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www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No.

6

STAT3-survivin signaling mediates a poor response to radiotherapy
in HER2-positive breast cancers
Jae-Sung Kim1,*, Hyun-Ah Kim2,*, Min-Ki Seong2, Hyesil Seol3, Jeong Su Oh4,
Eun-Kyu Kim5, Jong Wook Chang6, Sang-Gu Hwang1, Woo Chul Noh2
1
Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, Korea
2
Department of Surgery, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, Seoul, Korea
3
Department of Pathology, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, Seoul, Korea
4
Department of Genetic Engineering, Sungkyunkwan University, Suwon, Korea
5
Department of Surgery, Breast Cancer Center, Seoul National University Bundang Hospital, Seoul National University
College of Medicine, Gyeonggi-do, Korea
6
Stem Cell Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul, Korea
*These authors have contributed equally to this work
Correspondence to: Jae-Sung Kim, e-mail: jaesung@kirams.re.kr
Woo Chul Noh, e-mail: nohwoo@kcch.re.kr
Keywords: breast cancer, radioresistance, HER2, STAT3, survivin
Received: August 02, 2015      Accepted: January 01, 2016      Published: January 09, 2016

ABSTRACT

Although radiotherapy resistance is associated with locoregional recurrence and
distant metastasis in breast cancers, clinically relevant molecular markers and critical
signaling pathways of radioresistant breast cancer are yet to be defined. Herein, we
show that HER2-STAT3-survivin regulation is associated with radiotherapy resistance
in HER2-positive breast cancers. Depletion of HER2 by siRNA sensitized HER2-positive
breast cancer cells to irradiation by decreasing STAT3 activity and survivin, a STAT3
target gene, expression in HER2-positive breast cancer cells. Furthermore, inhibition
of STAT3 activation or depletion of survivin also sensitized HER2-positive breast
cancer cells to irradiation, suggesting that the HER2-STAT3-survivin axis is a key
pathway in radiotherapy resistance of HER2-positive breast cancer cells. In addition,
our clinical analysis demonstrated the association between HER2-positive breast
cancers and radiotherapy resistance. Notably, we found that increased expression
of phosphorylated STAT3, STAT3, and survivin correlated with a poor response to
radiotherapy in HER2-positive breast cancer tissues. These findings suggest that the
HER2-STAT3-survivin axis might serve as a predictive marker and therapeutic target
to overcome radiotherapy resistance in HER2-positive breast cancers.

INTRODUCTION sensitize radioresistant cells is essential for improving the
efficacy of radiotherapy in breast cancer. Accumulating
Breast cancer is the most common cancer in women evidence suggests that differences might exist in the
worldwide, and its incidence continues to rise [1, 2]. radiation susceptibility of each molecular subtype of
Radiotherapy is recommended for most patients for local breast cancer [4].
control following breast conserving surgery, as well as Human epidermal growth factor receptor 2
following mastectomy in patients who are at high risk of (HER2) is overexpressed in approximately 25–30% of
recurrence [3–5]. However, some patients are resistant breast cancer patients, and it plays a key role in both the
to radiotherapy and the failure of local control in breast progression and metastasis of breast cancer [8]. High levels
cancer decreases the overall survival rate of patients of HER2 are associated with poor prognosis and reduced
[6, 7]. Thus, identifying both a molecular signature survival rates [9]. Therefore, HER2 inhibition might
to predict the outcome of radiotherapy and targets to be an effective strategy to reduce tumor aggressiveness

www.impactjournals.com/oncotarget 7055 Oncotarget

we observed that HER2 depletion led of breast cancer is resistant to radiotherapy. we RESULTS found that HER2 promotes radiation-induced activation of STAT3. head.(54. results suggested that HER2 enhances radioresistance and HR-/HER2+ (11. Interestingly.breast cancer sensitizes breast cancer cells to irradiation both in vitro patients showed the highest locoregional recurrence- and in vivo [12–15]. one of the key signaling molecules in multiple HER2-positive breast cancer is associated with radioresistant cancers [21].6%. Several reports have shown that HER2 inhibition different among these groups. breast. In addition.693 depletion inhibits radiation-induced STAT3 activation patients). Figure 1A). 231 of 1. The majority the luciferase reporter assay also indicated that HER2 of these patients were HR+/HER2. Thus. a together. 20]. Furthermore. which has the STAT3- [PR]) and HER2 in their tumors [26. 347 of 1. Taken growth and survival of tumor cells [18]. different sensitivities to radiotherapy. we investigated the association siRNA significantly decreased the survival of SKBR3 between breast cancer subtypes and susceptibility to cells in response to various doses of radiation (Figure radiotherapy. four categories based on the molecular expression of HR the direct transcriptional activity of STAT3 was measured (estrogen receptor [ER] and/or progesterone receptor using a STAT3 reporter plasmid. a clonogenic survival analysis in response to various doses Signal transducer and activator of transcription 3 of irradiation was performed using various breast cancer (STAT3) is a transcription factor that transduces oncogenic cell lines.001. and lung [21. Our data shows that the HR-/HER2+ subtype 2A). binding element for luciferase expression [30]. breast cancer cells. Therefore. these patients). Patients were classified into of phosphorylated STAT3 and survivin [18]. rectum.693 patients). positive SKBR3 and MDA-MB453 breast cancer cells [28. to be evaluated. HR+/HER2+ (13. www.(20. we examined the signaling pathways of breast cancer. Next. and their susceptibility to radiotherapy needs molecular subtypes of breast cancer.9%. To test this possibility. (Figure 3C). we Therefore. Data from HR+/HER2+. 183 of 1. including breast that the HER2-positive (HR-/HER2+) breast cancer cell cancer [19.5%. survivin signaling. HR+/HER2. [18]. Constitutive activation of STAT3 is frequently and SKBR3 (HR-/HER2+). Among several oncogenic pathways. HER2 and hormone receptor with higher radiotherapy resistance compared to other (HR) status. is often leads to radioresistance in HER2-positive breast associated with tumor resistance to chemotherapy and cancer cells radiotherapy in the brain. and HR-/HER2-. neck. HR-/HER2+. siRNA-mediated cancers to irradiation [15. 22]. BT474 (HR+/HER2+).0%. This suggests that HER2 might be free survival rate. whereas HR-/HER2+ patients had the a predictive biomarker as well as a molecular target for lowest locoregional recurrence-free survival rate (P < radiotherapy in breast cancer patients [16]. As expected. we observed observed in a variety of human cancers. 25].and radio- resistant tumors [15. understanding silencing of HER2 was employed to test whether HER2 STAT3 signaling is crucial for predicting and overcoming is the key mediator of radioresistance in HER2-positive tumor resistance.com/oncotarget 7056 Oncotarget . However. 11]. as determined by the decreased level summarized in Table 1 . This suggests that different molecular HER-2 status alone cannot be used a predictive marker subtypes of breast cancer are inherently associated with for survival after postmastectomy radiotherapy [17]. including MCF7 and T47D (HR+/HER2-).impactjournals. This suggests that targeting HER2. be a promising target for treatment of chemo. signals from cytokines and growth factors to the nucleus MDA-MB231 (HR-/HER2-). followed by HR-/HER2. 27]: HR+/HER2-. such as survivin. 29] (Figure 2B and C). 21. The by activating STAT3 signaling in HER2-positive breast locoregional recurrence-free survival was significantly cancer cells. involved in HER2-mediated radioresistance of breast cancer cells. 0. and plays a role in tumor progression line SKBR3 exhibited the most radioresistant phenotype and resistance to anti-cancer treatments by regulating the of all breast cancer cells tested (Figure 1B and C).693 patients). [10. 21–24]. silencing of HER2 by In the present study. In addition. our clinical and pre-clinical results suggested that number of recent studies have shown that STAT3 might HER2-positive breast cancer is resistant to radiotherapy. the correlation between the molecular profile hypothesized that the HR-/HER2+ subtype is associated of breast cancers such as. Radiation-induced activation radiotherapy resistance of STAT3 was inhibited by HER2 depletion in HER2- positive SKBR3 and MDA-MB453 breast cancer cells The clinicopathologic features of the patients are (Figure 3A and B). 16].693 in irradiated cells. This suggests that HER2 is the STAT3-survivin signaling might be an effective strategy major regulator of radioresistance in HER2-positive breast for adjuvant radiotherapy in the HER2-positive subtype cancer cells. and that this to an increase in radiation-induced cell death in HER2- radio-resistant phenotype is mediated by HER2-STAT3. Further. colon. Inhibition of the STAT3 pathway Since HER2 expression is associated with often sensitizes radio-resistant tumor cells in various radioresistance in breast cancer [4. increased activation HER2-induced activation of STAT3 signaling of STAT3 and its target genes. 929 of 1. Taken together.

5%) ER.0%) T stage  Tis 132 (7.com/oncotarget 7057 Oncotarget . progesterone receptor.6%)  HR-/HER2+ 186 (11.0%)  HR-/HER2.impactjournals.7%) Breast operation  Mastectomy 1286 (76.9%)  Unknown 10 (0. estrogen receptor. HER2.3%)   ≥50 622 (36.76%)  T4 50 (3.8%)  T1 944 (55.0%)  Unknown 3 (0. 533 (31.2%) N stage  N0 1048 (61. PR.0%)   Breast conserving surgery 407 (24.5%)  I 723 (42.5%)  N3 116 (6.9%)  HR+/HER2+ 231 (13.8%)  T2 503 (29. 347 (20. Table 1: The clinicopathologic characteristics of patients Variable Total (n=1693) No.6%) Hormonal receptor   ER+ and/or PR+ 1160 (68.1%)  N2 229 (13.9%)  N1 290 (17.5%)   ER.5%) HER2  Negative 1276 (75. HR.7%)  T3 61 (3.6%) Stage  0 127 (7. 929 (54.7%)  II 464 (27.9%)  Unknown 10 (0.4%)  Positive 417 (24.6%) Molecular subtype  HR+/HER2. human epidermal growth factor receptor 2 www.3%)  III 370 (21. hormone receptor (ER or PR). (%) Age (yr) at the time of surgery  <50 1071 (63.and PR.

After 48 h.impactjournals. SKBR3.com/oncotarget 7058 Oncotarget .05 or **P < 0. the SKBR3 cells or MDA-MB453 cells either were left untreated (Ctrl) or treated with 10 Gy of radiation (IR) for 48 h. and BT-474 cells were analyzed by immunoblotting with anti-HER2 and anti-ER antibodies. Cell viability was determined with a FACScan flow cytometer. the cells were treated with different doses of radiation.1 30 β -actin ** Ctrl siRNA 20 Cleaved-PARP MDA-MB453 HER2 siRNA 10 HER2 β-actin 0. www. A. MCF7. and BT-474 cells were treated with different doses of radiation as indicated. C. T47D.01 0 0 2 4 6 HER2 siRNA HER2 siRNA Ctrl siRNA Ctrl siRNA IR Dose (Gy) 0 Gy 10 Gy Figure 2: HER2 depletion sensitized HR-/HER2+ breast cancer cells to irradiation. The clonogenic survival fraction was determined by clonogenic assay.001). The data are presented as the mean ± standard deviation of three independent experiments. and data are presented as the percentage of propidium iodide-positive cells (B).01 compared with irradiated siRNA control cells (A and B). P < 0. SKBR3 cells or MDA-MB453 cells were transfected with 100 nM control siRNA or HER2 siRNA. MDA-MB231. *P < 0. MDA-MB231. SKBR3. Kaplan–Meier event free survival curve in patients treated with breast-conservation therapy followed by adjuvant radiotherapy (log-rank test. T47D. A. β-actin was used as a loading control (C). After 48 h. A Loco-regional recurrence free survival (%) B 1 C MDA-MB231 Survival fraction (log) BT-474 SKBR3 MCF7 T47D 0. MCF7.01 0 2 4 6 Follow-up time (years) IR Dose (Gy) Figure 1: HR-/HER2+ subtype was associated with radioresistance in breast cancer patients as well as breast cancer cell lines. β-actin was used as a loading control. The data represent typical results and are presented as the mean ± standard deviation of three independent experiments. SKBR3 cells were transfected with 100 nM of control siRNA or HER2 siRNA. as indicated. The cells were analyzed by immunoblotting with anti-cleaved-PARP and anti-HER2 antibodies.1 MCF7 HER2 MDA-MB231 SKBR3 ERα T47D BT-474 β-actin 0. B. B and C. C 10 Gy A B 0 Gy HER2 siRNA HER2 siRNA Ctrl siRNA Ctrl siRNA 1 60 SKBR3 ** 50 MDA-MB453 Survival fraction (log) ** Cleaved-PARP % of cell death 40 SKBR3 * HER2 0.

Next. Treatment with lapatinib and S3I-201 increased radiation. After 48 h. In addition. STAT3. Further. phosphorylation (Figure 4D). that target HER2 and STAT3. A and B.5 0 HER2 siRNA HER2 siRNA Ctrl siRNA Ctrl siRNA 0 Gy 10 Gy Figure 3: HER2 depletion radiosensitized HR-/HER2+ breast cancer cells by modulating STAT3 activity. we found that increased that HER2-STAT3-survivin signaling is a key factor in expression of phosphorylated STAT3. survivin was positively associated with the group that implying that the HER2-STAT3-survivin axis could be was non-responsive to radiotherapy (Figure 5C–E). and survivin were we examined whether survivin inhibition with siRNA similar in the serial sections of relapsed HER2-positive enhanced the radiation sensitivity of HER2-positive breast cancer patients. Inhibition of the HER2-STAT3-survivin axis a potential target for adjuvant radiotherapy in HER2- increases radiation sensitivity in HER2-positive positive breast cancers. the strong HER2 and STAT3 inhibition. we used chemical inhibitors. indicative of STAT3 activation.impactjournals. These A 0 Gy 10 Gy B 0 Gy 10 Gy C HER2 siRNA HER2 siRNA HER2 siRNA HER2 siRNA Ctrl siRNA Ctrl siRNA Ctrl siRNA Ctrl siRNA 2. These results suggested right upper panels). to a significant reduction in the survival rate of HER2. Interestingly. and the radioresistance of HER2-positive breast cancer cells. breast cancer cells In vivo evidence for a positive correlation To investigate whether inhibition of HER2 and between the HER2-STAT3-survivin axis and STAT3 sensitizes HER2-positive SKBR3 breast cancer radiotherapy resistance in HER2-positive breast cells to irradiation. www. and survivin in relapsed (non-responder group. lapatinib cancer tissues and S3I-201. Cells were analyzed by immunoblotting with the indicated antibodies. the expression level of phosphorylated STAT3 (Tyr705). To further examine the physiological relevance induced cell death and decreased both radiation-induced of HER2-STAT3-survivin regulation in radiotherapy STAT3 phosphorylation and survivin expression in HER2.01 compared with irradiated siRNA control cells (C). but not in the recurrence-free SKBR3 breast cancer cells. when combined with radiation. SKBR3 cells or MDA-MB453 cells were transfected with 100 nM control siRNA or HER2 siRNA. were detected survival of HER2-positive SKBR3 breast cancer cells in relapsed HER2-positive breast cancer patients rather (Figure 4D and E). p(Y705)- 1. n = 8) lapatinib or S3I-201. β-actin was used as a loading control. led HER2-positive breast cancer patients after radiotherapy. resistance of HER2-positive breast cancers. we observed that the staining patterns positive SKBR3 breast cancer cells (Figure 4C).com/oncotarget 7059 Oncotarget . SKBR3 cells or MDA-MB453 cells were either left untreated (Ctrl) or treated with 10 Gy of radiation (IR) for 48 h.5 ** Luciferase activity (Relative value) SKBR3 MDA-MB453 2 HER-2 HER-2 ** p(Y705). respectively. STAT3. but it did not affect HER2 and STAT3 than in the recurrence-free patients (Figure 5A and B.5 MDA-MB453 STAT3 STAT3 SKBR3 STAT3 STAT3 1 Survivin Survivin β -actin β -actin 0. the rate of colony formation in STAT3. **P < 0. we evaluated positive SKBR3 breast cancer cells (Figure 4A and B). The data represent typical results and are presented as the mean ± standard deviation of three independent experiments. survivin depletion increased nuclear staining patterns of phosphorylated STAT3 and radiation-induced cell death and reduced clonogenic survivin. STAT3 activity in each sample was determined by a STAT3 activity assay. response to 3 Gy irradiation indicated that treatment with n = 7) or recurrence-free (responder group. In the survival analysis. Similar to the effects of patients (Figure 5A and B). C. of phosphorylated STAT3.

β-actin was used as a loading control (A and D). SKBR3 cells were transfected with 100 nM control siRNA or survivin siRNA. Moreover. the cells were treated with 10 Gy (D) or 3 Gy of radiation (E). many in vitro studies have shown that HER2 overexpression or DISCUSSION inhibition modulates radiation resistance in breast cancer cells [12–15]. 9] [17]. have shown survivin expression might confer poor outcomes in that HER2-STAT3 cross-talk increases the aggressiveness response to radiotherapy in patients with HER2-positive and radioresistance of breast cancer stem cells [15]. recent reports HER2-mediated regulation of STAT3-survivin signaling. Further.01 compared with irradiated control cells (B.subtype of breast apoptosis and promotion of mitosis in response to A B C D E 0 Gy 10 Gy Survivin siRNA Survivin siRNA 0 Gy 10 Gy Ctrl siRNA Ctrl siRNA Lapatinib Lapatinib 60 1. In contrast. cancer. Duru et al. SKBR3 cells were untreated (Ctrl) or treated with 10 Gy of radiation (IR) in the absence (Ctrl. our data suggests that increased HER2-STAT3.8 % of survival 0. Chung et al. After 48 h. C. postmastectomy radiotherapy confers Survivin plays a key role in the inhibition of survival benefits in the HR+/HER2.com/oncotarget 7060 Oncotarget . SKBR3 cells untreated (Ctrl) or treated with 3 Gy of radiation (IR) in the absence (Ctrl. such as observations are similar to those of several other groups PI3K/Akt and NF-kB. taken collectively. or 1 µM lapatinib plus 100 µM S3I-201. observations provided in vivo evidence that the HER2. breast cancer. Our data showed that HER2 promotes We have shown that the HR-/HER2+ subtype of breast radioresistance via STAT3-survivin regulation in HER2- cancer is associated with radiotherapy resistance via positive breast cancers. These observations. These findings subtype of breast cancer and increased risk of local or suggest that HER2-STAT3 regulation is crucial regulator regional recurrence following radiotherapy [4. C. distant metastasis after radiotherapy [17]. reported that STAT3 activation by HER2 Our clinical data showed that each molecular overexpression promotes cancer stem cell traits that subtype of breast cancer is associated with different correlate phenotypically with tumor resistance in HER2- locoregional recurrence rates in patients treated by expressing breast cancers [32]. The cells were analyzed by immunoblotting with the indicated antibodies.impactjournals.6 p-HER2 -STAT3 p(Y705)- STAT3 20 0.2 0. www. **P < 0.2 S3I-201 S3I-201 * Ctrl Ctrl 50 1 1 * Cleaved-PARP Cleaved-PARP % of cell death 40 0. HR-/HER2+ tumors are associated STAT3-survivin axis might confer radiotherapy resistance with an increased probability of local recurrence and in HER2-positive breast cancers. for tumor radioresistance of HER2-positive breast cancers.4 0. DMSO) or presence of 1 µM lapatinib or 100 µM S3I-201. crosstalk with STAT3 signaling in that have shown an association between the molecular resistant phenotypes of breast cancer [31].2 1. and survivin radiosensitized HR-/HER2+ SKBR3 breast cancer cells. Colony formation was quantified by automatic colony counter (C and E). have suggested that STAT3 could be the key downstream Further. Similar to our data. For instance. This study provides clinical and experimental indicate that the HR-/HER2+ subtype of breast cancer is evidence for the role of the HER2-STAT3-survivin axis in associated with radiotherapy resistance. it has been curative surgery followed by adjuvant radiotherapy. Our suggested that downstream pathways of HER2.2 β -actin * β -actin 0 0 0 S3I-201 S3I-201 IR+S3I-201 IR+Lapatinib+S3I-201 IR+Lapatinib Lapatinib Lapatinib Ctrl Ctrl Ctrl Ctrl siRNA IR IR+Survivin siRNA IR+Ctrl siRNA 0 Gy 10 Gy Figure 4: Inhibition of HER2. The data represent typical results and are presented as the mean ± standard deviation of four independent experiments.6 0. radiotherapy resistance of HER2-positive breast cancers. Cell viability was determined with a FACScan flow cytometer and data are presented as percentage of propidium iodide-positive cells (B). and E). STAT3. mediator of HER2 signaling [31]. and then incubated for 24 h.4 * STAT3 Survivin * STAT3 * 10 0. Clonogenic survival was determined by colony formation assay. 100 µM S3I-201.8 % of survival p-HER2 Survivin p(Y705) 30 0. D and E. A and B. DMSO) or presence of 1 µM lapatinib.

Representative microscopic images of relapsed (A) or recurrence-free (B) HER2-positive breast cancer tissues stained with anti-phosphorylated STAT3 (Y705) (left panel). Arrows indicate the nuclear staining pattern of the specific protein. STAT3. suggesting that STAT3-survivin is a potential is currently under investigation in clinical trials [36]. n = 8) and relapsed (non- responder. A and B. and that inhibition of HER2 increases radiation sensitivity of increased STAT3-survivin expression was associated with breast cancers [13-15. strong. Our apoptosis. 33].022085 D STAT3 P = 0. increased survivin by HER2. anti-STAT3 (middle panel). It also has the overcoming radiotherapy resistance in HER2-positive ability to promote mitosis as a regulator [34] or DNA breast cancers. Representative high-magnification images of relapsed (A) or recurrence-free (B) HER2-positive breast cancer tissues (upper right panel). and +3. STAT3 (D). Regarding the role of survivin in radioresistance. www. Data are represented by box-and-whisker plots. our pre-clinical evidence that inhibition of the cancers. 50 μm. work by our group as well as others has shown that A Relapsed patients after radiotherapy (Non-responder) B Recurrence-free patients after radiotherapy (Responder) p(Y705)-STAT3 STAT3 Survivin p(Y705)-STAT3 STAT3 Survivin Case #S10-7664 Case #S10-5056 Case #S10-12190 Case #S10-10332 C p(Y705)-STAT3 P = 0. and survivin expression and relapsed HER2- positive breast cancer after radiotherapy. weak. previous repair via Ku70 [25]. Scale bar. Staining intensity was scored as follows: 0. promoting mitosis. a combination treatment with a poor response to radiotherapy in HER2-positive breast radiation and HER2 inhibitors (trastuzumab or lapatinib) cancers. no staining.com/oncotarget 7061 Oncotarget . 000931 E Survivin P = 0. anticancer therapies [25. or anti- survivin antibody (right panel). Thus.05 compared with responder group. n = 7) breast cancer tissues. C-E. effecter of HER2-STAT3 regulation in response to On the basis of pre-clinic evidence indicating that irradiation of HER2-positive breast cancer cells. Quantification of phosphorylated STAT3 (C). *P < 0. moderate. +1.impactjournals. A previous study suggested STAT3 regulation might protect tumor cells from ionizing that STAT3 activation is correlated with survivin radiation through multiple mechanisms.011494 3 * 3 * * 3 2 2 2 IHC Score IHC Score IHC Score 1 1 1 0 0 0 Responder Non-responder Responder Non-responder Responder Non-responder (n = 8) (n = 7) (n = 8) (n = 7) (n = 8) (n = 7) Figure 5: Positive correlation between phosphorylated STAT3. or survivin (D) staining intensities in recurrence-free (responder. Related to this observation. 35]. and enhancing DNA repair present study showed that survivin is a downstream in radioresistant HER2-positive breast cancers. +2. biomarker for radioresistance in HER2-positive breast Similarly. such as inhibiting expression in high-risk breast cancer patients [19]. HER2-STAT3-survivin axis increases radiation sensitivity survivin inhibits apoptosis of tumor cells by directly or of HER2-positive breast cancers suggests that targeting indirectly regulating caspase-3/-7 or apoptosis-regulatory the HER2-STAT3-survivin axis may be important to factors such as HSP90 and AIF [25].

The resistant tumors. After 10–14 days the colonies were fixed with Patient population for locoregional methanol and stained with a Trypan blue solution. breast cancers.81 Madison. 22. Briefly. Therefore. Piscataway. SKBR3. Canada Ltd. MDA. A cell death analysis was performed as described Clonogenic assay previously [37]. FACScan flow cytometer (Becton Dickson. S3I-201 (100 µM. MO). and using G-Fectin (Genolution Pharmaceuticals Inc. STAT3 SDS-PAGE and transferred to a nitrocellulose membrane. and target for radiotherapy resistance in HER2-positive mouse monoclonal anti-β-actin from Sigma (St.. and luciferase activity was evaluated using a 137cesium (Cs) ray source (Atomic Energy of using the Dual Luciferase Reporter Assay Kit (Promega. TX) were used to inhibit STAT3 and HER2 activity. Norwalk. EMD Millipore. NJ). CA). MA). PR. rabbit polyclonal anti-phospho-STAT3 (Tyr705). survivin. Cell lines and treatments STAT3 activity assay Human breast cancer cell lines MCF7. Blots were developed using horseradish peroxidase- conjugated secondary antibody and an enhanced chemiluminescence detection system (Amersham Life MATERIALS AND METHODS Science. rabbit polyclonal anti-ERa. activation is a key pathway for the survival of various followed by detection using specific antibodies. Houston. antibodies were used included rabbit monoclonal anti- In conclusion. Korea Cancer Pharmaceuticals Inc. followed by incubation with propidium iodide Cell survival after irradiation was determined by (5 µg/mL) for 10 min. MA) and lapatinib (1 µM. The cells were analyzed with a a clonogenic assay as described previously [37].impactjournals. 24]. Mississauga. The clinical and pathologic data were obtained from siRNAs were synthesized at Genolution a database of the Breast Cancer Center. radiation were seeded in triplicate in 60-mm tissue culture dishes. 21pSTAT3-TA-Luc and control siRNA or HER2 siRNA VA) and grown in Dulbecco’s modified Eagle’s medium for 48 h using Lipofectamine 2000 (Invitrogen. the cells were co-transfected with from the American Type Culture Collection (Manassas. A non-targeting the molecular subtypes of tumors siRNA (Genolution Pharmaceuticals Inc. WI) on a Wallac Victor2 plate reader (Perkin Gy/min.com/oncotarget 7062 Oncotarget . The cells were irradiated a passive lysis buffer. Our Technology (Beverly. Briefly. Cell death analysis respectively. Louis. Chantilly. recently reported that trastuzumab resistance is regulated Western blotting was performed as described by STAT3-dependent feedback activation in HER2. CT). Elmer Corp. cells were trypsinized and washed. and anti-HER2 from axis is a predictive marker and a potential therapeutic Santa Cruz Biotechnology Inc.) HER2 expression in samples from each case by according to the manufacturer’s protocol. T47D. Billerica. Korea). STAT3-survivin regulation potentiated radiation and anti-cleaved-PARP (Asp214) from Cell Signaling resistance of HER2-positive breast cancer cells. Franklin various densities of cells treated with different doses of Lakes. STAT3 activity was determined as described MB231. supplemented with 10% fetal bovine serum (HyClone. Only recurrence-free survival analysis colonies containing more than 50 cells using a colony counter (Image Products. All patients were treated by RNA interference curative surgery and adjuvant radiotherapy prior to this study. Transfection of siRNA was performed A pathologist evaluated ER. proteins were separated by positive breast and gastric cancers [24]. this study showed that HER2. Selleckchem. VA) were counted as Between January 1980 and September 2010.693 primary breast cancer patients were included in this retrospective analysis. 5′-CUGGUGUAUGCAGAUUGCC-3′ and survivin.) was used as a negative control. Briefly.. 38]. Li et al. Carlsbad. (Santa Cruz. The cells were harvested after 24 h using humidified 5% CO2 atmosphere. UT) and penicillin/streptomycin at 37°C in a or no treatment. CA). Further. The following Center Hospital [39]. including breast cancer. immunohistochemistry (IHC) immediately after www. mouse monoclonal anti- work provides evidence that the HER2-STAT3-survivin STAT3. NJ). of 1. Classification of breast cancer patients based on 5′-AAGGAGAUCAACAUUUUCA-3′. and BT474 were purchased previously [30]. previously [37. (Seoul. Canada) at a dose rate of 3. This was followed by either irradiation with 10 Gy South Logan. a total surviving colonies. sequences were used for RNA interference: HER2. STAT3 inhibition is crucial for the treatment of various Western blot analysis radioresistant tumors [21. Briefly.

Breast Signaling Technology). Gray R. Basch E. Duane F. Pierce L. Eifel P. of Radiological and Medical Sciences. Positive staining for ER or PR was defined as CONFLICTS OF INTEREST staining of at least 10% of the nuclei in 10 high-power fields. 2014. 32:129-160. Peto R. surgery. Effect of radiother- first detection of locoregional recurrence by physical apy after breast-conserving surgery on 10-year recurrence examination or radiological imaging. www.(ER. Louis TA. Staining 5. Taylor C. Fulton S. Correa C. Peto R. We thank Yoon Y and Kim MO for technical 383:2127-2135. Gilbert MR. We matched patients from each group by Annual Report on Progress Against Cancer from the pathologic TNM staging and HR status. Roter D. Institutes of Health Consensus Development Conference Statement: adjuvant therapy for breast cancer. within 1 year after completion of radiotherapy. 2+ (moderate staining). negative. Dodwell DD.801 women in 17 randomised trials. and (d) HR-/HER2. PR. CA). HI14C1864).and/or PR-positive 50451–2015). PR. Wang Y.(ER. Naishadham D. 2013. Kemeny M. The Kaplan-Meier and 15-year breast cancer death: meta-analysis of individ- method with log-rank test was used for the statistical ual patient data for 10. intensity was scored as follows: 0 (no visible staining). Sharma S.and/or PR-positive and HER2. to the manufacturer’s instructions (Invitrogen). J Natl Cancer Inst. Lancet. 2011. Radiological Translational Research Program (No. and HER2: This work was supported by a grant from the (a) HR+/HER2. Clinical Cancer Advances 2013: of radiotherapy. Cutter D. Patel JD. J Clin Oncol. GeneTex. 2014. group of patients who showed locoregional recurrence CA Cancer J Clin. and HER2-negative). Correa C. Irvine. according radiosensitisation. P < 0. Wang Z. Costa J. locoregional recurrence. antibody (1:50 dilution. Horgan K. Masters GA.com/oncotarget 7063 Oncotarget . Jemal A. The non-responder group to radiotherapy was defined as the 1. Ministry of Health & Welfare (No. November Statistical analysis 1-3. Markman M. Deshler A. 378:1707-1716. Briefly. 2000. and HER2-positive). Whelan T. 2013. Effects of radiotherapy and surgery in early breast cancer. free survival was defined as the time from the first Clarke M. IHC experiments American Society of Clinical Oncology. Aghajanian C. staining intensity of 3+ or HER2 gene amplification by fluorescence in situ hybridization. Wang Y. For the survival analysis. Langlands FE. Kornblith 1+ (faint staining). 86:20120601. anti-phospho-STAT3 rabbit polyclonal Trialists’ Collaborative Group. Early Breast Cancer Santa Cruz). O’Day SJ. Patients were FUNDING classified into the following four molecular subtypes based on tumor expression of ER. 333:1444-1455. de Lima M. and HER2 positivity was defined by an IHC The authors declare no conflicts of interest. Darby S. analyze statistical differences between groups. The 2.impactjournals. funded by the Ministry of Science. Krilov L. Cell 4. Schwartz period. negative). Curran WJ. Ewertz M. which was at least 2 years from the completion GK. Axelson JA. A two-tailed Student’s t-test was performed to Lancet. 93:979-989. or anti. 1995. Immunohistochemistry 1711021781). Kris MG. Darby S. McGale P. Adams S. were performed as previously described [40. et al. Cancer statistics. N Engl J Med. Cutter D. The specimens for IHC were obtained from paraffin blocks of HER2 overexpressing primary breast cancer REFERENCES tissue that had been removed by curative surgery. Arriagada R. M. Crowley J. Project. Taylor C. 63:11-30. analysis. Gray diagnosis of primary breast cancer to the time of R. Mayer R. 2001. Whelan T. Immunostaining was performed cancer subtypes: response to radiotherapy and potential using the avidin-biotin-peroxidase method.05 7. who showed no evidence of disease during the follow-up Marshall JL. assistance. Davies C. Mannu G. survivin rabbit monoclonal antibody (1:100 dilution. PR-negative. (b) HR+/HER2+ (ER. Godwin J. responder group was defined as the group of patients Brose MS. National staining). Carroll WL. and 3+ (strong AB. (c) HR-/HER2+ (ER-negative. 41]. 2013. 6. Jr. Polite B. Br J Radiol. Siegel R. Smith L. immunohistochemical staining was performed using an 3. McGale P. and the National R&D Program of the Korea Institute negative. ICT & Future Planning (No. anti-STAT3 mouse monoclonal antibody (1:200 dilution. Effect of radiotherapy after mastectomy and axillary surgery on 10-year recurrence and 20-year breast ACKNOWLEDGMENTS cancer mortality: meta-analysis of individual patient data for 8135 women in 22 randomised trials. Ewertz was considered statistically significant. An overview of the randomized trials. Hendricks CB. the Korean Health Technology R&D and HER2-positive).

23:7350-7360. J. Lee HK. D. Liu HC. Kimple RJ. Lu Y. 90:2344-2348. of stat3 in primary tumors from high-risk breast cancer 32. Singh N. Jeffrey and radiosensitization of lapatinib-resistant cells restored SS. Menaa C. Frank DA. distinct cancer subtypes. 2005. LA. Jin W. Neve RM. Pollack JR. doi: 10. Chung SS. Raf>MEK>ERK mitogen-activated protein kinase cascade 26. Cancers (Basel). 2004. Yu JH. Sorlie T. Chin K. Nat Rev Cancer. 2012. 2013. of Survivin following irradiation. Sorensen FB. Wloch M. Lam KS. 2003. STAT3 activation patients is associated with elevated levels of activated SRC in HER2-overexpressing breast cancer promotes epithelial- and survivin expression. 31. Min JW. Overgaard M. Spano JP. 2006. 28:21-29. Wester K. Gritsko T. Drug Discov Today. Oncotarget. J Cancer Res Clin Oncol. J Clin Oncol. Seijo E. Eisen MB. Nielsen HM. Li JJ. Chromy BA. et al. Shields Kanwar JR. Mechanism of lapatinib-mediated radiosensitiza. Carey LA. Cox C. Guo Y. Voduc KD. Li W. Activation 140:1-14. 2006. Milas L. blocking the dimerization in DU145 prostate cancer cells. Carlsson J. Feedback activation of STAT3 mediates expressing this oncogene. breast cancer research. zumab.2135. Holliday DL. 20:578-587. negative breast cancer cells. Li JJ. Shin DS. Cryptotanshinone inhibits constitutive signal therapy in high-risk breast cancer: the Danish Breast Cancer transducer and activator of transcription 3 function through Cooperative Group.18632/ Sensitization of breast cancer cells to radiation by trastu. Wienands review. 2001. doi: 10. Jove R. 2014. Perou CM. Kim KW. Eur J Cancer. Fan M. cer: ready for clinical application? J Clin Oncol. Jove R. Breast cancer subtypes and 6:1986-2011. 26:1419-1426. 2009. Candas D. Sullivan D. Choe MH. Wang NJ. 5:8317-8329. J Clin Oncol. Duru N. Cao C. Tyldesley S. Blomqvist marker and therapeutic target in cancer. Wang H. Vadgama JV. Pietras RJ. An S. 16. 2:1113-1120. Albert JM. J. erogeneous tumor society. Spellman YJ. Wu Y. Wongvipat PN. Ross DT. Jeon HB. 2011. Caldas C. trastuzumab resistance via upregulation of MUC1 and MUC4 13. Breast cancer adaptive 19. and corresponding metastases. Ahmed AA. Hung MC. Zhao L. Williams C. Radiother Oncol. Eldridge A. Han DC. Zhu SX. mesenchymal transition and cancer stem cell traits. Speirs V. Minton S. Fluge O. 59:1347-1355. Lee JH. Akslen by direct inhibition of MEK. Original data and literature 21. Mutter RW. Clinical aspects for survivin: a crucial molecule JM. Diaz N. Kim HN. 93:639-644. Gallardo D. 14. 2013. Zhang L. Qian W. Raymond E. van de Rijn M. Candas D. 2006. 8. Suh Clark L. J Clin Oncol. Clin Cancer Res. Int J Oncol. The STATs of cancer--new molecular targets Cancer Res. vator of transcription 3 activity results in down-regulation 12. Livingston S. Cheang MC. Villman 20. Lee MR. Thery JC. Targeting 23. Bowman T. 2010. HER2 expression in breast cancer primary tumours 31:4560-4561. Lonning PE. Semin Oncol. Mol Cancer Ther. Mol Cancer Ther. 2015. Nordgren H. 11. Nature. Br J Cancer. Beam CA. Inhibition of signal transducer and acti- cancer patients. STAT3: A Novel Molecular Mediator of 9. Breast Cancer Res. Hwang SG. Ebner R. Ostenstad B. 16:266-273. Gelmon K. HER-2. Slamon DJ. 2014. Cho DH.com/oncotarget 7064 Oncotarget . expression. Cancer Res. 2009. Yoon YJ. Lee HJ. Sartor CI. Fevr T. 4:97-105. Coppe JP. 28. 2008. Wang SC. Clin Cancer Res. Grade M. Hou S. Pergamenschikov A. Duru N. Kennecke H. 28:1684-1691. Kwon BM. 22. C. Azria D. Johnsen H. Noh WC. 6:2654-2666. for targeting drug-resistant cancers. resistance: HER2 and cancer stem cell repopulation in a het- Eweis I. www. Cancer Cell. Kim HA. progesterone 30. Rees CA. classification and molecular forecasting of breast can- resistance of breast cancer stem cells isolated from HER2. 50:892-901. Resistance to human epidermal growth factor receptor a prognostic marker for laryngeal cancer. Spitz 27. 12:20-28. A collec- Factor Receptor 2 with Radiotherapy Resistance in Patients tion of breast cancer cell lines for the study of functionally with T1N0M0 Breast Cancer. Penault Llorca activity in radioresistant laryngeal cancer cells and serves as F. Jiang G. Shin KD. Spitzner M. Lee HG. 406:747-752. Kim EK. modulates repair of radiation-induced DNA damage and 24. Bengtsson NO. Liang K. Lapuk A. the risk of local and regional relapse. Cantor A. 2000. Monoclonal antibody to HER-2/neureceptor 5:2659-2665. Molecular portraits of 15. Borresen-Dale AL.impactjournals. 69:193-202. Kuo WL. Sambade MJ. 18:6634-6647. Giehl N. Krishnakumar S. Li S. 2014. Ghadimi BM. Yu H. Kim SJ. Fan K. 2014. Fan Z. Sjostrom J. Wicha MS. Camp JT. 2014. Oh JS. Dai enhances radiosensitivity of human breast cancer cells over. Resistance to Chemoradiotherapy. 29. ERp57 modulates STAT3 10. Wolff HA. Transcription factor STAT3 as a prognostic K. Wei H.18632/oncotarget.3042. Lundqvist H. and response to postmastectomy radio. Nantajit human breast tumours. Kanwar RK. HER2-associated radio. HER2: recent developments and future directions for breast Sekhar KR. Poen JC. tion of breast cancer cells is primarily by inhibition of the 2015. 18. Kyndi M. Tong F. Shinohara ET. Hortobagyi GN. oncotarget. Estrogen receptor. Kim JS. receptor. 10:515-527. et al. Ang KK. 1999. 2004. Brenton JD. Muro-Cacho CA. Kim MS. 25. Wen Y. Oncotarget. Xiao K. 13:215. Fridlyand J. Garcia R. Han YH. come of age. Choosing the right cell line for 17. Cheung CH. 44:403-411. Knudsen H. Nielsen TO. Li G. Speed T. Bayani N. type 2-targeted therapies. Yeh J. Lu B. DeVries S. Baehner FL. Molecular DR. Overgaard J. Association of Human Epidermal Growth PT. J Breast Cancer.

13:638-646. Noh WC. Um HD. expression and confers resistance to apoptosis in human 39. 2010. Turkson J. Kim JS. Yoder S. tance of laryngeal cancer cells via modulating p53. Kim EK. prognosis and treatment of breast cancer.com/oncotarget 7065 Oncotarget . Hwang SG. Cancer Res. Yao Q. Oh JS. 41. Kim EJ. breast cancer cells. Sohn YJ. Min JW. 2012. Wang L. hyde reductase expression mediates acquired radioresis- Enkemann S. Chang JW. Koh JS. Lee JH. www. INPP4B- 32:2886-2893. Byeon J. Interaction of radiation ther. Yun HS. Increased alde- Huang M. Eweis I. Breast Cancer. Cancer Persistent activation of stat3 signaling induces survivin gene Biol Ther. 2013. Dimou I. The role of sur. Williams A. Chloride intra. 15:1542-1550. CIP2A modu- C. Kim JS. Jeon HB. Park IC. Hwang SG. 2014. Clin Cancer Res. Nam S. Diaz N. Lee KH. apy with molecular targeted agents. Proteomics. Cho DH. Kim EK. cellular channel 1 identified using proteomic analysis plays 440:137-142. Dubos S. Tsezou A. 10:2589-2604. Ha JH. Kim glucose metabolism via hexokinase 2 regulation in laryn- DH. 2014. et al. Kim KI. Breast Cancer Recurrence in HER2-Positive Patients.impactjournals. J Clin Oncol. Cheng J. Kim JS. Oh JS. geal cancer cells Biochem Biophys Res Commun. 2006. 38. Hwang SG. Seol H. Park IC. Eschrich S. Lee SJ. Survivin regulation by HER2 lates cell-cycle progression in human cancer cells by regu- through NF-kappaB and c-myc in irradiated breast cancer lating the stability and activity of Plk1. Bowman T. Noh WC. 73:6667-6678 36. 2013. 35. Yang KM. Papanikolaou V. Kim HA. Park JK. Yu F. Gritsko T. Chang JW. Park IC. Seong MK. an important role in the radiosensitivity of HEp-2 cells via reactive oxygen species production. J Cell Mol Med. Lee JY. Sullivan D. mediated tumor resistance is associated with modulation of 37. 2010. Kim HA. J J Thorac Dis. Iliopoulos D. Chen JH. 17:33-39. Lv YG. 12:11-19. Morris ZS and Harari PM. Kaneko S. 2:100-110. Lee JK. 33. Kim JS. Hwang SG. Serial Serum HER2 Measurements for the Detection of vivin in diagnosis. 34. Kappas 40. Beam CA. 2011. Hong EH. cells. Kitsiou-Tzeli S.