Essential Role of Stat3 in PI3K-Induced Oncogenic Transformation

Essential role of Stat3 in PI3K-induced oncogenic transformation

Jonathan R. Harta, Lujian Liaob, John R. Yates IIIb, and Peter K. Vogta,1

aDepartment of Molecular and Experimental Medicine and bDepartment of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037

Contributed by Peter K. Vogt, June 28, 2011 (sent for review March 2, 2011) Cells transformed by the p110α-H1047R mutant of PI3K show in- generate a positive autocrine feedback leading to activation of creased tyrosine phosphorylation of Stat3. This activation of Stat3 Stat3 (16). is important for the transformation process, because a dominant- The PI3K signaling pathway is part of the core regulatory negative mutant of Stat3 interferes with PI3K-induced oncogene- networks in the cell and affects virtually all cellular activities, sis. GDC-0941, a speciﬁc inhibitor of PI3K reduces the level of Stat3 including growth, replication, movement, differentiation, and phosphorylation. The effect of PI3K on Stat3 appears to be medi- metabolism. PI3K signaling is elevated in most human cancers. ated by a member of the Tec kinase family. The Tec kinase inhib- This aberrant activity can result from differential regulation of itor LFM-A13 blocks Stat3 phosphorylation in H1047R-transformed PI3K itself or members of the pathway; it can also be caused by cells. The Janus kinase inhibitor AG490 and the Src kinase inhibitor gain-of-function mutations in the pathway or loss-of-function Src-1, as well as rapamycin, have no effect on Stat3 phosphoryla- mutations in the PI3K antagonist PTEN. The PI3K pathway tion in H1047R-transformed cells. The H1047R-transformed cells connects to numerous other signaling nodes including Ras, p53, also release a factor that induces Stat3 phosphorylation in normal Hif1α, and Lkb1. However, crosstalk between PI3K and Stat cells with possible effects on the cellular microenvironment. In signaling has not been reported. Here we describe a unique link some human tumor cell lines, the enhanced phosphorylation of between Stat3 and PI3K. In PI3K-transformed cells, Stat3 is Stat3 is inhibited by both PI3K and by Tec kinase inhibitors, sug- activated. This activation is essential for the process of trans- ﬁ gesting that the link between PI3K and Stat3 is signi cant in formation. Inhibition of PI3K prevents Stat3 phosphorylation, human cancer. and dominant-negative Stat3 interferes with PI3K-induced on-

cogenic transformation. MEDICAL SCIENCES oncogenic signaling | tumor stroma | tyrosine kinase Results tat3 is a member of a transcription factor family that was Stat3 Is Activated in C3H 10T1/2 Mouse Fibroblasts Transformed by Sdiscovered during the analysis of IFN-induced transcription the PI3K Mutant p110α-H1047R. We have used stable isotope la- (1–6). Stats are transcriptional regulators controlled by a path- beling with amino acids in cell culture (SILAC) in conjunction way that can be activated by growth factor as well as cytokine with tandem mass spectrometry to analyze the changes to the receptors (7). Stat3 is activated in response to the epidermal global proteome induced by the expression of the oncogenic growth factor and to IL-6 (2). Activation by IL-6 is primarily H1047R mutant of p110α in C3H 10T1/2 cells (26). The up- mediated by receptor-associated kinases of the Janus kinase (Jak) regulated PI3K signaling in the H1047R-transformed cells is family, which phosphorylate cytoplasmic Stats at tyrosine, effect- documented in Fig. 1A. Several of the proteins up-regulated by ing dimerization, translocation into the nucleus, sequence-specific p110α-H1047R are known targets of Stats (Table 1) (26–32). The DNA binding, and transcriptional activation (8). corresponding genes contain IFN-stimulated response elements The role of Stats, however, goes far beyond the IFN response. or IFN-γ activation sites (GAS). These binding sites interact with Stat3 is an important and often essential factor in oncogenic the IFN-stimulated gene factor 3 complex which contains both cellular transformation and in cancer. It is a required target of IFN response factor and Stat proteins. We therefore investigated the Src oncoprotein (9). Expression of dominant-negative Stat3 possible activation of Stat proteins by phosphorylation (Fig. 1B). blocks Src-induced cellular transformation. Stat3 is frequently The activation of Stat proteins by phosphorylation was evaluated and persistently activated in a wide variety of cancers (10). Mu- by Western blotting. The p110α-H1047R-transformed 10T1/2 rine cells in which Stat3 has been genetically inactivated are re- cells show enhanced phosphorylation of Stat3 and Stat6 and a sistant to oncogenic transformation (11, 12). Constitutively active decrease of phosphorylation in Stat1 (Fig. 1B). Because of the mutants of Stat3 are sufficient to convert normal cells into cancer prominent role of Stat3 in cancer (9–13), we decided to inves- cells (13). tigate its significance in PI3K-induced oncogenic transformation. The canonical kinases of Stat are members of the Jak family. There is no tyrosine kinase in the canonical PI3K signaling However, several other tyrosine kinases can phosphorylate and pathway. However, activated TOR (target of rapamycin) can activate Stats, including both receptor tyrosine kinases, such as phosphorylate S727 of Stat3 (33, 34) and this phosphorylation Egfr (14), Fgfr (15), Met (16), and Erbb2 (17), and nonreceptor enhances the activity of Stat3. However, in the two 10T1/2 cell tyrosine kinases, such as the Src and Fak kinase families. Such lines, S727 is constitutively phosphorylated, regardless of the ex- noncanonical Stat kinases are activated either through mutation pression of p110α-H1047R (Fig. 1B). It is therefore unlikely that – or through the aberrant expression of cytokines (18 20). In the enhanced expression of Stat targets in PI3K-transformed chronic myelogenous leukemia, the BCR-ABL fusion kinase also cells is mediated by TOR. In the context of the following ex- mediates Stat3 phosphorylation, and the leukemic cells are dependent upon this activity for sustained proliferation (21).

Activation of Stat transcription factors induces a variety of Author contributions: J.R.H., J.R.Y., and P.K.V. designed research; J.R.H., L.L., J.R.Y., and proliferative and prosurvival proteins as they suppress immune P.K.V. performed research; J.R.H., L.L., and J.R.Y. contributed new reagents/analytic tools; responses (22). Stat proteins enhance the expression of the J.R.H., L.L., J.R.Y., and P.K.V. analyzed data; and J.R.H. and P.K.V. wrote the paper. antiapoptotic Bcl2 and Bcl-XL (13) and repress the expression of The authors declare no conﬂict of interest. proapoptotic proteins, such as p53 (23). Many growth factors are 1To whom correspondence should be addressed. E-mail: [email protected]. under Stat3 transcriptional control, including VEGF (24) and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. HGF (25). Constitutive expression of these growth factors can 1073/pnas.1110486108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1110486108 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 phosphorylation of Y705 on Stat3 indicating that PI3K activity is essential for this phosphorylation event.

Cells Expressing p110α-H1047R Release a Stat3-Activating Factor. The reduction of Stat3 phosphorylation by a PI3K inhibitor does not rule out an IFN response to the retroviral vector. To explore a potential role of IFN further, we transfected 10T1/2 cells with the TLR3/RIG-I agonist poly-I:C to trigger an authentic IFN response. Cell-free conditioned medium from these cells and from nontransfected H1047R-expressing cells was then used to stimulate 10T1/2 cells. The cells were lysed 2 h after exposure to the conditioned medium and analyzed by Western blot. IFN produced in response to poly-I:C transfection triggered a large increase in the expression of Isg15, but did not affect phosphorylation of Stat3 (Fig. 1D). In contrast, medium conditioned by H1047R-transformed cells strongly stimulated the phosphorylation of Stat3, but had no effect on the production of Isg15. These data suggest that H1047R-transformed cells do not release detectable quantities of IFN, but release a factor that can stimulate the phosphorylation of Stat3. The nature of this factor Fig. 1. (A) PI3K signaling in cells transformed by the H1047R mutant of is not known but is currently under investigation. p110α. The Western blot shows expression of p110α and phosphorylation of Akt and of S6 in the presence and in the absence of serum. H1047R-trans- Dominant-Negative Mutant of Stat3 Interferes with PI3K-Induced formed cells show constitutive activation of PI3K signaling in the absence of Oncogenic Transformation. To determine the functional signifi- serum. (B) Phosphorylation of Stat proteins. 10T1/2 and 10T1/2-H1047R cells cance of Stat3 activation in H1047R-induced transformation, we were lysed and probed for the indicated tyrosine phosphorylations of Stat examined a dominant negative, DNA binding-defective form of proteins. Transformation by p110α-H1047R induces an increase in Stat3 and Stat3 for its ability to affect the oncogenic potency of p110α- Stat6 phosphorylation and a decrease in Stat1 phosphorylation. S727 of fi Stat3, a known TOR phosphorylation site, is unaffected. (C) The PI3K in- H1047R. Chicken embryonic broblasts were transfected with hibitor GDC-0941 blocks tyrosine phosphorylation of Stat3. The 10T1/2- RCAS(B)-Stat3DB (9) or RCAS(B) as a vector control. After H1047R cells were treated with 20 μM GDC-0941 for 0, 1, 6, 24, or 48 h as 6 d to allow full expression of the dominant-negative Stat3, the indicated and then analyzed by Western blot. (D) IFN stimulation. 10T1/2 cultures were superinfected with serial dilutions of RCAS(A) ex- cells were exposed to conditioned medium from either 10T1/2-H1047R or pressing the oncogenic H1047R mutant of p110α or with ASV17 from normal 10T1/2 cells transfected with poly-I:C. The IFN produced by expressing v-Jun, a presumably Stat3-independent oncoprotein. normal 10T1/2 cells in response to poly-I:C causes a sharp rise in the ex- Potencies of oncogenic transformation were determined by focus pression of Isg15 but does not induce phosphorylation of Stat3. In contrast, counts 10 d after challenge infection. Stat3DB inhibited trans- H1047R-conditioned medium leads to Stat3 phosphorylation but does not α change Isg15 expression. formation induced by p110 -H1047R, but not v-Jun induced transformation (Fig. 2). Under these conditions, the efficiency of transformation by H1047R was reduced ∼10-fold, whereas periments, we will use “phosphorylation of Stat3” to refer spe- the oncogenic activity of v-Jun remained at control levels. In an cifically to the phosphorylation of residue Y705, not of S727. expansion of this experiment, we tested the ability of Stat3DB to interfere with oncogenic transformation induced by the E545K Inhibition of PI3K Reduces Phosphorylation of Stat3. The p110α- mutant of p110α and by the wild-type proteins of p110β, p110γ, H1047R protein is expressed in the 10T1/2 cells with the retro- and p110δ. The transforming activity of all these oncogenic PI3K viral vector RCAS (35). It is therefore conceivable that the viral constructs were reduced by Stat3DB as summarized in Table 2. sequences of the vector could elicit an IFN response through recognition of double stranded RNA sequences of RCAS LTR Inhibition of Stat3 Phosphorylation Induces Resistance to PI3K-Induced Transformation. The signaling chain leading to Stat3 phosphory- by TLR3 (Toll-like receptor 3) or by Ddx58 (DEAD/H box lation in H1047R-transformed cells has not been identified. Our polypeptide RIG-I) (36). In this case, PI3K activity would be ir- observations with conditioned medium suggest that activation relevant for the phosphorylation of Stat3. To test this possibility, of PI3K can lead to the release of a cytokine or growth factor μ we treated 10T1/2-H1047R cells with 1 M GDC-0941, a potent that interacts with a receptor linked to Stat3. The activation of PI3K inhibitor (37). Phosphorylation in the PI3K pathway in- Stat3 could also involve a tyrosine kinase that is controlled cluding T308 and S473 of Akt and S235/236 of S6 was inhibited by PI3K. Candidates for such PI3K-stimulated activity are the (Fig. 1C). The PI3K inhibitor GDC-0941 also interfered with the members of the Tec kinase family (38). The Tec family tyrosine

Table 1. Enhanced expression of Stat3-regulated proteins in 10T1/2 cells transformed by PI3K (p110α-H1047R)* Name Description Fold overexpressed Conﬁdence interval

Isg15 ISG15 ubiquitin-like modifier 13.54 7.08 25.87 Oas2 2’-5′ oligoadenylate synthetase 2 8.95 7.80 10.27 Ifit1 IFN-induced protein with tetratricopeptide repeats 1 5.19 4.42 6.10 Igtp IFN-γ induced GTPase 3.73 2.89 4.81 Ddx58 DEAD/H box polypeptide RIG-I 2.44 1.96 3.05 Ccnd1 G1/S-specific cyclin-D1 2.17 Eif2ak2 IFN-induced, double-stranded RNA-activated protein kinase 1.79 1.64 1.95

*As determined by SILAC analysis (26).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1110486108 Hart et al. Downloaded by guest on October 2, 2021 Fig. 3. (A) LFM-A13, a Tec kinase inhibitor, blocks Stat3 phosphorylation. 10T1/2-H1047R cells were treated with 20 μM LFM-A13 for 0, 1, 6, 24, or 48 h as indicated. Cells were lysed and analyzed by Western blotting using the indicated antibodies. (B) LFM-A13 inhibits transformation by RCAS(A)- p110α-H1047R. CEF were infected with RCAS(A)-p110α-H1047R and either not treated or treated with varying concentrations of LFM-A13. Treatment with 20 μM LFM-A13 is shown.

tion of Stat3. The results obtained with the Tec family kinase Fig. 2. Dominant-negative Stat3 inhibits transformation by p110α-H1047R. inhibitor and with conditioned medium support the conclusion Chicken embryonic fibroblasts were transfected with RCAS(B)-Stat3DB, a that both intracellular and intercellular mechanisms are respon- dominant-negative, DNA-binding defective form of Stat3 or RCAS(B) as an sible for the activation of Stat3 in H1047R-transformed cells. empty vector control. These cells were then superinfected with dilutions MEDICAL SCIENCES of Avian sarcoma virus 17, expressing (A) v-jun or (B) RCAS(A)-p110α-H1047R. Stat3-PI3K Connection in Human Cancer Cell Lines. The 10T1/2 cells Focus formation induced by p110α-H1047R was strongly inhibited by Stat3DB. transformed by p110α-H1047R can be regarded as a model for human PI3K-driven cancer. We tested four human cancer cell lines for their responses to the inhibition of Tec family kinases kinases (Btk, Itk, Txk, Bmx, and Tec) contain a PH domain that and of PI3K signaling (Fig. 4). Three of the cell lines carry gain- is selective for binding to PIP3, their activity is consequently of-function mutations in p110α (T-47D and HCC1954 carry stimulated by PI3K (39), and they are known to phosphorylate the H1047R mutation, and MCF7 carries the E545K mutation). Stat3 (40). Btk, Itk, and Txk are restricted to hematopoietic cells. fi fi The fourth cell line, SK-BR-3, shows ampli ed HER2 with up- In the 10T1/2 mouse embryonic broblasts used in this study, we regulated signaling by wild-type PI3K. In two of these cell lines, detected the expression of Bmx and Tec. Of these expressions, SK-BR-3 and MCF-7, the phosphorylation of STAT3 is sensitive only Bmx was phosphorylated on tyrosine and, hence, activated to the Tec family kinase inhibitor as well as the PI3K inhibitor. (Fig. S1). LFM-A13 is a selective Tec family kinase inhibitor (41, These results suggest that in some human cancers the activation 42). We treated 10T1/2-H1047R cells with 20 μM LFM-A13 and A of STAT3 is PI3K-dependent and appears to be mediated by a analyzed critical proteins by Western blot (Fig. 3 ). LFM-A13 Tec kinase. reduced the phosphorylation of Stat3 as effectively as did the PI3K inhibitor GDC-0941. In contrast, the Janus kinase inhibitor Discussion AG490 and the Src kinase inhibitor Src-1 failed to affect Stat3 PI3K and Stat proteins represent two distinct cellular regulatory phosphorylation in these cells. Rapamycin also did not affect the systems that are not known to share components. An unexpected phosphorylation of Stat3 in H1047R-transformed 10T1/2 cells. link between PI3K and Stat-dependent transcription was recently The reduction in Stat3 phosphorylation by LFM-A13 is accom- revealed by a SILAC analysis of PI3K-transformed cells (26). panied by a stimulation of Akt and S6 phosphorylation. How- The current series of experiments confirms this unique connec- ever, despite the increased phosphorylation of PI3K signaling tion. Dominant-negative Stat3 interferes with PI3K-induced on- proteins, p110α-H1047R-induced oncogenic transformation was cogenic transformation but does not affect transformation ini- inhibited by LFM-A13 (Fig. 3B). These observations suggest tiated by the Jun oncoprotein. The finding suggests a role of Stat3 a role of a Tec family kinase in the PI3K-induced phosphoryla- in the PI3K-dependent transformation process but does not rule out the involvement of other Stats. Stat3 does not strictly homodimerize (43), and the overexpression of dominant-negative Table 2. Dominant-negative Stat3 (Stat3DB) reduces the fi Stat3 could therefore affect other proteins. Oncogenic PI3K ef ciency of oncogenic transformation by all class I PI3K isoforms stimulates phosphorylation of Stat3 and of Stat6; the possible Oncoprotein EOT* significance of the latter still remains to be explored. Stat6 is activated in certain cancers (44–46), and its increased phosphory- p110α-H1047R 0.16 ± 0.05 α ± lation in PI3K-transformed cells could be important for the p110 -E545K 0.17 0.05 oncogenic phenotype. p110β 0.33 ± 0.10 γ ± Could the activation of Stat3 in the 10T1/2-H1047R cells be an p110 0.12 0.03 artifact related to an IFN response that resulted from the use of p110δ 0.05 ± 0.01 ± a retroviral vector? Although this possibility cannot be ruled out v-Src 0.19 0.06 with absolute certainty, there are two sets of data that argue *EOT, efficiency of transformation = focus count in Stat3DB-transfected CEF strongly against it. First, inhibiting PI3K activity with a small- over focus count in CEF transfected with empty vector. molecule inhibitor blocks the phosphorylation of Stat3. This

Hart et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 Fig. 4. Phosphorylation of Stat3 in human cell lines. MCF-7, HCC-1954, T-47D, and SK-BR-3 cells were treated with 1 μM GDC-0941, 5 nM rapamycin, or 20 μM LFM-A13 for 24 h, as indicated. Cells were then lysed and analyzed by Western blotting. MCF-7 and SK-BR-3 show signiﬁcant reduction in Stat3 phosphorylation in response to both PI3K and to Tec kinase inhibition, similar to the sensitivity of 10T1/2-H1047R cells.

result clearly shows that PI3K is required for the activation of oncogenic activity (47, 48), and the result with the Tec family Stat3. Second, IFN produced in response to poly-I:C fails to in- kinase inhibitor suggests that phosphorylation of Akt and of S6 duce phosphorylation of Stat3 in 10T1/2 cells when up-regulating are not sufficient for PI3K-induced oncogenic transformation. the expression of Isg15. Testing a possible effect of the empty Critical factors in the transformation process clearly remain to vector on Stat3 phosphorylation directly was not possible be- be identified. cause cells transfected with the empty RCAS vector cannot be In human cancer cell lines, the link between PI3K and Stat3 is selected for. indicated by a sensitivity of Stat3 tyrosine phosphorylation to two A small molecule inhibitor of PI3K blocks the enhanced defining inhibitors: inhibitors of PI3K and inhibitors of Tec phosphorylation of Stat3, but there is no tyrosine kinase in the kinases. This criterion of dual sensitivity can now be applied to canonical PI3K pathway that could accomplish Stat3 activation. a larger number of cancer cell lines to evaluate the incidence of However, the Tec tyrosine kinases can link to PI3K through their the PI3K-Stat3 connection in human tumors of different tissue PIP3-specific PH domain and are candidates for such a function. origin and genetic make-up. Of particular interest will be cancers The ability of the selective Tec family kinase inhibitor LFM-A13 that show enhanced PI3K signaling, either because of a gain-of- to interfere with PI3K-induced phosphorylation of Stat3 supports function in a PI3K pathway component or a loss-of-function in this possibility. LFM-A13 also interferes with PI3K-induced on- PTEN. The importance of the Tec kinase Bmx in bridging PI3K cogenic transformation, possibly by blocking the PI3K-dependent signaling to Stat3 has also recently been convincingly documented activation of Stat3. LFM-A13 inhibits Tec family kinases with for the tumor-propagating stem cells of glioblastoma (49). an IC50 of 2.7 μM (42), but it also inhibits Polo-like kinases with H1047R-transformed 10T1/2 cells release a factor that acti- an IC50 of 60 μM (41). The concentrations of LFM-A13 used vates Stat3 without up-regulating Isg15 in normal 10T1/2 cells. in our experiments are insufficient to affect Polo-like kinases, This factor does not appear to be IFN because IFN produced in which therefore are unlikely to play a role in the PI3K-dependent response to poly-I:C by 10T1/2 cells causes up-regulation of phosphorylation of Stat3. The phosphorylation of Stat3 in PI3K- Isg15 but does not lead to enhanced Stat3 phosphorylation. The transformed cells is also unresponsive to the Janus kinase in- nature of this factor is currently unknown but is likely a growth hibitor AG490 and the Src kinase inhibitor Src-1, suggesting that factor or cytokine. One possibility is IL-1. A limiting determinant these canonical activators of Stat3 are not involved. Further- in the release of this proinflammatory cytokine is caspase-1 more, rapamycin fails to interfere with Stat3 phosphorylation in which processes and thereby activates the IL-1 precursor (50). H1047R-transformed cells, placing the critical signaling compo- Our SILAC study of H1047R-transformed 10T1/2 cells revealed nent for the PI3K-dependent Stat3 activation upstream of the a large increase in caspase-1 levels, which could enhance the TOR kinase. Of the five members of the Tec kinase family—Btk, generation of IL-1 (26). Another likely candidate is IL-6. Al- Itk, Txk, Bmx, and Tec—only Bmx and Tec are expressed in the though antibody to IL-6 fails to inactivate the factor, we continue fibroblast cell line studied, and only Bmx is phosphorylated on to pursue the possibility of IL-6 involvement using different tyrosine and, hence, activated. Bmx is therefore the likely me- approaches. The release of a Stat3-activating factor by trans- diator between PI3K and Stat3. The molecular details of the formed cells is of relevance for an understanding of the tumor connection between Bmx and Stat3 still remain to be explored, in microenvironment. The identification of this factor and studies particular the question whether Bmx acts on Stat3 directly or of its possible occurrence in human cancer are clearly important indirectly. The role Tec family kinases as link between PI3K and topics for future work. Stat3 identifies these kinases as complementary targets in PI3K- driven cancer and suggests the possibility of including Tec kinase Materials and Methods inhibitors in combination therapy for such cancers. Cell Lines, Cell Culture, and Transformation Assays. C3H10T1/2 cell lines were A surprising finding is the stimulating effect of the Tec kinase established as previously described (26). C3H10T1/2 cells were cultured in inhibitor on PI3K signaling. Phosphorylation of Akt and of S6 4.5 g/L glucose DMEM (Invitrogen) supplemented with 10% FBS, 2 mM μ μ are significantly increased in the presence of this inhibitor. This L-glutamine, 100 U/mL penicillin, 100 g/mL streptomycin (Sigma), and 200 g/ mL G418 (Invitrogen). HCC-1954, MCF-7, SK-BR-3, and T-47D were obtained observation suggests a possible negative regulatory effect of Tec from the ATCC and cultured in 4.5 g/L glucose DMEM (Invitrogen) supple- kinases on PI3K signaling. The apparent up-regulation of PI3K mented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL signaling is paradoxical in view of the fact that the Tec family streptomycin (Sigma), and 0.1 U/mL insulin (Sigma). Cell lines were used at low kinase inhibitor interferes with PI3K-induced transformation. passage numbers not exceeding 30 passages. Chicken embryonic fibroblasts The phosphorylation of Akt is not always correlated with PI3K (CEF) were prepared from pathogen free White Leghorn embryos (Charles

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1110486108 Hart et al. Downloaded by guest on October 2, 2021 River). For transformation assays, CEF were seeded into six-well plates and was prepared by transfection of 10T1/2 wild-type cells with 1 μg Poly-I:C and infected with RCAS(A) expressing the H1047R mutant of p110α, ASV17, or 60 μL lipofectamine or the medium from confluent 10T1/2-H1047R cells. Of PRA as indicated and as previously described (51–53). In focus assays, inhib- these media, 500 μL were added to 60-mm dishes of wild-type 10T1/2 cells fi fi itors are added at the speci ed concentration in the rst nutrient agar for 2 h. Cells were lysed using RIPA buffer (50 mM Tris pH 8.0, 150 mM so- overlay and in all subsequent feeding overlays. For interference assays, CEF dium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) were transfected with either RCAS(B) or RCAS(B)-Stat3DB by DMSO shock. containing Complete Protease Inhibitor mixture (Roche), 1 mM PMSF, 1 mM Cells were passed three times, during which the transfected retroviral con- Na VO . Protein concentrations were determined by the BCA method struct spread throughout the cell culture. The cells were then seeded into six- 3 4 well plates and superinfected with the indicated tumor viruses. All trans- (Pierce) and concentrations normalized before separation with 4% to 12% formation assays were performed in media containing: 0.715× F-10, 0.20× gradient SDS/PAGE using Bis-Tris/MOPS buffer system under reducing con- Earle’s balanced salt solution, 0.6% SeaPlaque Agarose, 3% FBS, 1% heat- ditions (Invitrogen). Separated proteins were blotted onto Immobilon-P inactivated chicken serum, 9% tryptose phosphate broth, 1.8 mM glutamine, PVDF membranes (Millipore). Membranes were blocked using 5% BSA in 89 U/mL penicillin, 89 μg/mL streptomycin, 1.1% DMSO. This mixture was TBS-T, incubated with primary antibodies, washed three times with TBS-T, applied every 2 to 3 d for 10 d at which point the overlay was removed and incubated with secondary antibody-HRP (Pierce) in 5% dry milk TBS-T, the cell layer stained with crystal violet. washed three times with TBS-T and developed using Superpico West ECL RCAS(B)-Stat3DB. Stat3DB was cloned from RcCMV-Stat3DB (kindly pro- reagent (Pierce), and imaged using a Biorad Chemidoc XRS. Primary anti- vided by Jacqueline Bromberg, Memorial Sloan-Kettering Cancer Center, bodies include PY-Stat1 (#9171), PY-Stat2 (#4441), PY-Stat3 (#9132), PY-Stat6 New York) to pBSfi by BamHI restriction, gel purification, and ligation to (#9361), PS-Stat3 (#9134), Stat3 (#9132), ISG15 (#2743), actin (#4967), PS-S6 form pBSfi-Stat3DB. Stat3DB was cloned from pBSfi-Stat3DB to RCAS(B). (#2211), PT308-Akt (#9275), PS473-Akt (#9271), and p110α (#4255) (Cell Sfi by restriction with SfiI followed by gel purification and ligation to form RCAS(B)-Stat3DB. Orientation was confirmed by AccI digestion. Signaling).

Western Blotting. Cells were cultured in the presence of 10% FBS unless ACKNOWLEDGMENTS. The C3H 10T1/2 tva cells were generated in our laboratory by Masa Aoki, who kindly shared this resource with the authors otherwise indicated. Cells were starved by culturing in 4.5 g/L glucose DMEM of this article. RCAS(B)-Stat3DB was made by Qing Sun using materials from containing 0.5% FBS for 22 h followed by 4.5 g/L glucose DMEM without Jacqueline Bromberg. This work was supported by National Institutes of serum for 2 h. Inhibitors, LFM-A13 (Tocris), GDC-0941 (Toronto Research Health Grants R01 CA078230 and P01 CA078045 (to J.R.H. and P.K.V.), and Chemicals), AG490 (Cayman), and Src-I (Sigma), were introduced into the P30 NS057096 and RR011823 (to L.L. and J.R.Y.). This is manuscript 21144 of medium 24 h before lysis unless otherwise indicated. Conditioned medium The Scripps Research Institute.

1. Shuai K, Stark GR, Kerr IM, Darnell JE, Jr. (1993) A single phosphotyrosine residue of 22. Yu H, Kortylewski M, Pardoll D (2007) Crosstalk between cancer and immune cells:

Stat91 required for gene activation by interferon-gamma. Science 261:1744–1746. Role of STAT3 in the tumour microenvironment. Nat Rev Immunol 7:41–51. MEDICAL SCIENCES 2. Zhong Z, Wen Z, Darnell JE, Jr. (1994) Stat3: A STAT family member activated by ty- 23. Niu G, et al. (2005) Role of Stat3 in regulating p53 expression and function. Mol Cell rosine phosphorylation in response to epidermal growth factor and interleukin-6. Biol 25:7432–7440. Science 264:95–98. 24. Niu G, et al. (2002) Constitutive Stat3 activity up-regulates VEGF expression and tumor 3. Khan KD, et al. (1993) Induction of the Ly-6A/E gene by interferon alpha/beta and angiogenesis. Oncogene 21:2000–2008. gamma requires a DNA element to which a tyrosine-phosphorylated 91-kDa protein 25. Wojcik EJ, et al. (2006) A novel activating function of c-Src and Stat3 on HGF tran- – binds. Proc Natl Acad Sci USA 90:6806 6810. scription in mammary carcinoma cells. Oncogene 25:2773–2784. 4. Schindler C, Shuai K, Prezioso VR, Darnell JE, Jr. (1992) Interferon-dependent tyrosine 26. Hart JR, Liao L, Ueno L, Yates, JR, 3rd, Vogt PK (2011) Protein expression profiles of – phosphorylation of a latent cytoplasmic transcription factor. Science 257:809 813. C3H 10T1/2 murine fibroblasts and of isogenic cells transformed by the H1047R mu- 5. Shuai K, Schindler C, Prezioso VR, Darnell JE, Jr. (1992) Activation of transcription by tant of phosphoinositide 3-kinase (PI3K). Cell Cycle 10:971–976. IFN-gamma: Tyrosine phosphorylation of a 91-kD DNA binding protein. Science 258: 27. Su ZZ, Sarkar D, Emdad L, Barral PM, Fisher PB (2007) Central role of interferon 1808–1812. regulatory factor-1 (IRF-1) in controlling retinoic acid inducible gene-I (RIG-I) ex- 6. Schindler C, Fu XY, Improta T, Aebersold R, Darnell JE, Jr. (1992) Proteins of tran- pression. J Cell Physiol 213:502–510. scription factor ISGF-3: One gene encodes the 91-and 84-kDa ISGF-3 proteins that are 28. Dupuis S, et al. (2003) Impaired response to interferon-alpha/beta and lethal viral activated by interferon alpha. Proc Natl Acad Sci USA 89:7836–7839. disease in human STAT1 deficiency. Nat Genet 33:388–391. 7. Sadowski HB, Shuai K, Darnell JE, Jr. Gilman MZ (1993) A common nuclear signal 29. Nakaya T, et al. (2001) Gene induction pathways mediated by distinct IRFs during viral transduction pathway activated by growth factor and cytokine receptors. Science 261: – 1739–1744. infection. Biochem Biophys Res Commun 283:1150 1156. 8. Schindler C, Darnell JE, Jr. (1995) Transcriptional responses to polypeptide ligands: The 30. Sinibaldi D, et al. (2000) Induction of p21WAF1/CIP1 and cyclin D1 expression by the fi JAK-STAT pathway. Annu Rev Biochem 64:621–651. Src oncoprotein in mouse broblasts: Role of activated STAT3 signaling. Oncogene 9. Bromberg JF, Horvath CM, Besser D, Lathem WW, Darnell JE, Jr. (1998) Stat3 activa- 19:5419–5427. tion is required for cellular transformation by v-src. Mol Cell Biol 18:2553–2558. 31. Kuhen KL, Samuel CE (1997) Isolation of the interferon-inducible RNA-dependent 10. Yu H, Jove R (2004) The STATs of cancer—New molecular targets come of age. Nat protein kinase Pkr promoter and identification of a novel DNA element within the 5′- Rev Cancer 4:97–105. flanking region of human and mouse Pkr genes. Virology 227:119–130. 11. Chiarle R, et al. (2005) Stat3 is required for ALK-mediated lymphomagenesis and 32. Shenoy AR, et al. (2007) Emerging themes in IFN-gamma-induced macrophage im- provides a possible therapeutic target. Nat Med 11:623–629. munity by the p47 and p65 GTPase families. Immunobiology 212:771–784. 12. Poget M, Meyer R (1976) Modification of a technic of reconstruction after total or 33. Zhou J, et al. (2007) Activation of the PTEN/mTOR/STAT3 pathway in breast cancer subtotal resection of the lower lip (Translated from French) ORL J Otorhinolaryngol stem-like cells is required for viability and maintenance. Proc Natl Acad Sci USA 104: Relat Spec, 38(Suppl 1):127–132 . 16158–16163. 13. Bromberg JF, et al. (1999) Stat3 as an oncogene. Cell 98:295–303. 34. Yokogami K, Wakisaka S, Avruch J, Reeves SA (2000) Serine phosphorylation and 14. David M, et al. (1996) STAT activation by epidermal growth factor (EGF) and am- maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin phiregulin. Requirement for the EGF receptor kinase but not for tyrosine phosphor- target mTOR. Curr Biol 10:47–50. – ylation sites or JAK1. J Biol Chem 271:9185 9188. 35. Oh J, Julias JG, Ferris AL, Hughes SH (2002) Construction and characterization of 15. Dudka AA, Sweet SM, Heath JK (2010) Signal transducers and activators of tran- a replication-competent retroviral shuttle vector plasmid. J Virol 76:1762–1768. fi scription-3 binding to the broblast growth factor receptor is activated by receptor 36. Abujamra AL, et al. (2006) Leukemia virus long terminal repeat activates NFkappaB amplification. Cancer Res 70:3391–3401. pathway by a TLR3-dependent mechanism. Virology 345:390–403. 16. Zhang YW, Wang LM, Jove R, Vande Woude GF (2002) Requirement of Stat3 signaling 37. Raynaud FI, et al. (2009) Biological properties of potent inhibitors of class I phos- for HGF/SF-Met mediated tumorigenesis. Oncogene 21:217–226. phatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC- 17. Ren Z, Schaefer TS (2002) ErbB-2 activates Stat3 alpha in a Src- and JAK2-dependent 0941. Mol Cancer Ther 8:1725–1738. manner. J Biol Chem 277:38486–38493. 38. Qiu Y, Kung HJ (2000) Signaling network of the Btk family kinases. Oncogene 19: 18. Gao SP, et al. (2007) Mutations in the EGFR kinase domain mediate STAT3 activation – via IL-6 production in human lung adenocarcinomas. J Clin Invest 117:3846–3856. 5651 5661. 19. Iliopoulos D, Hirsch HA, Struhl K (2009) An epigenetic switch involving NF-kappaB, 39. Qiu Y, Robinson D, Pretlow TG, Kung HJ (1998) Etk/Bmx, a tyrosine kinase with ′ Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation. Cell 139: a pleckstrin-homology domain, is an effector of phosphatidylinositol 3 -kinase and is 693–706. involved in interleukin 6-induced neuroendocrine differentiation of prostate cancer 20. Iwamoto T, et al. (2000) The JAK-inhibitor, JAB/SOCS-1 selectively inhibits cytokine- cells. Proc Natl Acad Sci USA 95:3644–3649. induced, but not v-Src induced JAK-STAT activation. Oncogene 19:4795–4801. 40. Tsai YT, et al. (2000) Etk, a Btk family tyrosine kinase, mediates cellular trans- 21. Spiekermann K, et al. (2002) Constitutive activation of STAT3 and STAT5 is induced by formation by linking Src to STAT3 activation. Mol Cell Biol 20:2043–2054. leukemic fusion proteins with protein tyrosine kinase activity and is sufficient for 41. Uckun FM, et al. (2007) Anti-breast cancer activity of LFM-A13, a potent inhibitor of transformation of hematopoietic precursor cells. Exp Hematol 30:262–271. Polo-like kinase (PLK). Bioorg Med Chem 15:800–814.

Hart et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 42. Mahajan S, et al. (1999) Rational design and synthesis of a novel anti-leukemic agent 48. Kang S, Denley A, Vanhaesebroeck B, Vogt PK (2006) Oncogenic transformation in- targeting Bruton’s tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta- duced by the p110beta, -gamma, and -delta isoforms of class I phosphoinositide methyl-N-(2, 5-dibromophenyl)propenamide]. J Biol Chem 274:9587–9599. 3-kinase. Proc Natl Acad Sci USA 103:1289–1294. 43. Wehinger J, et al. (1996) IL-10 induces DNA binding activity of three STAT proteins 49. Guryanova OA, et al. (2011) Nonreceptor tyrosine kinase BMX maintains self-renewal (Stat1, Stat3, and Stat5) and their distinct combinatorial assembly in the promoters of and tumorigenic potential of glioblastoma stem cells by activating STAT3. Cancer Cell selected genes. FEBS Lett 394:365–370. 19:498–511. 44. Wang CG, et al. (2010) EZH2 and STAT6 expression profiles are correlated with co- 50. Thornberry NA, et al. (1992) A novel heterodimeric cysteine protease is required for lorectal cancer stage and prognosis. World J Gastroenterol 16:2421–2427. interleukin-1 beta processing in monocytes. Nature 356:768–774. 45. Zhang WJ, et al. (2008) IL-4-induced Stat6 activities affect apoptosis and gene ex- 51. Kang S, Bader AG, Vogt PK (2005) Phosphatidylinositol 3-kinase mutations identified pression in breast cancer cells. Cytokine 42:39–47. in human cancer are oncogenic. Proc Natl Acad Sci USA 102:802–807. 46. Das S, Roth CP, Wasson LM, Vishwanatha JK (2007) Signal transducer and activator of 52. Bos TJ, et al. (1990) Efficient transformation of chicken embryo fibroblasts by c-Jun transcription-6 (STAT6) is a constitutively expressed survival factor in human prostate requires structural modification in coding and noncoding sequences. Genes Dev 4: cancer. Prostate 67:1550–1564. 1677–1687. 47. Vasudevan KM, et al. (2009) AKT-independent signaling downstream of oncogenic 53. Duff RG, Vogt PK (1969) Characteristics of two new avian tumor virus subgroups. PIK3CA mutations in human cancer. Cancer Cell 16:21–32. Virology 39:18–30.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1110486108 Hart et al. Downloaded by guest on October 2, 2021