STAT5A-Mediated SOCS2 Expression Regulates Jak2 and STAT3 Activity Following C-Src Inhibition in Head and Neck Squamous Carcinoma

Published OnlineFirst November 16, 2011; DOI: 10.1158/1078-0432.CCR-11-1889

Clinical Cancer Cancer Therapy: Preclinical Research

STAT5A-Mediated SOCS2 Expression Regulates Jak2 and STAT3 Activity Following c-Src Inhibition in Head and Neck Squamous Carcinoma

Banibrata Sen1, Shaohua Peng1, Denise M. Woods2, Ignacio Wistuba2, Diana Bell2, Adel K. El-Naggar2, Stephen Y. Lai3, and Faye M. Johnson1,4

Abstract Purpose: The inhibition of c-Src results in a striking reduction in cancer cell invasion, but the effect on cell survival is modest. Defining mechanisms that limit apoptosis following c-Src inhibition could result in an ideal therapeutic approach that both inhibits invasion and leads to apoptosis. In this regard, we discovered a novel feedback loop that results in STAT3 reactivation following sustained c-Src inhibition. Here we define the mechanism underlying this feedback loop and examine the effect of inhibiting it in vivo. Experimental Design: We measured levels and activity of pathway components using PCR, Western blotting, and kinase assays following their manipulation using both molecular and pharmacologic approaches. We used a heterotransplant animal model in which human oral squamous cancer is maintained exclusively in vivo. Results: Following c-Src inhibition, STAT5 is durably inhibited. The inhibition of STAT5A, but not STAT5B, subsequently reduces the expression of suppressors of cytokine signaling 2 (SOCS2). SOCS2 inhibits Janus kinase 2 (Jak2) activity and Jak2–STAT3 binding. SOCS2 expression is necessary for STAT3 inhibition by c-Src inhibitors. Overexpression of SOCS2 is adequate to prevent STAT3 reactivation and to enhance the cytotoxic effects of c-Src inhibition. Likewise, the combination of Jak and c-Src inhibitors led to significantly more apoptosis than either agent alone in vivo. Conclusions: To our knowledge, ours is the first study that fully defines the mechanism underlying this feedback loop, in which sustained c-Src inhibition leads to diminished SOCS2 expression via sustained inhibition of STAT5A, allowing activation of Jak2 and STAT3, Jak2–STAT3 binding, and survival signals. Clin Cancer Res; 18(1); 127–39. 2011 AACR.

Introduction reduction in invasion of cancers in vitro and in vivo (2, 3). However, despite c-Src expression and activation in One potential and promising therapeutic cancer target epithelial tumors and c-Src’s robust inhibition by clini- is c-Src, given its well-defined roles in promoting cell cally relevant agents, the effect of c-Src inhibition on migration and metastasis as well as regulating prolifera- epithelial cancer cell survival and proliferation has been tion, survival, and angiogenesis. The Src family kinases modest (3). A clinical trial of the SFK inhibitor dasatinib (SFK) are nonreceptor tyrosine kinases involved in signal as a single agent in head and neck squamous cell carci- transduction in both normal and cancer cells (1). c-Src is noma (HNSCC) did not show significant activity (4). the SFK that is most often implicated in cancer progres- Current treatment for HNSCC includes a combination sion. Inhibition of c-Src results in a nearly universal of cytotoxic chemotherapy, radiotherapy, and surgery. Cetuximab enhances the efficacy of chemotherapy and radiotherapy, but no kinase inhibitors are currently a Authors' Affiliations: Departments of 1Thoracic/Head and Neck Medical Oncology, 2Pathology, and 3Head and Neck Surgery, The University of standard of care for HNSCC. Although invasion is impor- Texas MD Anderson Cancer Center; and 4The University of Texas Graduate tant in the pathophysiology of many cancers, local inva- School of Biomedical Sciences at Houston, Houston, Texas sion is a critical determinant of both morbidity and Note: Supplementary data for this article are available at Clinical Cancer mortality for HNSCC and is associated with worse locor- Research Online (http://clincancerres.aacrjournals.org/). egional control and decreased survival. There is a great Corresponding Author: Faye M. Johnson, Department of Thoracic/Head need to improve systemic therapy to treat both local and Neck Medical Oncology, Unit 432, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. recurrence and distant metastatic disease. Thus, defining Phone: 713-792-6363; Fax: 713-792-1220; E-mail: mechanisms that limit the proapoptotic effects of c-Src [email protected] inhibitors could result in an ideal combination of ther- doi: 10.1158/1078-0432.CCR-11-1889 apeutic agents that both inhibit local invasion and lead to 2011 American Association for Cancer Research. significant cytotoxicity.

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location. In HNSCC cells, Jak inhibition or knockdown Translational Relevance completely and durably blocked both basal activation of STAT3 and subsequent reactivation of STAT3 following Despite c-Src expression in epithelial tumors and the c-Src inhibition (9, 10). Consistent with the effects of c-Src availability of potent inhibitors, the effects of c-Src inhibition on STAT3 activity, c-Src inhibition resulted in inhibition on cancer cell survival have been modest. initial inhibition and then recovery of Jak2 kinase activity, Our current study defines the molecular mechanism that confirming that the reactivation of STAT3 is mediated by Jak underlies a compensatory pathway that ultimately leads reactivation. to STAT3 activation and survival despite sustained c-Src Although there are no known positive feedback loops inhibition; the combination of c-Src and Janus kinase leading to Jak activation after its inhibition, loss of a (Jak) inhibitors leads to significant cancer cell apoptosis negative feedback loop could play such a role. There are in vivo. Our study could have a direct clinical application 3 canonical negative feedback loops that regulate Jak/STAT because previous results show that STAT3 reactivation is function after cytokine signaling: SH-2–containing phos- likely to occur in a broad range of cancers and inhibitors phatases (SHP), which inactivate Jak by dephosphoryla- of c-Src and Jak are well tolerated in humans. Specific tion; protein inhibitors of activated STAT (PIAS), which are STAT3 inhibitors are being developed and may be more negative regulators of STAT transcription downstream; and specific and less toxic than Jak inhibitors. Our long-term suppressors of cytokine signaling (SOCS), which inhibit Jak goal is to use these results to design clinical trials com- kinase activity, facilitate proteasomal degradation of Jak, bining c-Src inhibitors with Jak2 or STAT3 inhibitors to and reduce STATs binding to cytokine receptors (5). improve the survival of patients with cancer. The mechanism by which sustained c-Src inhibition allows Jak reactivation is unknown. We observed changes in Jak activity and Jak–STAT binding following c-Src inhibition that suggest SOCS proteins to be the most likely Because signal transducers and activators of transcription candidates for regulating Jak/STAT function in this setting. (STATs) are known to be c-Src substrates and can mediate Our hypothesis is that the inactivation of STAT5 caused by c-Src’s biologic effects (5), we explored the potential role of sustained c-Src inhibition suppresses the expression of one STATs in modulating the biologic effects of c-Src inhibition. or more of the SOCS proteins. This loss allows recovery of The STAT family of transcription factors, especially STAT3 Jak2–STAT3 binding and Jak2 kinase activity and relieves and STAT5, regulates oncogenic signaling in many different STAT3 inhibition, thereby reactivating proliferative signals tumor types. In HNSCC cells, c-Src’s inhibition results in through Jak2 and STAT3. In addition, the 2 STAT5 isoforms reduced STAT3 and STAT5 activation and reduced cell (A and B) are known to have distinct roles in cancer and in proliferation (6). Correspondingly, inhibition of STAT3 in embryonic development, but the roles of these isoforms in HNSCC leads to increased apoptosis, decreased prolifera- this feedback loop have never been explored (5, 11). Under- tion, and decreased tumor size (7, 8). However, we found standing the basis for STAT3 reactivation is essential to that whereas inhibition of c-Src led to durable inhibition of maximizing the proapoptotic effect of c-Src inhibitors. STAT5, c-Src’s inhibition of STAT3 was only transient, with To test our hypothesis, we measured the levels of all levels of phospho-STAT3 (pSTAT3, Y705) returning to known SOCS family members following c-Src knockdown baseline or above by 7 hours. We confirmed this finding or inhibition with the ATP-competitive SFK inhibitor, dasa- by reducing c-Src specifically with siRNAs (siRNAs) and by tinib, and found that SOCS2 expression was consistently measuring STAT3 activity using DNA-binding and tran- decreased. To further define this novel feedback loop, we scriptional activity assays (9). We also established the bio- manipulated the levels of SOCS2, STAT3, STAT5A, and logic importance of this feedback loop by showing that STAT5B to show that c-Src inhibition leads to STAT5 inac- abrogation of STAT3 reactivation enhanced the cytotoxicity, tivation, that STAT5A drives SOCS2 protein expression, and cell-cycle arrest, and apoptosis caused by c-Src inhibition in that SOCS2 inhibits Jak2–STAT3 binding, Jak activity, and vitro. These findings established that the STAT3 compensa- STAT3 activation. We previously showed that c-Src inhibitory pathway is important for maintaining cancer cell tion did not affect total levels of Jak2 protein (9). Moreover, proliferation and survival after sustained c-Src inhibition. SOCS2 loss caused increased resistance to dasatinib, and Furthermore, the depletion of STAT3 by an siRNA reduced SOCS2 overexpression led to increased sensitivity to c-Src the 50% inhibitory concentration (IC50) of the c-Src inhib- inhibitors. We confirmed the biologic importance of this itor dasatinib from 23 to 4 nmol/L, increasing sensitivity to feedback pathway using a heterotransplant model of levels comparable with those observed after inhibition of HNSCC and clinically relevant inhibitors of Jak and c-Src. Bcr-Abl in leukemia. In addition to regulation by c-Src, STAT3 can be activated Materials and Methods by the nonreceptor tyrosine kinases Jaks. Following activation, Jak molecules phosphorylate cytokine receptors, thus Cells and reagents allowing the binding of the monomeric inactive STATs Dasatinib was purchased from Selleck Chemicals and the present in the cytoplasm. STATs then become Jak substrates clinical pharmacy. INCB16562 was provided by Incyte and the pSTATs undergo dimerization and nuclear trans- Corporation. Both were prepared as 10 mmol/L stock

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solutions in DMSO. Antibodies used included c-Src, pSFK amer, 500 mmol/L deoxyribonucleotide triphosphates, 100 (Y416), pSTAT3 (Y705), pJak2 (Y221), pJak2 (Y1007/ mg/mL BSA, and 1.5 mL Moloney murine leukemia virus 1008), pSTAT5 (Y694) XP, and SOCS2 (Cell Signaling (MMLV) reverse transcriptase enzyme. The final reaction Technology); total phosphotyrosine and total STAT5B volume was 20 mL. The reaction mixture was incubated at (Upstate Biotechnology); SOCS1 and total Jak2 (BD Bios- 42C for 2 hours, and the reaction was terminated by ciences); total STAT5A (Abcam); and b-actin (Sigma heating the mixture at 99C for 5 minutes and cooling it Chemical). at 5C for 5 minutes. Human HNSCC cell lines were obtained from Dr. Jeffrey The level of mRNA for the SOCS genes was measured with Myers and maintained as described previously (9). All cell SYBR green–based real-time PCR in triplicate. The primers lines were validated by cross-comparing their allelic short were designed by using Primer Express (Applied Biosystems; tandem repeat profiling (Johns Hopkins Fragment Analysis Supplementary Table S1). Each cDNA sample was amplified Core Facility) and patterns generated with the PowerPlex by using SYBR Green PCR Master Mix (Applied Biosystems) 1.2 platform (Promega) to those from the American Type according to the manufacturer’s suggested protocol. The Culture Collection repository database. PCR products and their dissociation curves were detected using the ABI Prism 7500 fast real-time PCR system. The Western blot analysis and immunoprecipitation level of the housekeeping gene L32 ribosomal gene (Rpl32) Western blot analysis and immunoprecipitation were was used as an internal control. Individual data sets were carried out as previously described (3, 9). Briefly, for immu- normalized with control vehicle-treated cells; absolute noprecipitation, cells were lysed and equal amounts of quantities were normalized with L32 as internal control. protein cell lysates (800 mg) were precleared with protein A–G-sepharose beads (Invitrogen) for 1 hour. The pre- In vitro kinase assay cleared lysate was incubated with 5 mg agarose-conjugated Purified recombinant Jak2 (Abcam) and SOCS2 primary antibody overnight. The immunocomplexes were (Abnova) proteins were incubated at a 1:1 molar stoichio- washed and resolved by SDS-PAGE. Following transfer to metric ratio with 15 mCi [g-32P]ATP (3,000 Ci/mmol), and nitrocellulose membranes, immunoblots were probed with kinase activity was assayed as described previously (9). primary antibody and proteins detected with horseradish peroxidase–conjugated secondary antibody (Bio-Rad Lab- Xenograft nude mouse models oratories) and enhanced chemiluminescent reagent (Pierce All animal procedures were in accordance with the pol- Biotechnology). icies of MD Anderson’s Institutional Animal Care and Use Committee. For the orthotopic models, the tongues of five Cytotoxicity assay 6-week-old female Swiss nu/nu mice were injected with 5 MTT assay was used to assess cytotoxicity as previously 105 Osc19 cells. For the heterotransplant studies, residual described (3). Eight wells were treated for each experimental tumor from a patient with untreated oral squamous carci- condition. noma was identified by a head and neck pathologist (A. El- Nagar) at the time of surgical resection and implanted Transfection with siRNA and recombinant plasmids directly into the flank of a nude mouse. The resulting tumor The siRNAs were predesigned sets of 4 independent was divided and transplanted into subsequent mice until 40 sequences (siGENOME SMARTpool, Dharmacon). Con- fifth-generation tumors were produced. The heterotrans- trols included cells that were mock transfected (no siRNA) plant tumors were never cultured in vitro. Dasatinib (20 mg/ and those transfected with a nontargeting (scrambled) kg), INCB16562 (60 mg/kg), both, or vehicle was admin- siRNA. The pUSE STAT5A 16, pUSE STAT5B 16 recom- istered by oral gavage daily for 7 days (orthotopic) or 17 binant plasmids, and pMet7-FLAG-mSOCS2 constructs days (heterotransplant). Mice were killed 2 hours after the were used to achieve overexpression of STAT5A and/or last drug dose, tumors were dissected, and the mice were STAT5B and mouse full-length SOCS2, respectively, in cells. examined for distant metastases. The tumors were homog- Mouse SOCS2 shows 94% identity and 95% amino acid enized and subjected to Western blot analysis as described sequence similarity with human SOCS2. Cells were har- previously (12). vested, washed, and suspended (106/100 mL) in Nucleo- fector V solution (Lonza Group). siRNA (200 pmol/100 Immunohistochemical analysis mL), DNA (5 mg/100 mL), or controls were added and Immunohistochemical staining was carried out as pre- electroporated using the U-31 Nucleofector program viously described using the following specific conditions: (Lonza) as described previously (9). antigen retrieval was carried out using a Dako-Target retrieval at pH 6.0 for proliferating cell nuclear antigen Quantitative PCR (PCNA),CD31(Abcam),andpSFK (Y416; Cell Signaling Total RNA was isolated from cells that had been either Technology). Peroxide blocking was carried out using 3% transfected with siRNAs or incubated with dasatinib (as methanol and hydrogen peroxide (PCNA and CD31) or indicated in the figure legends) by using an RNeasy mini kit 3% water and hydrogen peroxide (pSFK Y416; ref. 13). (Qiagen). Total RNA (2 mg) was converted into cDNA using Primary antibody dilutions were: PCNA (1:100), CD31 1 MMLV buffer, 1 mL RNasin, 10 mmol/L random hex- (1:50), and pSFK (1:50). Slides were examined by a

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blinded observer for the intensity and extent of immu- experiments show that c-Src activation is upstream of nostaining by light microscopy using a 20 magnifica- SOCS2 transcription. tion objective. Nuclear PCNA expression was quantified Given that STAT5 can regulate SOCS2 expression, we using a 3-value intensity: 0, none; low (1þ,weakand2þ, investigated whether c-Src could regulate STAT5 activation moderate); and high (3þ,strongandþ4verystrong). in HNSCC cell lines. We incubated cells with dasatinib for 7 CD31-positive vessels were counted in 5 high-powered hours and measured pSTAT5 (Y694). c-Src inhibition ren- fields by a blinded observer. dered STAT5 durably inactive which is consistent with our previous results showing STAT5 inhibition from 2 to 24 TUNEL assay hours following dasatinib treatment (Fig. 1F; ref. 3). Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining was carried out using SOCS2 expression is regulated by STAT5A but not the DeadEnd Colorimetric TUNEL system from Promega STAT3 or STAT5B per the manufacturer’s instructions as previously described Previous reports showed that STAT5 can act as a tran- (13). TUNEL-positive nuclei were counted for each repre- scriptional regulator for SOCS family proteins in hemato- sentative treatment group. poietic cells (14, 15). We sought to determine whether the modulation of STAT5 activity regulates SOCS2 expression Statistical methods in HNSCC cells. HNSCC cell lines express both isoforms of All experiments in which error bars and P values are STAT5 (STAT5A and STAT5B) and their roles may be distinct provided were done in at least triplicate. The Student t test (11). Likewise, we found that selective STAT5A knockdown was used to determine if the mean values of these contin- using siRNA led to a considerable decrease in SOCS2 uous variables were different in the various treatment expression, whereas STAT5B depletion alone had little effect groups. on SOCS2 expression (Fig. 2A). In contrast, selective STAT3 depletion with siRNA did not affect SOCS2 expression Results (Fig. 2B). To further elucidate the function of the STAT5 isoforms in c-Src inhibition leads to decreased SOCS2 expression the regulation of SOCS2 expression and STAT3 activation, and STAT5 inactivation we selectively overexpressed constitutively active forms of We postulated that the loss of one of the SOCS proteins both STAT5 isoforms. STAT5A activation led to increased could contribute to STAT3 reactivation after sustained c-Src expression of SOCS2 but not SOCS1 (Fig. 2C). Likewise, inhibition. To test this hypothesis, we determined the STAT5A overexpression resulted in decreased activation of expression level of all members of the SOCS family after STAT3, thus supporting our hypothesis that STAT5A reg- 7 hours of c-Src inhibition with dasatinib using quantitative ulates SOCS2 expression, which subsequently acts as a PCR analysis in a panel of 6 different HNSCC cell lines negative regulator of STAT3 activation. In contrast, STAT5B (Fig. 1A). Among the 8 family members of SOCS proteins, overexpression alone did not significantly alter basal SOCS2 only SOCS2 showed consistent downregulation in all 6 cell protein levels or pSTAT3 (Y705) expression. lines. We also measured the expression of the 4 PIAS family members but found no significant alteration in PIAS expres- Selective knockdown of SOCS2 leads to STAT3 sion following dasatinib treatment (representative data, activation Supplementary Fig. S1). STAT3 reactivation was not medi- To determine whether SOCS2 downregulation could lead ated by an autocrine mechanism such as cytokine release to STAT3 activation, we selectively decreased SOCS2 expres- (Supplementary Fig. S2). sion in HNSCC cell lines using siRNA. Upon SOCS2 knock- To characterize the effect of c-Src inhibition on SOCS2 down, STAT3 phosphorylation increased markedly by 4.6- protein expression, we examined the effect of dasatinib in 2 and 4.8-fold in TU167 and Osc19 cell lines, respectively, representative HNSCC cell lines, that grow well both in vitro over that in control cells (Fig. 3A). This result supports our and in vivo, using Western blot analysis (Fig. 1B). As hypothesis that SOCS2 has a negative regulatory role in the expected, c-Src phosphorylation was rapidly and durably Jak2–STAT3 signaling pathway. Total Jak2 protein levels inhibited at a site associated with its activation (pSFK, were also increased by SOCS2 knockdown, a result consis- Y416). SOCS2 protein expression was significantly down- tent with the known role of SOCS in promoting Jak protein regulated after sustained c-Src inhibition. degradation. In our previous work, however, we did not To determine whether SOCS2 expression is downstream observe changes in total Jak2 levels following dasatinib of c-Src specifically, we transfected HNSCC cells with siR- treatment or c-Src knockdown (9). NAs specific to c-Src and examined the effect on SOCS family members’ mRNA (Fig. 1C and D) and protein (Fig. SOCS2 depletion results in sustained STAT3 activation 1E) expression. Upon c-Src depletion, the levels of SOCS2 despite acute c-Src inhibition mRNA and protein decreased significantly. In addition to Our previous experiments have shown that acute c-Src SOCS2, CIS1 expression was decreased following c-Src inhibition results in transient STAT3 inactivation (10). knockdown (Fig. 1C and D), but CIS1 was not consistently We hypothesized that early SOCS2 depletion would allow affected by incubation with dasatinib (Fig. 1A). These STAT3 to remain activated despite acute c-Src inhibition.

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STAT5A Mediates SOCS2 Expression and Jak2/STAT3 Activity

AB# 3 TU167 TR146 HN5 UMSCC14a Osc19 2 TU138

† 1 * Expression (fold control) (fold Expression

CS6 IS1 OCS4 C SOCS1 SOCS2 SOCS3 S SOCS5 SO SOCS7

C D Control Control TU167 Scrambled siRNA Scrambled siRNA 1.5 Src siRNA 1.5 Osc19 Src siRNA * * * 1 * 1

0.5 0.5 Fold control Fold control

0 0 6 7 5 S S S2 S3 S S6 S7 IS1 CS5 C C C C C C CIS1 OC O O O O S SO SOCS4 SOC S SO SOCS2 SOCS3 SOCS4 S S S

Figure 1. c-Src inhibition leads to decreased SOCS2 expression and STAT5 activation. A, six HNSCC cell lines were incubated with 100 nmol/L dasatinib for 7 hours, and mRNA levels of all the known SOCS molecules were measured by quantitative PCR and expressed as fold control (vehicle treatment). #, 6.2-fold control; †, SOCS5 was not detectable in 4 cell lines; , P < 0.05. B, TU167 and Osc19 cell lines were incubated with 100 nmol/L dasatinib for the indicated times, lysed, and analyzed by Western blot analysis with the indicated antibodies. C–E, TU167 and Osc19 cells were transfected with nontargeting siRNA or c-Src– speciﬁc siRNA. The mRNA levels of all the SOCS molecules were measured by quantitative PCR analysis (C and D) or Western blot analysis (E). F, TU167 and Osc19 cell lines were treated with 100 nmol/L dasatinib for 7 hours, and lysates were subjected to immunoprecipitation with the total STAT5 antibody. Immunoprecipitates were resolved on 8% SDS-PAGE and blotted with the pSTAT5 (Y694) monoclonal antibody. The data in A, C, and D are presented as an average of 3 replicates; bars indicate the SD. , P < 0.05. Con, control; KD, knockdown.

To test this hypothesis, we examined the effect of dasa- phosphorylation 30 minutes after treatment (SOCS2 tinib on STAT3 reactivation in cells with depleted SOCS2. levels were not affected at this early time point). In As we showed previously, TU167 cells incubated with contrast, SOCS2-depleted TU167 cells had incomplete dasatinib showed signiﬁcant downregulation of STAT3 inhibition of STAT3 phosphorylation at 30 minutes after

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Figure 2. STAT5A regulates SOCS2 expression in HNSCC cells. A, TU167 and Osc19 cells were transfected with nontargeting scrambled siRNA or STAT5A- or STAT5B-specific siRNA either alone or in combination (KD, knockdown). Cells were harvested and lysed after 72 hours of transfection, and the indicated molecules were visualized using Western blot analysis. SOCS1 and SOCS2 expression was normalized with that of b-actin (bar graph). B, TU167 cells were transfected with STAT3-specific siRNA and analyzed by Western blot analysis 72 hours later. C, TU167 and Osc19 cells were transiently transfected with empty vector, CA-STAT5A or CA-STAT5B (OE, overexpression), or both. Cells were harvested and lysed after 72 hours of transfection, and the indicated molecules were visualized using Western blot analysis. The terms lighter and darker refer to film exposure. SOCS1 and SOCS2 expression was normalized with that of b-actin (bar graph). , P < 0.05.

dasatinib treatment (Fig. 3B). This result shows that SOCS2 expression mediates sensitivity and resistance SOCS2 expression is required for STAT3 inhibition by to c-Src inhibition c-Src. In contrast, STAT5 was inhibited by dasatinib To determine the biologic significance of SOCS2 in independently of SOCS2 expression. this feedback loop, we transiently overexpressed or knocked down SOCS2 and estimated cytotoxicity in the SOCS2 overexpression leads to STAT3 inhibition presence of the c-Src inhibitor dasatinib (Fig. 4). SOCS2 To further explore the role of SOCS2 as a negative knockdown led to increased resistance to dasatinib in regulator of STAT3, we transiently overexpressed SOCS2, both HNSCC cell lines compared with results in controls which resulted in significant sustained decreases in both (Fig. 4A and C). In contrast, overexpression of SOCS2 in STAT3 and Jak2 activation while leaving total STAT3, either line led to increased sensitivity to c-Src inhibition SOCS1, and pSFK levels unchanged (Fig. 3C). To determine (Fig. 4B and D). The basal differences in dasatinib the effect of forced SOCS2 expression following sustained c- sensitivity between Osc19 and TU167 cells are likely Src inhibition, we transfected Osc19 and TU167 cells with because of distinct interactions between c-Src and c-Met either SOCS2 or empty vector and exposed them to dasa- (16). tinib for 30 minutes to 7 hours (Fig. 3D and E). The over- Although the manipulation of SOCS2 expression expression of SOCS2 significantly diminished the basal affected sensitivity to c-Src inhibition in a predictable activation and reactivation of STAT3 compared with manner, we were concerned that the biologic effects of controls. STAT5 modulation might not parallel what we observed

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Figure 3. SOCS2 expression negatively regulates STAT3 activation. A and B, TU167 and Osc19 cells were mock-transfected with no siRNA, nontargeting (scrambled), or SOCS2- speciﬁc siRNA (KD, knockdown). Cells were subjected to no further treatment (A) or incubated with 100 nmol/L dasatinib for 30 minutes before lysis (B) and then harvested and lysed. The indicated molecules were visualized using Western blot analysis. C, TU167 and Osc19 cell lines were transiently transfected with the pMet7-FLAG- mSOCS2 overexpression construct (SOCS2 OE) or vector control. Cells were harvested and lysed 72 hours after transfection, and the indicated molecules were analyzed by Western blot analysis.

with direct SOCS2 manipulation, because STAT5 itself SOCS2 inhibits Jak2–STAT3 binding and Jak2 kinase can promote cancer cell survival and proliferation in activity HNSCC (17). We transfected cells with constitutively Previous reports have shown that SOCS family members active STAT5A or B or both and then measured cytotox- bind to Jaks and inhibit their kinase activity, as well as icity in the presence of dasatinib. HNSCC cells that compete with STAT molecules for recruitment to the recep- overexpressed STAT5A (and had increased SOCS2 and tor complex (18). To determine whether SOCS2 affects decreased STAT3 activation, Fig. 2C) were slightly more Jak2–STAT3 binding in HNSCC cells, we overexpressed sensitive to dasatinib. However, those cells overexpres- SOCS2 in TU167 cells and immunoprecipitated total Jak2; sing STAT5B (no change in SOCS2) or both isoforms immunocomplexes were analyzed by immunoblotting (Fig. (increased SOCS2) were more resistant to dasatinib 5A and Supplementary Fig. S4). When SOCS2 was over- (Supplementary Fig. S3A), suggesting that STAT5B pro- expressed, Jak2–STAT3 binding was significantly decreased. motes cancer survival through an independent mecha- To determine whether SOCS2 can directly affect Jak2 nism. In TU167 cells, STAT5A and B knockdown led to activity, we carried out an in vitro kinase assay in which a modest increase in sensitivity to dasatinib, whereas purified Jak2 and SOCS2 proteins were incubated together in Osc19 cells, this observation was reversed (Supple- at a 1:1 molar stoichiometric ratio with ATP; we detected mentary Fig. S3B). Because dasatinib causes STAT5 phosphorylated molecules by autoradiography (Fig. 5B). In inhibition, it is not surprising that STAT5 knockdown the presence of SOCS2, Jak2 autophosphorylation and does not have a striking effect on dasatinib-induced activity toward an exogenous substrate (enolase) were sig- cytotoxicity. nificantly inhibited. As expected, SOCS2 alone showed no

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Figure 3. (Continued) TU167 (D) and Osc19 (E) cells were transfected with SOCS2 OE. Cells were treated with 100 nmol/L dasatinib for the indicated times starting 48 hours after transfection, lysed, and analyzed by Western blot analysis.

kinase activity. These observations confirm that SOCS2 acts tumor was transplanted directly into a mouse. The resulting as a negative regulator of Jak2–STAT3 signaling by inhibit- tumor was divided and serially passaged into mice; the ing Jak2 activity as well as Jak2–STAT3 binding. tumors were never cultured in vitro. The resulting tumors maintained the histologic characteristics of the primary Jak inhibition enhances the antitumor effects of c-Src tumor from which they were derived (Supplementary Fig. inhibition in vivo S5). Heterotransplants maintain the gene expression pro- To determine whether the reactivation of STAT3 is bio- files of the original tumors and their pattern of response to logically significant in vivo, we used a heterotransplant chemotherapy resembles those observed in the clinic (19), model of HNSCC in which an oral squamous carcinoma suggesting that this model may be superior to other

Figure 4. SOCS2 expression levels affect sensitivity to c-Src inhibition. TU167 (A and B) and Osc19 (C and D) cells were mock-transfected (no siRNA) or transfected with either nontargeting siRNA or SOCS2- speciﬁc siRNA (A and C), empty vector, or pMet7-FLAG-mSOCS2 overexpression construct (B and D). Twenty-four hours after transfection, cells were seeded in 96-well plates for the MTT assay. Cell viability was assayed in the presence of increasing concentrations of dasatinib for 48 hours (B and D) or 72 hours (A and C). Data are presented as the average of 8 replicates; bars indicate the SD. KD, knockdown; O.D, optical density; OE, overexpressed.

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Discussion

Our current findings define the mechanism underlying a novel feedback loop in which sustained c-Src inhibition or knockdown leads to diminished SOCS2 expression via the sustained inhibition of STAT5A. This relieves the negative constitutive inhibition of SOCS2 on the Jak2–STAT3 pathway, specifically allowing the activation of Jak2 kinase activity, Jak2–STAT3 binding, and STAT3 activation. Although SOCS2 can affect Jak2 protein levels by promoting protein degradation, in our previous studies we observed no changes in total Jak2 expression following c- Src inhibition or knockdown. Ultimately, the loss of SOCS2 expression leads to the reactivation of proliferative signals through STAT3 despite sustained c-Src inhibition (Supple- mentary Fig. S7). Although it is well established that SOCS proteins can inhibit Jak/STAT function, we are aware of only one other study in which altered signaling led to the loss of SOCS function with subsequent Jak/STAT activation and cancer – Figure 5. SOCS2 regulates Jak2 STAT3 binding and Jak2 kinase activity. promotion (20). Jak1 activation is important for v-Abl- A, lysates from control and SOCS2-overexpressing cells were immunoprecipitated (IP) with Jak2 antibodies, and immunoprecipitated induced transformation of pre-B cells. In nontransformed complexes were resolved on SDS-PAGE. The indicated molecules were cells, the induction of SOCS1 acts as a negative feedback visualized using Western blot analysis. B, purified recombinant SOCS2 loop to suppress Jak/STAT function, but v-Abl phosphor- and Jak2 proteins were incubated at room temperature in the presence of ylates SOCS1 and inhibits its targeting of Jak1 for degrada- 15 mCi g32-ATP and the exogenous substrate enolase for 30 minutes. Reactions were terminated with the addition of sample buffer, boiled for 5 tion. Thus, v-Abl’s inhibition of SOCS1 allows sustained minutes, and separated on an 8% SDS-PAGE. Radiolabeled proteins Jak1 and STAT5 activation, contributing to cytokine inde- were detected by autoradiography. OE, overexpressed. pendence in the transformed cells. Our study showed a distinct role for a SOCS protein in regulating Jak/STAT function; in HNSCC, SOCS2 was regulated at the transcrip- tional level and not by posttranslational modification and xenograft approaches for therapeutic studies. Both dasa- degradation. tinib and the Jak inhibitor INCB16562 modestly inhib- SOCS proteins have been most extensively studied in ited tumor growth; the combination was significantly normal immune function and hematologic malignancies, more effective than the single agents (Fig. 6A). Likewise, where they function as classic mediators of a negative the tumors treated with the combination had significantly feedback loop downstream of cytokine receptors (21). more apoptosis (Fig. 6B) and less proliferation (Fig. 6C The roles of SOCS proteins in epithelial cancers are not as and Supplementary Table S2). Consistent with our in vitro well known, although studies support a tumor-suppressor results, c-Src inhibition did not result in STAT3 inhibi- role for SOCS proteins via Jak/STAT suppression in non- tion, but Jak inhibition abrogated STAT3 activation (Fig. hematologic malignancies. In this context, SOCS1 and 6D); c-Src was inhibited in vivo by dasatinib (Fig. 6E). SOCS3 are the most extensively studied, although the loss Tumor microvessels were stained with CD31 and of SOCS2 can promote intestinal growth, polyp forma- counted; the tumors from mice treated with dasatinib, tion, and colon cancer progression (22–24). The expres- INCB16562, and the combination had lower microvessel sion of SOCS1, which is downregulated via methylation density than controls (0.68-, 0.65-, and 0.64-fold control in about a third of HNSCC tumors, can inhibit STAT3 levels, respectively), but the differences were not statisti- activation by Jak in HNSCC cell lines (25). In those cell cally significant (P values of 0.12, 0.11, and 0.06, lines with SOCS1 expression, STAT3 was shown to be respectively). activated via epidermal growth factor receptor (EGFR); in We also used an orthotopic HNSCC model in which those lines lacking SOCS1, STAT3 was activated via inter- Osc19 cells were implanted into the tongue. Mice were leukin-6 and Jak. The effects of SOCS1 on STAT5 were not treated with dasatinib or INCB16562 or the combination examined (25). SOCS3 is commonly hypermethylated for 7 days. Tumors consisted primarily of HNSCC cells and downregulated in HNSCC tumors; its overexpression (>90%) with no distant metastases. As expected, dasatinib in HNSCC cell lines leads to apoptosis (26). SOCS3 is treatment inhibited c-Src, and STAT3 remained activated also hypermethylated in lung cancer cell lines and tissues (1.7-fold) over the control level. In the presence of (27). In melanoma, the SOCS1 expression was decreased INCB16562, pSTAT3 reactivation upon dasatinib treatment and STAT3 and Jak2 expression increased compared with was significantly reduced to 0.2-fold (Supplementary primary tumor cells. Restoration of SOCS1 expression Fig. S6). leads to STAT3 inactivation and inhibition of brain

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Sen et al.

A 3.0 B Control Dasatinib 50 * 2.5 INCB16562 Combination 40

2.0 30 20 * 1.5 10

1.0 0 TUNEL-positive cells (% total) Tumor size (fold baseline) Control Dasatinib INCB16562 Combination 0.5 Control Dasatinib INCB16562 Combination 0 6 12 18 Time (D) Percent decrease in tumor size (vs. control) Dasatinib 37.29 INCB16562 32.75 Combination 47.59 C D E

60 High Intermediate Low *

40 * Control Dasatinib 20 (% of total) *

PCNA staining intensity 0 Control Dasatinib INCB16562 Combination INCB16562 Combination Control Dasatinib INCB16562 Combination

High Intermediate Low

Figure 6. The combination of SFK and Jak inhibition resulted in increased cytotoxic effects in vivo. A, mice bearing heterotransplanted oral squamous carcinoma were treated with vehicle alone, dasatinib alone, INCB16562 alone, or the 2 agents combined, and tumor size was measured on the indicated days. B, TUNEL assay was carried out with heterotransplanted tumor tissues, and TUNEL-positive nuclei were counted and calculated as the percentage of total nuclei. C, tumor tissues were stained for PCNA. Nuclear staining was classiﬁed as low, intermediate, or high grade. D and E, tumors from treated mice were subjected to Western blot analysis with the indicated antibodies (D) or immunohistochemical staining with an anti-pSFK (Y416) Ab (E). , P 0.05.

metastasis (28). Similarly, exogenous expression of html; ref. 31). STAT5A and STAT5B share similar binding SOCS1, SOCS3, or SOCS5 in thyroid cancer cells reduces sequences (31). Given the high level of homology between STAT3 phosphorylation and sensitizes cells to chemo- STAT5A and STAT5B, it is not clear how the 2 isoforms could therapy in vitro and in vivo (29). be differentially regulating SOCS2 expression based exclu- In our experiments, SOCS2 had a function distinct from sively on sequence data. Another layer of complexity in the its classically understood role described in hematopoietic regulation of SOCS function is that SOCS2 may compete cells (21). SOCS2 did inhibit Jak2 kinase activity but does with or regulate other SOCS proteins. SOCS2 can lead to not contain the classic kinase inhibitory region that SOCS1 proteasome-dependent SOCS3 degradation (32). Such a and SOCS3 proteins possess (21). However, our study was complex system of interregulation may explain why we limited in that we used isolated recombinant proteins that observed diverse effects on the levels of multiple SOCS may function differently from native proteins in an intact proteins in HNSCC cell lines following c-Src inhibition. cell. SOCS2 also is classically understood to promote the Although STAT5A and STAT5B may possess some func- degradation of Jak2, yet we did not observe changes in total tional redundancy, their roles in both normal physiology STAT3 or Jak2 levels in HNSCC cells following prolonged c- and cancer biology are distinct. Their separate roles in Src inhibition or knockdown (9). However, we did observe normal physiology are shown by discrete tissue expression that SOCS2 knockdown led to increased Jak2 expression, patterns, distinct phenotypes of the knockout mice, and showing that SOCS2 is capable of this classical function in different roles in cell signaling [reviewed in (5)]. STAT5 has HNSCC cells. been studied in multiple cancer types, but the distinction SOCS2 expression is dependent upon STAT5 (30). between STAT5A and STAT5B has been examined only There are at least 5 STAT5A-binding sites in the SOCS2 infrequently in epithelial tumors (11, 33, 34). STAT5A and promoter (intron 1; www.cbrc.jp/research/db/TFSEARCH. STAT5B have differential regulatory roles in HNSCC, breast

136 Clin Cancer Res; 18(1) January 1, 2012 Clinical Cancer Research

STAT5A Mediates SOCS2 Expression and Jak2/STAT3 Activity

cancer, glioblastoma, and hepatocellular carcinoma (35– soluble growth factor or cytokine in this feedback loop 39). In HNSCC, STAT5 activation led to increased cell and (ref. 10 and Supplementary Fig. S2) and our previous work tumor growth and increased invasion and induced epithe- did not support the role for the kinase activity of a growth lial-to-mesenchymal transition (17). Activated and total factor receptor (10), these experiments do not preclude the STAT5B, but not STAT5A, was found to increase in HNSCC role of such a receptor as a scaffold for the complex. Future tumors compared with normal-appearing mucosa. Like- studies will be needed to address these issues. wise, in a xenograft model of HNSCC, STAT5B antisense Our study could have a direct clinical application. We was found to inhibit tumor growth in mice, whereas have found STAT3 reactivation in cell lines from lung STAT5A antisense did not affect tumor size (40). Cells cancer, mesothelioma, and squamous carcinoma of the containing a dominant-negative STAT5B construct fail to skin (10, 51). We have also observed STAT3 reactivation proliferate in vitro (11). Erythropoietin mediates invasion in in vivo, after specific c-Src knockdown and using 3 different HNSCC through the activation of STAT5A; STAT5A did not pharmacologic inhibitors (9, 10, 12); the combination of c- promote tumor proliferation (41). These studies support a Src and Jak inhibitors leads to significant cancer cell apo- role for STAT5B, but not STAT5A, in the progression of ptosis in vivo. The reciprocal regulation of c-Src and STAT3 HNSCC. Although we did not study the differential roles of activation in tumors from patients with lung cancer suggests STAT5A and STAT5B in HNSCC cells with unperturbed c- that this pathway functions in human tumors (12). These Src, our model would support a role for STAT5A as a tumor results show that STAT3 reactivation is likely to occur in suppressor (by driving SOCS2 expression and subsequent patients with a broad range of cancers that are treated with suppression of Jak2–STAT3 activation). Also consistent any c-Src inhibitor. Specific and potent kinase inhibitors of with the finding that STAT5B promotes HNSCC cancer c-Src and Jak are well tolerated in humans (1, 5). Specific progression, we found that activation of STAT5B resulted SOCS mimetics are being developed and may be more in resistance to c-Src inhibition (Supplementary Fig. S3A). specific and presumably less toxic than Jak inhibitors Although STAT5 contributes to the progression of HNSCC, (52). STAT3 inhibitors also are being developed, but none activation of STAT5 correlates with improved survival in have completed clinical trials (5). breast cancer, where it may promote differentiation rather Despite the finding of c-Src expression in epithelial than progression (36, 42). tumors and the availability of agents to sustain its inhibi- Our study has shown that STAT3 and STAT5 are regulated tion, the effects of c-Src inhibition on cell survival and independently. STAT5 activity was predominantly depen- proliferation have been moderate and inconsistent. c-Src dent upon c-Src, as the reactivation of Jak activity did not mediates its effects on cancer cell survival and proliferation result in STAT5 reactivation. In contrast, STAT3 activation via diverse substrates including STATs. We have discovered a was predominantly Jak dependent, as STAT3 was reactivated heretofore unknown compensatory pathway culminating in the presence of c-Src inhibition (9). Moreover, acute c-Src in STAT3 reactivation and cancer cell survival. Our long- inhibition alone did not result in complete STAT3 inhibi- term goal is to use these results to design clinical trials tion unless SOCS2 was present (Fig. 3B). Jaks are the classic combining these or other more specific c-Src inhibitors with regulators of STAT5 and STAT3, but they are not the only Jak2 or STAT3 inhibitors or SOCS mimetics to improve the kinases that can do so. ErbB receptor-induced activation of survival of patients with HNSCC and other cancers. STAT1, STAT3, and STAT5 was found to be mediated by c- Src and independent of Jak (43). Likewise, c-Src can directly Disclosure of Potential Conflicts of Interest phosphorylate STAT5A (Y694) and activate STAT3 (44–46). c-Src can activate STAT5B directly by phosphorylation No potential conflicts of interest were disclosed. or indirectly by phosphorylating EGFR (Y845; ref. 47). In Acknowledgments HNSCC specifically, c-Src inhibition using both molecular and pharmacologic agents leads to STAT3 and STAT5 inhi- The vector control plasmid, pUSEamp(þ), and plasmids carrying the bition downstream of EGFR (6). EGFR possesses a STAT- activated mutants of STAT5A and STAT5B (pUSE STAT5A 16 and pUSE STAT5B 16 plasmids) were kind gifts from Dr. Jennifer Grandis. Mouse full- binding capacity and can activate STATs in a Jak-indepen- length SOCS2 construct (pMet7-FLAG-mSOCS2) was a kind gift from Dr. dent manner (48, 49). EGFR, though an important medi- Julie Piessevaux. Drs. Jeffrey Myers and Gary Gallick provided technical ator of both c-Src and STAT3 activation in HNSCC, does not assistance with cell line authentication and in vitro kinase assays, respectively. The authors thank Brenda Robinson and Michael Worley for editorial function in STAT3 reactivation following sustained c-Src assistance. inhibition (10). The functions of Jaks, c-Srcs, and growth factor receptors are not independent, as they can cooperate Grant Support to enhance STAT3 activation during oncogenesis (47, 50). One unanswered question is what mechanism leads to This study was supported by R01-CA143369-01 (F.M. Johnson). S.Y. Lai was supported by NIH Mentored Career Development Award K08 Jak kinase inhibition. Our previous studies showed that DE018061. c-Src inhibition led to a rapid and significant inhibition of The costs of publication of this article were defrayed in part by the payment Jak kinase activity (9). However, Jak is not a known c-Src of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. substrate. Another unresolved issue is the potential role for a cytokine or growth factor receptor as a scaffold for the Jak2/ Received July 21, 2011; revised November 4, 2011; accepted November 4, STAT3/SOCS2 complex. Although there is no role for a 2011; published OnlineFirst November 16, 2011.

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STAT5A-Mediated SOCS2 Expression Regulates Jak2 and STAT3 Activity Following c-Src Inhibition in Head and Neck Squamous Carcinoma

Banibrata Sen, Shaohua Peng, Denise M. Woods, et al.

Clin Cancer Res 2012;18:127-139. Published OnlineFirst November 16, 2011.

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