KR20170071393A - Composition for diagnosis of radioresistance and use thereof - Google Patents

Abstract

The present invention relates to a biomarker composition for the diagnosis of radiation resistance of breast cancer cells, which comprises gene mRNA consisting of STAT3 and survivin or a protein group thereof as an active ingredient, and its use, and more particularly to a biomarker composition for HR- / HER2 + Have been shown to be due to increased activity or expression of STAT3 and survivin, the STAT3 and survivin genes or their proteins can be provided as biomarkers for the prediction of radiation resistance in HR- / HER2 breast cancer patients , And the effect of radiation therapy on breast cancer patients can be improved by controlling their expression.

Description

Composition for Diagnosis of Radiation Resistance and Use Thereof

The present invention relates to a biomarker for measuring radiation resistance, which is excluded from breast cancer cells showing radiation resistance, and its medical use.

Breast cancer is the most common cancer in women worldwide and its incidence is steadily increasing. Therapeutic methods for such breast cancer include surgical procedures, radiation therapy, estrogen receptors, hormone therapy targeting HER2, or target treatment methods .

Most breast cancers are mainly hormone - positive breast cancers and are treated with surgical procedures or drug therapies such as tamoxifen, but the chemotherapy method of breast cancer is easily tolerated and easily transformed into a metastatic form.

Radiation therapy is a recommended treatment method for effective local site management of patients who have received high-risk mastectomy as well as breast conservation surgery, but some patients show decreased survival due to radiation therapy resistance and local site management failure.

HER2 is a gene that is overexpressed in approximately 20-30% of breast cancer patients and plays an important role in the progression and metastasis of breast cancer. HER2 overexpression has been reported to be associated with poor prognosis and decreased survival rate of patients, HER2 has been shown to be an effective strategy for reducing aggression and confirming the increased sensitivity of breast cancer cells to radiation when HER2 is inhibited. HER2 is a biomarker for predicting target molecules for radiation therapy in breast cancer patients and radiation therapy (HER2), it is difficult to predict the survival rate after radiation therapy (Kyndi M, Sorensen FB, Knudsen H, Overgaard M, Nielsen HM and Overgaard J. Estrogen receptor , progesterone receptor, HER-2, and response to postmastectomy radiotherapy in high-risk breast cancer: the Danish Breas t Cancer Cooperative Group. J Clin Oncol. 2008; 26 (9): 1419-1426.).

In order to solve the problems of radiation therapy, it is necessary to study molecular signals to increase sensitivity to radiation resistant cells as well as to predict the results of radiation therapy.

Published Patent Application No. 2016-0029053 (published March 14, 2016)

The present invention provides a biomarker for predicting the result of radiotherapy of one or more genes selected from STAT3 or survivin or its protein for effective radiotherapy in a breast cancer patient, And the like.

The present invention provides a composition for diagnosing radiation resistance of cancer cells, comprising an mRNA of one or more genes selected from STAT3 or survivin, or an agent for measuring the protein level thereof.

The present invention provides a radiation resistance diagnostic kit for cancer cells comprising the composition.

The present invention provides a method for detecting a protein comprising the steps of: measuring the level of one or more gene mRNA or protein thereof selected from STAT3 or survivin from a biological sample; And comparing the level of the gene mRNA or protein to the level of one or more gene mRNA or protein thereof selected from STAT3 or survivin measured from a normal control, to provide information necessary for diagnosis of a radiation resistant prognosis .

The present invention provides a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, which comprises, as an active ingredient, an agent for inhibiting the expression or activity of at least one gene selected from STAT3 or survivin.

The present invention also provides a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, which comprises, as an active ingredient, an agent for inhibiting the expression or activity of at least one protein selected from STAT3 or survivin.

According to the present invention, it has been confirmed that the radiation resistance observed in HR- / HER2 breast cancer patients is due to an increase in the activity or expression of STAT3 or survivin, the STAT3 or survivin gene or proteins thereof can be detected in HR- / HER2 breast cancer And can be provided as a biomarker for predicting patient radiation resistance, and the effect of radiation therapy of breast cancer patients can be improved through their expression control.

FIG. 1 shows the correlation between HR- / HER2 + subtypes and radiation resistance in breast cancer patients and breast cancer cell lines. FIG. 1A shows the survival rate of patients receiving breast conserving treatment with adjuvant radiation therapy, Kaplan (Log-rank test, P < 0.001). Fig. 1B shows the results of direct irradiation of various doses of radiation to MCF7, MDA-MB231, SKBR3, T47D and BT- FIG. 1C shows immunoblot analysis using anti-HER2 and anti-ER antibodies in MCF7, MDA-MB231, SKBR3, T47D and BT-474 cells. As a result, β-actin was used as a loading control. Data are presented as the mean ± standard deviation of the results of triplicate experiments.
Figure 2 shows the results of confirming the radiation resistance of HR- / HER2 + breast cancer cells deficient in HER2. Figure 2A shows that SKBR3 cells were transfected with 100 nM control siRNA or 100 nM HER2 siRNA for 48 hours and then treated with various doses of radiation FIG. 2B shows the results of confirming the cell survival rate. FIG. 2B shows that SKBR3 cells or MDA-MB453 cells were irradiated with 10 Gy radiation after transfection with 100 nM control siRNA or HER2 siRNA for 48 hours, FIG. 2C is a graph showing cell viability of untreated cell group (Ctrl) by FACScan flow cytometer. FIG. 2C is a graph showing cell proliferation of anti-SKBR3 cells or MDA-MB453 cells irradiated with 100 nM control siRNA or HER2 siRNA, As a result of immunoblot analysis using -cleaved-PARP and anti-HER2 antibody, β-actin was used as a loading control, and each experiment data was repeated three times, Were tanae, the value * P <0.05 or ** P <0.01 compared with irradiated siRNA control cells (A and B).
FIG. 3A and FIG. 3B show that SKRNA3 cells or MDA-MB453 cells were treated with 100 nM of control siRNA or HER2 siRNA for 48 hours in HR- / HER2 + breast cancer cells deficient in HER2 Actin was treated with 10 Gy of radiation (IR) or not treated (Ctrl) after 48 hours, and the result of immunoblotting using β-actin, which is a loading control, and each antibody, As a result of confirming STAT3 activity in each cell, the experimental data are shown as the mean ± standard deviation of repeated experiments performed three times, and ** P <0.01 compared with irradiated siRNA control cells (C).
FIG. 4 shows the results of confirming the radiation sensitivity of HR- / HER2 + breast cancer cells according to HER2, STAT3 and survivin inhibition. FIG. 4A shows that SKBR3 cells were treated with 10 Gy radiation in the presence of 1 μM lapatinib or 100 μM S3I- FIG. 4B shows the cell viability of SKBR3 cells treated with the above process by flow cytometry, and the percentage of PI-positive cells FIG. 4C shows the results of confirming the survival rate of SKBR3 cells treated or not treated with 3 Gy radiation in the presence of 1 μM lapatinib, 100 μM S3I-201 or 1 μM lapatinib + 100 μM S3I-201, Immunoblotting results were obtained by treating 10 Gy radiation with 100 nM control siRNA or survivin siRNA transfected SKBR3 cells for 48 hours and confirming the amount of protein expression using each antibody. 4E was treated with 10 Gy radiation to 100 nM control siRNA or survivin siRNA-transfected SKBR3 cells for 48 hours, and colony formation was checked by colony formation assay, and β-actin loading control was used A and D), and the experimental data are shown as the mean ± standard deviation of the experiment performed three times repeatedly. ** P <0.01 compared with irradiated siRNA control cells (B, C and E).
FIG. 5 shows a statistical correlation between HER2-positive breast cancer recurred after radiation therapy and phosphorylated STAT3, STAT3 and survivin expression, and FIG. 5A shows the results of the anti-phosphorylated STAT3 FIG. 5B is a micrograph showing the anti-STAT3 (Y705), anti-STAT3 and anti-survivin staining in HER2-positive breast cancer tissues without recurrence, (Scale bar, 50 μm). Fig. 3C shows the staining intensity of phosphorylated STAT3 in recurrent (n = 7) and non-recurrent (n = 7) FIG. 3E shows the results of quantifying the STAT3 staining intensity of the recurrent group (n = 7) and non-receding group (n = 7) (n = 7), the results are representative of box-and-whisker plots , And the staining intensity was evaluated as follows: 0, not stained; +1, weak staining; +2, normal; And +3, strong staining; * P <0.05 compared with responder group.

The present invention can provide a composition for diagnosing radiation resistance of cancer cells, which comprises an mRNA of one or more genes selected from STAT3 or survivin, or an agent for measuring the protein level 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 include a primer or a probe that specifically binds to the gene.

The agent for measuring the protein level may comprise an antibody or an aptamer specific for the protein.

According to one embodiment of the present invention, it was found that the recurrence-free survival rate was localized according to the molecular subtypes of breast cancer patients. Referring to FIG. 1A, HR + / HER2- Recurrence survival rate, whereas HR- / HER2 + patients showed a very low survival rate when relapsed. In order to confirm the above results, a breast cancer cell line containing MCF7 and T47D (HR + / HER2-), MDA-MB231 (HR- / HER2-), BT474 (HR + / HER2 +) and SKBR3 (HR- / HER2 +) breast cancer cell line SKBR3 as compared to other breast cancer cells as shown in FIGS. 1B and 1C.

From these results, it was found that radiation therapy sensitivity is related to the difference in molecular subtype of breast cancer, and especially the prognosis of radiation therapy for HR- / HER2 breast cancer patients is very bad.

As a result of confirming the signaling process of radiation resistance in the HR- / HER2 breast cancer patients confirmed in the above results, the decrease of the phosphorylated STAT3 and survivin in HER2-deficient HER2-positive SKBR3 and MDA-MB453 breast cancer cells, In order to confirm whether STAT3 inhibition could increase the radiation sensitivity of HER2-positive SKBR3 breast cancer cells, the inhibitors of HER2 and STAT3, lapatinib and S3I-201, were administered to HER2-positive SKBR3 breast cancer cells And irradiated with 10 Gy dose of radiation.

As a result, it was confirmed that radiation-induced cell death was significantly increased in HER2-positive SKBR3 breast cancer cells treated with lapatinib and S3I-201 as shown in FIG. 4A, Induced STAT3 phosphorylation and survivin expression was significantly reduced.

In addition, after the 3Gy irradiation, the colony formation rate was confirmed and the survival rate was analyzed. As a result, the survival rate of HER2-positive SKBR3 breast cancer cell line treated with lapatinib or S3I-201 and radiation was significantly decreased .

From the above results, it was confirmed that the radiation resistance in HR- / HER2 breast cancer patients was due to the activity or expression of STAT3 and survivin.

Therefore, the STAT3 and survivin gene or proteins thereof can be used as a biomarker for predicting radiation sensitivity of HR- / HER2 breast cancer patients, and their effect on the radiation therapy of breast cancer patients can be improved.

The present invention can provide a radiation resistance diagnostic kit for cancer cells comprising the composition.

The kit may be an RT-PCR kit, a DNA chip kit, a microarray, or a protein chip kit, but is not limited thereto.

The present invention provides a method for detecting a protein comprising the steps of: measuring the level of one or more gene mRNA or protein thereof selected from STAT3 or survivin from a biological sample; And comparing the level of the gene mRNA or protein to the level of one or more gene mRNA or protein thereof selected from STAT3 or survivin measured from a normal control, to provide information necessary for diagnosis of a radiation resistant prognosis Can be provided.

The biological sample may be selected from the group consisting of tissue and blood, and the gene mRNA or its protein level may be measured by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA) For example, one selected from the group consisting of immunohistochemistry, Western blotting and flow cytometry (FACS).

The present invention also provides a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, which comprises, as an active ingredient, an agent that inhibits the expression or activity of at least one gene 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, siRNA, and shRNA complementarily binding to mRNA of STAT3 or survivin gene, but is not limited thereto.

In addition, the present invention can provide a pharmaceutical composition for enhancing radiation sensitivity to cancer cells, which comprises, as an active ingredient, an agent that inhibits the expression or activity of at least one protein 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 that specifically bind STAT3 or survivin proteins, peptides, aptamers, antibodies, and natural products.

The above-mentioned &quot; enhancement of radiation sensitivity &quot; means improving the sensitivity of the cells to radiation in the treatment of diseases using radiation. This can increase the radiation treatment efficiency. In particular, when the cancer treatment is performed in parallel, the radiation sensitivity of the cancer cells is enhanced, and the killing effect and proliferation inhibiting effect of the cancer cells can be exhibited.

The composition for promoting radiation sensitivity of the present invention may further comprise a pharmaceutically acceptable carrier and may be formulated together with a carrier.

"Pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound.

Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The composition for enhancing radiation sensitivity of the present invention can be applied to any formulations and can be manufactured into oral or parenteral formulations and can be formulated into unit dosage forms for ease of administration and uniformity of dosage. The pharmaceutical formulations of the present invention may be administered orally, rectally, nasal, topical (including under the ball and tongue), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous And intravenous), or forms suitable for administration by inhalation or insufflation.

Such oral dosage forms may be formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. A binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrating agent such as corn starch or sweet potato starch, a disintegrant such as magnesium stearate , Calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned materials, a liquid carrier such as a fatty oil may be further contained.

The formulation for parenteral administration may be formulated for injection such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection system, or aerosol formulation which enables inhalation through a respiratory apparatus. For formulation into injectable formulations, the compositions of the present invention may be formulated as solutions or suspensions in water with stabilizers or buffers in water, and formulated for unitary administration of ampoules or vials. For injection into suppositories, it may be formulated into rectal compositions such as suppositories or enema preservatives, including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying, such as an aerosol formulation, a propellant or the like may be formulated with the additive such that the water-dispersed concentrate or wet powder is dispersed.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example  1> Cell culture and irradiation

Human breast cancer cells MCF7, MDA-MB231, SKBR3, T47D and BT474 were purchased from ATCC (American Type Culture Collection; Manassas, Va.).

The purchased cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS; HyClone, South Logan, UT) and penicillin / streptomycin at 37 ° C and 5% CO ₂ .

(EMD Millipore, Billerica, MA) and 1 μM of lapatinib (Selleckchem, Inc.) were irradiated to the cells at a dose of 3.81 Gy at a dose of ¹³⁷ Cs (Cs) (Atomic Energy of Canada Ltd., Mississauga, Canada) ., Houston, Tex.) To inhibit STAT3 and HER2 activity, respectively.

< Experimental Example  2> Colony formation  Method ( Clonogenic  assay)

In order to confirm the cell survival rate after irradiation, colony formation analysis was performed with the previously reported method (Kim JS et al., Proteomics. 2010; 10 (14): 2589-2604.).

First, irradiated cells of different doses were inoculated into 60 mm culture dishes at varying densities. After 10-14 days, the colonies were fixed with methanol and stained with tryptophan blue.

Only colonies containing more than 50 cells were measured with survival colonies using a FACS (Image Products, Chantilly, VA).

< Experimental Example  3> RNA interference

siRNA was purchased from Genolution Pharmaceuticals Inc. (Seoul, Korea). The sequences used for RNA interference are as follows: HER2, 5'-CUGGUGUAUGCAGAUUGCC-3 'and survivin, 5'-AAGGAGAUCAACAUUUUCA-3'.

Non-targeting siRNA (Genolution Pharmaceuticals Inc.) was used as a negative control, and siRNA transfection was performed using G-Fectin (Genom Pharmaceutical Co., Inc.) according to the manufacturer's instructions.

< Experimental Example  4> Western Blot  analysis

Western blot analysis was performed with the previously reported method (Kim JS et al., Proteomics. 2010; 10 (14): 2589-2604.).

Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and detected by 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) Monoclonal anti-STAT3, rabbit polyclonal anti-ERa and anti-HER2 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.) And mouse monoclonal anti-beta-actin was purchased from Sigma (St. Louis, Mo.).

Blots were detected using an HRP-conjugated secondary antibody and an enhanced chemiluminescence detection system (Amersham Life Science, Piscataway, NJ).

< Experimental Example  5> STAT3  Activity analysis

STAT3 activity was confirmed by previously reported method (Shin DS et al., Cancer Res. 2009; 69 (1): 193-202).

Cells were transfected with 21pSTAT3-TA-Luc and control siRNA or HER2 siRNA for 48 hours using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) And treated or untreated with 10 Gy of radiation.

 After 24 hours, the cells were harvested using passive lysis buffer and luciferase activity was confirmed using a Dual Luciferase Reporter Assay Kit (Promega, Madison, WI) and a Wallac Victor2 plate reader (Perkin Elmer Corp., Norwalk, Conn.).

< Experimental Example  6> Cell death analysis

The apoptosis analysis was performed with the previously reported method (Kim JS et al., Proteomics. 2010; 10 (14): 2589-2604).

After washing the trypsin-treated cells, they 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).

< Experimental Example  7> Local region Disease-free survival rate  Patients for analysis

A total of 1,693 primary breast cancer patients who underwent curative surgery and adjuvant radiotherapy between January 1980 and September 2010 were analyzed.

All clinical and pathological data were obtained from the Center for Breast Cancer in Korea Cancer Center Hospital.

< Experimental Example  8> Molecules of the tumor In subtypes (molecular subtypes)  Classification of breast cancer patients

Immediately after surgery, immunohistochemistry (IHC) was performed to evaluate the expression of ER, PR, and HER2 in tumor tissues. ER or PR positive was classified as HER2 positive by HER2 gene amplification through IHC staining or fluorescence in situ hybridization.

The patients were classified according to the following four subtypes based on the degree of tumor expression of ER, PR and HER2.

(a) Hormone receptor HR + / HER2-: ER- and / or PR-positive and HER2-negative

(b) HR + / HER2 +: ER- and / or PR-positive and HER2-positive

(c) HR- / HER2 +: ER-negative, PR-negative and HER2-positive

(d) HR- / HER2-: ER-negative, PR-negative and HER2-negative

< Experimental Example  9> Immunohistochemistry ( Immunohistochemistry )

A sample for IHC was obtained from HER2 overexpressed primary breast cancer tissue paraffin block removed by radical surgery.

Patients with local recurrence within 1 year after radiation therapy were classified as sensitive response group. Patients who did not show symptoms of disease during the follow - up period of at least 2 years after radiotherapy were classified as non - sensitive group, The pathological TNM staining and HR status were matched.

Min JW et al., Biochem Biophys Res Commun. 2013: 440 (1): 137-142). , IHC experiments were performed.

Briefly, an anti-STAT3 mouse monoclonal antibody (1: 200 dilution; Santa Cruz), anti-phospho-STAT3 rabbit polyclonal antibody (1:50 dilution; GeneTex, Irvine, Calif.), Immunohistochemical staining was performed using a clone antibody (1: 100 dilution; Cell Signaling Technology).

Immunostaining was performed using avidin-biotin-peroxide according to the manufacturer's instructions (Invitrogen).

The staining intensity was evaluated according to the following criteria: 0 (no staining observed), 1+ (slight staining), 2+ (normal staining intensity), and 3+ (strong staining confirmed).

Example 1 Confirmation of Radiation Resistance and HER2-Positive Breast Cancer Association

Radiation resistance and HER2-positive breast cancer were confirmed in 1693 primary breast cancer patients who received appropriate adjuvant radiotherapy after radical surgery.

These patients were divided into four categories (HR + / HER2-, HR + / HER2 +, HR- / HER2 +, and HR- / HER2 +) based on the expression of HER2 and HR (estrogen receptor [ER] and / or progesterone receptor - / HER2-), and clinicopathological features were as shown in Table 1 below.

HR + / HER2- (20.5%, 347 out of 1,693 patients), HR + / HER2- (54.9%, 929 of 1,693 patients) (13.6%, 231 out of 1,693 patients) and HR- / HER2 + (11.0%, 183 out of 1,693 patients).

In addition, as shown in FIG. 1A, the grouping based on the molecular subtype of breast cancer patients showed a significant difference in the recurrence-free survival rate according to the subtype. HR + / HER2- And high relapse survival rates were higher in the HR / HER2 + patients.

From these results it can be suggested that radiation therapy sensitivity may be related to molecular subtype differences in breast cancer.

To confirm the association between these HR- / HER2 + subtypes and high radiotherapy resistance, radiotherapy effects were compared with other subtypes of breast cancer.

For clonal survival analysis, breast cancer cell lines containing MCF7 and T47D (HR + / HER2-), MDA-MB231 (HR- / HER2-), BT474 (HR + / HER2 +), and SKBR3 To confirm the reactivity.

As a result, it was confirmed that SKBR3, a HER2-positive (HR- / HER2 +) breast cancer cell line, showed a very high radiation resistance phenotype as compared to other breast cancer cells as shown in Figs. 1B and 1C.

From these results, it was confirmed that radiation resistance appears in HER2-positive breast cancer.

< Example  2> HER2 - Identification of radiation-resistant signal processes in benign breast cancer cells

Previous studies have confirmed that HER2 expression is related to radiation resistance of breast cancer, and HER2 is a major mediator of radiation resistance through HER2 siRNA - induced gene suppression in HER2 - positive breast cancer cells.

As a result, as shown in FIG. 2A, the SKBR3 cell group in which HER2 expression was inhibited by siRNA showed low survival rate due to sensitivity to various doses of radiation, and the HER2-positive SKBR3 and MDA-MB453 breast cancer cells When HER2 was deficient, it was confirmed that cell death due to radiation was increased.

From these results, it was confirmed that HER2 is a major factor controlling the radiation resistance in HER2-positive breast cancer cells.

Next, through a variety of carcinogenic pathways, HER2 promotes the activity of STAT3 induced by radiation, and this signaling process has been identified as one of the major signaling processes in complex radiation resistant cancer.

As a result, as shown in FIG. 3A, 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 radiation-induced STAT3 activity was suppressed there was.

In addition, STAT3 reporter plasmid with STAT3 binding element was used to perform luciferase analysis to confirm the direct transcriptional activity of STAT3. As a result, it was confirmed that when HER2 was deficient in the irradiated cells, the radiation-induced STAT3 activity was inhibited there was.

From the above results, it was confirmed that HER2 enhances radiation resistance through activation of STAT3 signal in HER2-positive breast cancer cells.

< Example  3> HER2 - in positive breast cancer cells HER2 - STAT3 - Survivin  Confirmation of increase of radiation sensitivity through suppression of signal process

We confirmed that inhibition of HER2 and STAT3 could increase the radiation sensitivity of HER2-positive SKBR3 breast cancer cells.

HER2 and STAT3 inhibitors lapatinib and S3I-201 were each treated with HER2-positive SKBR3 breast cancer cells and irradiated with 10 Gy dose.

As a result, it was confirmed that radiation-induced cell death was significantly increased in HER2-positive SKBR3 breast cancer cells treated with lapatinib and S3I-201 as shown in FIG. 4A, Induced STAT3 phosphorylation and survivin expression was also effectively reduced.

In addition, after the 3Gy irradiation, the colony formation rate was confirmed and the survival rate was analyzed. As a result, the survival rate of HER2-positive SKBR3 breast cancer cell line treated with lapatinib or S3I-201 and radiation was significantly decreased .

Next, we confirmed that when the survivin was inhibited via siRNA in HER2-positive SKBR3 breast cancer cells, the radiation sensitivity was improved.

As a result, referring to FIGS. 4D and 4E, similarly to the HER2 and STAT3 inhibitory effects confirmed above, it was confirmed that radiation-induced apoptosis was increased regardless of HER2 and STAT3 phosphorylation in cells lacking survivin.

From the above results, it was confirmed that the HER2-STAT3-survivin signaling process is a major factor of radiation resistance in HER2-positive SKBR3 breast cancer cells, so that the HER2-STAT3- May be proposed as a major target of treatment.

< Example  4> HER2 - In positive breast cancer tissue HER2 - STAT3 - Survivin  Confirmation of positive correlation between signal and radiation therapy resistance

To confirm the physiological relevance between HER2-STAT3-survivin regulation and resistance to radiation therapy in HER2-positive breast cancer, recurrent HER2-positive breast cancer patients (nonsensitive group; n = 7) Expression levels of phosphorylated STAT3 (Tyr705), STAT3 and survivin in HER2-positive breast cancer patients (sensitivity group; n = 8) were determined.

As a result, staining patterns of phosphorylated STAT3, STAT3 and survivin were similar in the serial sections of recurrent HER2-positive breast cancer patients as shown in Figs. 5A and 5B, but they were different in the non-relapsed patients.

In addition, strong nucleic acid staining patterns of phosphorylated STAT3 and survivin showing STAT3 activity were confirmed.

As a result, a strong nucleic acid staining pattern of phosphorylated STAT3 and survivin in the relapsed HER2-positive breast cancer patient group was confirmed by referring to the upper right image of FIGS. 5A and 5B. 5C to 5E, it was confirmed that the expression of phosphorylated STAT3, STAT3 and survivin was positively correlated with the group not showing sensitivity to radiation therapy.

From these results, it was confirmed that the resistance to radiation therapy in HER2-positive breast cancer was due to the HER2-STAT3-survivin signaling process.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (18)

MRNA of one or more genes selected from STAT3 or survivin, or an agent for measuring the protein level thereof. The composition for diagnosing radiation resistance of cancer cells according to claim 1, wherein the cancer cells are breast cancer cells. The composition for diagnosing radiation resistance of cancer cells according to claim 1, wherein the breast cancer cell is a breast cancer cell of HR- / HER2 + subtype. [Claim 2] 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. 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 an aptamer specific to the protein. A kit for the diagnosis of radiation resistance of a cancer cell comprising the composition of claim 1. [Claim 7] The kit according to claim 6, wherein the kit is an RT-PCR kit, a DNA chip kit, a microarray, or a protein chip kit. Measuring at least one gene mRNA or protein level thereof selected from STAT3 or survivin in the biological sample;
Comparing said gene mRNA or its protein level with at least one gene mRNA or protein level thereof selected from STAT3 or survivin as measured from a normal control, to provide information necessary for diagnosis of a radiation resistant prognosis . 9. The method of claim 8, wherein the biological sample is selected from the group consisting of tissue and blood. [8] The method according to claim 8, wherein the gene mRNA or protein level thereof is selected from the group consisting of Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Enzyme Immunoassay (ELISA), Immunohistochemistry, Western Blotting ) And flow cytometry (FACS). &Lt; Desc / Clms Page number 20 &gt; STAT3, or survivin as an active ingredient. The pharmaceutical composition for promoting radiation sensitivity to cancer cells, [Claim 12] The pharmaceutical composition for promoting radiation sensitivity to cancer cells according to claim 11, wherein the cancer cells are breast cancer cells. [Claim 12] The pharmaceutical composition for promoting radiation sensitivity to cancer cells according to claim 12, wherein the breast cancer cell is HR- / HER2 + subtype. [Claim 11] The pharmaceutical composition according to claim 11, wherein the preparation is selected from the group consisting of antisense nucleotides, siRNA and shRNA complementarily binding to mRNA of STAT3 or survivin gene. Composition. STAT3, or survivin as an active ingredient. The pharmaceutical composition for promoting radiation sensitivity to cancer cells, [Claim 16] The pharmaceutical composition for promoting radiation sensitivity to cancer cells according to claim 15, wherein the cancer cells are breast cancer cells. [Claim 16] The pharmaceutical composition for promoting radiation sensitivity to cancer cells according to claim 16, wherein the breast cancer cell is HR- / HER2 + subtype. 16. The method of claim 15, wherein the agent is selected from the group consisting of a compound that specifically binds STAT3 or a survivin protein, a peptide, an aptamer, an antibody, and a natural product. &Lt; / RTI &gt;

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