청구항 15에 있어서, 상기 제제는 STAT3 또는 서바이빈(survivin) 단백질에 특이적으로 결합하는 화합물, 펩티드, 앱타머, 항체 및 천연물로 이루어진 군에서 선택되는 것을 특징으로 하는, 암세포에 대한 방사선 민감성 증진용 약학조성물.The method of claim 15, wherein the agent is selected from the group consisting of compounds, peptides, aptamers, antibodies and natural products that specifically bind to STAT3 or survivin proteins, enhancing radiation sensitivity to cancer cells. Pharmaceutical composition.
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