PDGFRβ reverses EphB4 signaling in alveolar rhabdomyosarcoma M. Imran Aslama,b,c,d, Jinu Abrahamc, Atiya Mansoore, Brian J. Drukera,b,f,1, Jeffrey W. Tynera,g,1,2, and Charles Kellerc,1,2 a Knight Cancer Institute, Oregon Health and Science University, Portland, OR 97239; bDivision of Hematology and Medical Oncology, Department of Medicine, Oregon Health and Science University, Portland, OR 97239; cPediatric Cancer Biology Program, Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239; dHoward Hughes Medical Institute Medical Research Fellows Program, Chevy Chase, MD 20815; eDepartment of Pathology, Oregon Health and Science University, Portland, OR 97239; fHoward Hughes Medical Institute, Portland, OR 97239; and gDepartment of Cell and Developmental Biology, Oregon Health and Science University, Portland, OR 97239
Alveolar rhabdomyosarcoma (aRMS) is an aggressive myogenic childhood malignancy, not infrequently presenting as incurable metastatic disease. To identify therapeutic targets, we performed an unbiased tyrosine kinome RNA interference screen in primary cell cultures from a genetically engineered, conditional mouse model of aRMS. We identified ephrin receptor B4 (EphB4) as a target that is widely expressed in human aRMS and that portends a poor clinical outcome in an expression level-dependent manner. We also uncovered cross-talk of this ephrin receptor with another receptor tyrosine kinase, PDGFRβ, which facilitates PDGF ligand-dependent, ephrin ligand-independent activation of EphB4 converging on the Akt and Erk1/2 pathways. Conversely, EphB4 activation by its cognate ligand, EphrinB2, did not stimulate PDGFRβ; instead, apoptosis was paradoxically induced. Finally, we showed that smallmolecule inhibition of both PDGFRβ and EphB4 by dasatinib resulted in a significant decrease in tumor cell viability in vitro, as well as decreased tumor growth rate and significantly prolonged survival in vivo. To our knowledge, these results are the first to identify EphB4 and its cross-talk with PDGFRβ as unexpected vital determinants of tumor cell survival in aRMS, with EphB4 at the crux of a bivalent signaling node that is either mitogenic or proapoptotic. sarcoma
progression in the malignancy. Particularly in colorectal cancer, loss of EphB4 expression was shown to be critical at the adenoma-carcinoma transition (11). The platelet derived growth factor receptors (PDGFRs) α and β have been shown to be expressed both on tumor cells and stromal cells of mesenchymal origin in the tumor microenvironment (12, 13). In turn, the PDGFRs are involved in multiple malignant processes through activating mutations or driving oncogenic signaling pathways in both epithelial and hematologic malignancies (14–16). In aRMS, PDGFRα is a transcriptional target of a translocation-mediated fusion gene of PAX3 and FOXO1 (PAX3:FOXO1) and a therapeutic target in preclinical studies using the small molecule inhibitor imatinib or antibodymediated receptor blockade (17). Although cross-talk between the PDGFRs and the Eph receptors has not been directly investigated in tumorigenesis, evidence of an existing functional interface between the receptors has been implicated in select nonmalignant biological systems (18). Furthermore, targeting RTKs Significance Effective targeted therapies to complement already intensive chemotherapy are much needed for the childhood muscle cancer rhabdomyosarcoma, yet few targeted agents have been identified that improve long-term survival. In particular, the alveolar subtype of rhabdomyosarcoma accounts for disproportionate mortality. Herein, the receptor tyrosine kinase EphB4 is identified as a potential two-way switch for alveolar rhabdomyosarcoma. Whereas the typical EphB4 ligand, EphrinB2, drives tumor cells toward apoptosis, the interaction between EphB4 and another receptor tyrosine kinase, PDGFRβ, drives tumors to proliferate in the presence of the PDGFRβ ligand, PDGF-BB. The Food and Drug Administration-approved dual EphB4-PDGFRβ inhibitor, dasatinib, is found to have significant preclinical activity, which is clinically relevant because EphB4 and PDGFRβ are independent poor prognostic factors in this childhood disease.
| pediatric | muscle
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he childhood cancer alveolar rhabdomyosarcoma (aRMS) is a malignancy for which molecularly targeted therapies are in great need. More than half of aRMS patients present with unresectable or metastatic disease, yet despite maximally intensive chemotherapy, survival of these patients has remained unchanged for four decades (1). For adults with aRMS, survival probability is even more dismal (2). Thus, viable targeted therapies to incorporate into clinical trials for aRMS are greatly anticipated. We have previously identified receptor tyrosine kinases (RTKs) to be high-value target candidates in aRMS based on clinical survival data (3), which has led to the present studies performing an unbiased examination of the tyrosine kinome. Ephrin receptors (Eph proteins) are the largest family of RTKs and bind to Eph receptor-interacting (ephrin) ligands, which are glycosylphosphatidylinositol-linked or transmembrane ligands at sites of cell–cell contact. This receptor-ligand axis is atypical for RTKs in that it generates a bidirectional signaling cascade in both the cell expressing the receptor and the cell presenting the ligand. Although both Eph receptors and ephrins are demonstrated to be widely expressed in a variety of malignancies, their contribution to tumorigenesis has been controversial. This disparity has been because of the ability of Eph receptors, in particular, to either contribute to tumorigenesis or to perform a role in tumor suppression, depending upon the cellular context (4–8). In cancer, these signaling proteins may support progression, not necessarily through association with their respective ligands, but through cross-talk using other oncogenic signaling pathways, such as Akt, the Src family kinases, or EGFR (4, 9, 10). Another interesting and important aspect of the Eph receptors’ function in cancer is the regulation of their expression with respect to the stage of www.pnas.org/cgi/doi/10.1073/pnas.1403608111
Author contributions: M.I.A., J.A., J.W.T., and C.K. designed research; M.I.A., J.A., and C.K. performed research; M.I.A., J.W.T., and C.K. contributed new reagents/analytic tools; M.I.A., J.A., A.M., B.J.D., J.W.T., and C.K. analyzed data; and M.I.A., B.J.D., J.W.T., and C.K. wrote the paper. Conflict of interest statement: B.J.D. is principal investigator or coinvestigator on Novartis and Bristol-Myers Squibb (BMS) clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. B.J.D. does not derive salary, nor does his laboratory receive funds from these contracts. Oregon Health and Science University (OHSU) and B.J.D. have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. C.K. previously employed a technician who is a family member of a BMS employee. Freely available online through the PNAS open access option. 1
To whom correspondence may be addressed. E-mail: [email protected], tynerj@ohsu. edu, or [email protected].
2
J.W.T. and C.K. contributed equally to this work.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1403608111/-/DCSupplemental.
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Contributed by Brian J. Druker, March 21, 2014 (sent for review January 28, 2013)
with small-molecule inhibitors is a major clinical trial interest area of the Children’s Oncology Group (19). Thus far, the importance of EphB4 in tumorigenesis has been interrogated by methods involving inhibition of extracellular domain interaction with its ligand, as well as in vivo silencing of the receptor in murine tumor xenograft models (4, 8); however, the potential benefit of clinical small-molecule inhibitors targeting EphB4 kinase activity in cancer is yet to be explored. In this report, we have identified EphB4 as a druggable target in aRMS, a result obtained after investigating a surprising dichotomy for the signaling role of EphB4 through EphrinB2 versus through crosstalk with PDGFRβ and PDGF ligand. Results RNA-Interference–Assisted Protein Identification Screen. With the aim of identifying novel tyrosine kinase gene targets in aRMS, we first established primary tumor cell cultures from our genetically engineered, conditional mouse model of aRMS, as previously described (20, 21). Primary cell culture U23674 was derived from a primary limb tumor of a mouse comparable to Intergroup Rhabdomyosarcoma Study Group (IRSG) surgical stage T2b/N1/M0, and culture U48484 was derived from a pulmonary metastasis of a different tumor-bearing animal comparable to IRSG surgical stage T2b/N0/M1. We performed an RNA-interference assisted protein target identification (RAPID) screen of validated siRNAs to individually silence each member of the mouse tyrosine kinome (22, 23). Cell viability was assessed by MTS assay to determine the siRNAs that have deleterious effects on cell viability. The statistical threshold used to classify a significant decrease in tumor cell viability after siRNA gene silencing was as previously described (22, 23). This method identified Ntrk4, Pdgfrβ, and EphB4 in U23674, and Pdgfrβ, EphB4, and EphB6 in U48484. Interestingly, Pdgfrβ and EphB4 were both identified as targets in both cell lines (Fig. 1A). Expression of these RTKs at the protein level was validated by immunoblotting (Fig. 1B), and knockdown confirmed at the protein level (Fig. 1C). The average knockdown of Pdgfrβ and EphB4 in U23674 cells was ∼98% and 78%, respectively, and ∼95% and ∼98% in U48484, respectively (Fig. S1 A and B). We further validated the RAPID screen results using pooled EphB4 and Pdgfrβ siRNA in aRMS compared with a control mouse myoblast cell line (C2C12) (Fig. 1D). To determine if decrease in tumor cell viability from EphB4 silencing was a ligand-dependent or -independent phenotype, we silenced EphrinB2 and saw no appreciable decrease in tumor cell viability (Fig. 1D), suggesting EphB4’s oncogenic effect is independent of its cognate ligand, EphrinB2. There was no cooperative decrease in tumor cell viability when both receptors were silenced concurrently (Fig. 1D). Results of all MTS assays were confirmed by flow cytometry (Fig. S1C). To control for potential off-target effects causing the observed decrease in cell viability, we used the individual duplex siRNAs that comprise the Pdgfrβ and EphB4 siRNA pools used in the aforementioned studies to transfect U23674 and U48484 cells. The decrease in relative tumor cell viability we observed correlated with the individual duplex siRNA that most efficiently silenced Pdgfrβ or EphB4 (Fig. S1 D–G). EphB4 and EphrinB2 Is Expressed in Human and Mouse aRMS and EphB4 Is a Poor Prognostic Indicator in Fusion-Positive Human aRMS. No previous studies have examined expression or func-
tional significance of EphB4 or its ligand in aRMS. We examined expression of EphB4 and EphrinB2 in human and mouse aRMS. By quantitative RT-PCR (RT-qPCR), EPHB4 and EPHRINB2 expression were elevated in human aRMS compared with normal skeletal muscle, whereas in mouse aRMS only EphB4 levels were elevated (Fig. S2A). In both human skeletal muscle and aRMS, we saw a direct correlation between EphB4 and PDGFRβ expression level, with PAX3:FOXO1+ aRMS demonstrating a higher Pearson’s correlation coefficient (r = 0.91) compared 6384 | www.pnas.org/cgi/doi/10.1073/pnas.1403608111
Fig. 1. RNA-interference screen identifying EphB4 and PDGFRβ are required for tumor cell survival. (A) Cell viability result for the RNAi-assisted protein identification screen of primary (U23674) and metastatic (U48484) mouse aRMS tumor cultures. Dashed lines are ±2 SD with respect to median viability of cells. (B) Expression of EphB4, PDGFRβ, and EphrinB2 in U23674 and U48484 in addition to C2C12, an immortalized murine myoblast cell line. (C) Representative immunoblot of U23674 cells showing expression and knockdown efficiency of EphB4 and PDGFRβ in conditions identical to RNAi screen in A. White bar separates lane cropped from a lane on same gel. (D) Cell viability assessed by MTS assay on day 4 of murine aRMS cells and C2C12 transfected with nonspecific targeting siRNA, in addition to siRNA targeting EphB4 or Pdgfrß alone or in combination, EphrinB2 and Plk1 (+ control). *P < 0.05 compared with nonspecific siRNA by Student t test. For all experiments we included an siRNA targeted to Plk1 to serve as a positive control.
with PAX3:FOXO1− aRMS (r = 0.82) or human skeletal muscle (r = 0.80) (Fig. 2A). Immunoblot of PDGFRβ and EphB4 in four previously described human RMS cell lines, Rh5, Rh30, Rh3 (a subclone of Rh28), and Rh18 was also performed (Fig. 2B). Rh5, Rh30, and Rh3 are PAX3:FOXO1 fusion-positive aRMS, whereas Rh18 is a fusion-negative rhabdomyosarcoma. Both RTKs were present at the protein level in Rh5, Rh30, and Rh18, although the cell line Rh3 did not express EphB4. Immunohistochemistry on a human aRMS tissue microarray consisting of 19 unique cases with 31 sections confirmed expression of EphB4 (scores of 0–3) (Fig. S2B) and PDGFRβ (scores of 0–3) (Fig. S3A) at the protein level. The majority of tumor sections demonstrated strong staining of both EphB4 and PDGFRβ, although there were sections with weak expression of either receptor. Scoring by staining intensity is summarized in SI Methods. Fig. 2C includes representative images of human aRMS tissue stained for EphB4 and PDGFRβ, as well as their ligands EphrinB2 and PDGF-BB, respectively. In addition, wildtype human skeletal muscle was stained for EphB4 and PDGFRβ (Fig. S3 B and C). Additional images stained for the receptors and their corresponding ligands can be found in Figs. S4 and S5. To control for antibody specificity, antibodies were preincubated with blocking peptides before sections were stained (Figs. S4 and S5). PDGFRβ expression has been shown to be a strongly negative prognostic factor in rhabdomyosarcoma, reducing 5-y survival probability by >35% when adjusting for known clinical covariates (3). A similar analysis of this IRSG-IV microarray dataset (3) demonstrated that EPHB4 expression showed a >70% decrease in 5-y survival between high and low EPHB4 levels in fusion positive aRMS (Fig. 2D). Taken together, these results are consistent with a role of potential clinical significance for EphB4 in aRMS. Furthermore, Aslam et al.
unidentified target of PAX3:FOXO1. An indirect relationship is likely because previous ChIP-Seq performed on global/genomewide PAX3:FOXO1 binding sites in human aRMS, as well as various PAX3 isoform binding sites, did not identify EphB4 or PDGFRβ as direct targets of these transcription factors (25, 26). In our murine aRMS primary cell culture U23674, we observed that decreased Pax3:Foxo1 levels further decreases cell viability when silencing PDGFRβ or EphB4 individually (Fig. S6E). To confirm that EphB4 is a target of Pax3:Foxo1, we introduced a pCDNA3 vector expressing the Pax3:Foxo1 fusion transcript, or an empty vector, into murine myoblast (C2C12) cells. Accordingly, we observed increased expression of EphB4 at both the mRNA and protein level in C2C12 cells transfected with the fusion transcript compared with control C2C12 cells (Fig. S7). Pax3:Foxo1 expression was confirmed at the protein level in these cells (Fig. S7B). These data are consistent with EphB4 being either a direct or indirect target of Pax3:Foxo1a.
significant correlating expression of PDGFRβ and EPHB4 suggested potential interactions for further investigation.
status of EphB4 and Pdgfrβ in U23674 and U48484 cells by immunoprecipitating both RTKs from cell lysates and probing with antiphospho-tyrosine (p-Tyr) antibody, revealing that both receptors are phosphorylated at baseline (Fig. 4A). The primary cell cultures (U23674 and U48484) were treated with imatinib (targets PDGFRβ) and dasatinib (targets PDGFRβ and EphB4) for 72 h and cell viability assessed. These inhibitors decreased tumor cell viability and phosphorylation of their targeted receptors (Fig. 4 A and B). Dasatinib proved more potent; IC50s were achieved in the nanomolar range, whereas the imatinib IC50 was in the micromolar range. The IC50 for dasatinib in Rh5 and Rh30 (human aRMS which express PDGFRβ and EphB4) (Fig. 2B) were 35 and 200 nM, respectively (Fig. S8A).
Knockdown of PAX3:FOXO1 Regulates EphB4 Levels in Murine and Human aRMS. Previously, other RTKs such as PDGFRα and
siRNA-Mediated Knockdown of Pdgfrβ or EphB4 Induces Apoptosis and Abrogates Anchorage-Independent Colony Formation. To determine
c-Met have been established as downstream transcriptional targets of PAX3:FOXO1 (17, 24). To investigate whether EphB4 and PDGFRβ are directly or indirectly regulated by the PAX3: FOXO1 fusion protein, we silenced the fusion gene in both U23674 and U48484 using siRNA targeted to eYFP. This approach can be taken in our conditional mouse model because eYFP is expressed by means of an internal ribosomal entry site on a bicistronic transcript with Pax3:Foxo1 (Pax3:Foxo1-IRESeYFP). We are thus able to knock down Pax3:Foxo1 in tumor cells by targeting eYFP. Silencing of Pax3:Foxo1 led to decreased levels of EphB4 protein (Fig. 3A), but Pdgfrβ expression surprisingly increased. This result for Pdgfrβ is the opposite of what we have observed for Pdgfrα (17). The same phenomenon was observed in Rh5 (human aRMS) when PAX3:FOXO1 was silenced (Fig. 3A). Furthermore, silencing of PAX3:FOXO1 in murine and human aRMS resulted in a decrease and increase in EphB4 and PDGFRβ levels, respectively (Fig. S6 A and B). We hypothesized that the increase observed in PDGFRβ protein may represent a compensatory response to loss of EphB4 after silencing of PAX3:FOXO1 or a tumor cell response to stress. To test the former hypothesis, we studied the effect on PDGFRβ protein level after direct silencing of EphB4 in murine and human aRMS cells. Indeed, we observed that EphB4 silencing resulted in a significant increase in PDGFRβ protein (Fig. 3B), but had no effect on PDGFRβ mRNA levels in murine and human aRMS (Fig. S6 C and D); this suggests a posttranslational mechanism may be responsible for increased PDGFRβ protein. Silencing of PDGFRβ in both murine and human aRMS resulted in a significant decrease of EphB4 at the protein (Fig. 3B) and mRNA level (Fig. S6 C and D). Decreased EphB4 expression from PAX3:FOXO1 knockdown suggests EphB4 is a previously
whether the decrease in cell viability observed after knockdown
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Fig. 3. Effect of PAX3:FOXO1 on EphB4 and PDGFRβ expression and sensitivity to siRNA mediated silencing. (A) Effect of Pax3:Foxo1 silencing by eYFP-targeted siRNA on expression of EphB4 and PDGFRβ by immunoblot in U23674 and U48484 murine aRMS cells, and PAX3:FOXO1 silencing in human aRMS cell line Rh5 demonstrates an identical phenomenon. (B) Effect on PDGFRβ expression after EphB4 silencing in U23674, U48484, and Rh5 cells.
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Fig. 2. EphB4 and EphrinB2 levels in mouse aRMS are similar to human aRMS and tumor microarray of human aRMS shows strong expression of EphB4 and PDGFRβ by immunohistochemistry. (A) Correlation of PDGFRβ and EphB4 between human normal skeletal muscle (SKM) and aRMS tumors. r indicates calculated Pearson’s correlation coefficient. (B) Immunoblot of previously established human aRMS cell lines positive for PAX3:FOXO1: Rh5, Rh30, Rh3, and fusion negative Rh18 show expression of PDGFRβ and EphB4. (C) Immunohistochemistry of human aRMS tumors stained for EphB4, EphrinB2, PDGFRβ and PDGF-BB. (Scale bar, 50 μm.) (D) EPHB4 expression portends a poor overall survival for patients in PAX3:FOXO1 fusion positive aRMS (P = 0.032). Histogram signifies frequency of varying EPHB4 expression level in fusion positive aRMS patients.
Activation of PDGFRβ and EphB4 in Murine aRMS Can Be Blocked by Tyrosine Kinase Inhibitors. We next determined the activation
of each receptor individually, we observed a reduction in phosphorylation of Erk 1/2 and Akt. FAK phosphorylation was reduced after silencing of EphB4, but not Pdgfrβ in both U23674 and U48484 cells (Fig. 4E). To further interrogate signaling from Pdgfrβ and EphB4 receptor stimulation, cells were serumstarved then stimulated with their respective ligands. PDGF-BB stimulated phosphorylation of Pdgfrβ, Akt, FAK, and Erk 1/2 (Fig. 5A). EphrinB2 stimulation of either cell line did not yield an increase in FAK, Akt, or Erk 1/2 phosphorylation (Fig. 5A). These results were also observed in the human aRMS cell lines Rh5 and Rh30 (Fig. 5B). Because EphrinB2 stimulation did not result in activation of Akt, Erk, or FAK, we immunoprecipitated EphB4 and probed with an anti–p-Tyr antibody to confirm EphB4 phosphorylation upon EphrinB2 stimulation (Fig. 6A). Taken together, these data suggest that with PDGF-BB ligand stimulation, PDGFRβ activates the common downstream effectors Akt and Erk1/2, whereas EphB4 signaling through Akt and Erk 1/2 appears to be independent of its cognate ligand, EphrinB2, in human and murine aRMS. Therefore, to test if Pdgfrβ could activate EphB4 (and thus, hypothetically, downstream mediators Akt and Erk 1/2), we looked for evidence of the ability of Pdgfrβ to phosphorylate EphB4. We immunoprecipitated EphB4 with and without stimulation of Pdgfrβ with PDGF-BB and showed that phosphorylation of EphB4 increases after PDGF-BB treatment (Fig. 6B). Imatinib was able to inhibit this effect despite EphB4 not being a direct target of imatinib. This result suggested
Fig. 4. PDGFRβ and EphB4 inhibition abrogates growth of mouse tumor primary cell cultures. (A) Small molecule inhibition of Pdgfrβ phosphorylation by imatinib or dasatinib and EphB4 phosphorylation by dasatinib. DMSO was used as a vehicle control. (B) A 72 h MTS assay on cells treated with increasing concentrations of imatinib and dasatinib. IC50 for each drug and cell line are shown. (C) Knockdown of Pdgfrβ and EphB4 results in tumor cells undergoing apoptosis as measured by Annexin V. Early apoptotic cells are Annexin V+ only, late apoptotic cells are Annexin V and 7-AAD+. (D) Anchorage-independent colony formation is decreased significantly in murine aRMS U23674 and U48484 cells when Pdgfrβ and EphB4 are silenced by siRNA. Relative number of colonies normalized to nonspecific siRNA. (E) Immunoblot showing signaling downstream EphB4 and PDGFRβ shown after siRNA mediated silencing of each kinase in U23674 and U48484 cells. For PDGFRβ and EphB4 levels after corresponding siRNAs, see Fig. 3B. *P < 0.05 compared with nonspecific siRNA by Student t test.
of Pdgfrβ and EphB4 was from either cell cycle redistribution and/or apoptosis, we performed cell cycle analysis of U23674 and U48484 cells by FACS after transfection with nonspecific Pdgfrβor EphB4-targeted siRNA, revealing cell cycle distribution was not significantly altered (Fig. S8B). Silencing of EphB4 or Pdgfrβ individually or simultaneously resulted in significant apoptosis as determined by FACS (Fig. 4C). Silencing of Pax3:Foxo1 was used as a comparator of tumor cell apoptosis (Fig. 4C). The ability of murine aRMS cells to form colonies in an anchorage-independent colony formation assay was significantly inhibited upon Pdgfrβ and EphB4 silencing (Fig. 4D and Fig. S8C). Taken together, these data indicate a vital role of Pdgfrβ and EphB4 in tumor cell survival and transformation. Akt, Erk 1/2 Are Downstream of PDGFRβ and EphB4, Whereas FAK Is Downstream of EphB4 and EphB4 Is Phosphorylated upon PDGF-BB Stimulation. We next assessed the downstream signaling pathways
affected by silencing of Pdgfrβ and EphB4. Upon knockdown 6386 | www.pnas.org/cgi/doi/10.1073/pnas.1403608111
Fig. 5. PDGFRβ but not EphB4 stimulation by their respective ligands activates downstream cell survival and proliferative pathways in murine and human aRMS. (A) Immunoblots showing U23674 and U48484 cells stimulated (after overnight serum starvation) with PDGF-BB (10 ng/mL, 15 min) alone or in the presence of imatinib (100 nM, 1 h) or with EphrinB2 (2 μg/mL, 15 min) alone or in the presence of dasatinib (100 nM, 1 h). Fc only (Fc portion lacking clustered ligand, 2 μg/mL for 15 min) lane represents control for EphrinB2. (B) Immunoblots showing Rh5 and Rh30 cells stimulated (after overnight serum starvation) with PDGF-BB (10 ng/mL, 15 min) alone or in the presence of imatinib (100 nM, 1 h) or with EphrinB2 (2 μg/mL, 15 min) alone or in the presence of dasatinib (100 nM, 1 h).
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that EphrinB2 was similarly stimulating a distinct signaling cascade. Because Crkl is a monogamous substrate for phosphorylation by Abl (6), we examined Crkl phosphorylation upon EphrinB2 treatment. p-Crkl was stimulated by EphrinB2 treatment in both murine and human aRMS cells, U48484 and Rh5, respectively (Fig. 6E). The murine aRMS primary cell cultures U23674 and U48484 along with human aRMS cell lines Rh5 and Rh30 in the presence of Fc only control or EphrinB2 for a 3 d period showed that all cell lines underwent apoptosis upon EphrinB2 treatment (Fig. 6 F and G, respectively). These results indicate that although EphrinB2 does stimulate EphB4, the signaling cascade initiated by EphrinB2 is distinct from that stimulated through cross-talk with PDGFRβ.
Fig. 6. PDGFRβ and EphB4 cross-talk; EphrinB2 signals through EphB4 to induce apoptosis in murine and human aRMS. (A) Representative immunoblot indicating EphrinB2 treatment (2 μg/mL, 15 min) results in phosphorylation of EphB4 and (B) PDGF-BB treatment (10 ng/mL, 15 min) results in EphB4 phosphorylation in murine aRMS (U48484) and human aRMS (Rh30). PDGF-BB mediated phosphorylation is inhibited by imatinib. (C) A graded increase in PDGF-BB concentrations of 0, 100, 200, 500, and 1,000 ng/mL serves as a mitogen in U23674 and U48484 cells. (D) A graded increase in PDGF-BB concentrations of 0, 100, 200, 500, and 1,000 ng/mL serves as a mitogen in Rh5 and Rh30 cells. (E) In murine aRMS U48484 and human aRMS Rh5, EphrinB2 (2 μg/mL, 15 min) treatment stimulates p-Crkl, which dasatinib inhibits. (F) EphrinB2 (20 μg/mL) treatment versus Fc only for 3 d of mouse aRMS and (G) human aRMS cells results in tumor cells undergoing apoptosis as measured by Annexin V. Early apoptotic cells are Annexin V+ only, late apoptotic cells are Annexin V and 7-AAD+. *P < 0.01, **P < 0.05 by Student t test compared with respective control.
dasatinib’s increased efficacy relative to imatinib was conserved in vivo, we orthotopically engrafted (SCID/hairless/outbred) mice with U23674 cells and treated these mice with dasatinib (15 or 50 mg/kg) or imatinib (100 mg/kg). Once tumors were 0.2 cc3, we commenced daily treatment with drug or vehicle control. Survival was significantly extended with dasatinib at a dose of 50 mg/kg compared with all other treatment groups (P < 0.05) (Fig. 7A). To confirm drug administration in vivo inhibited Pdgfrβ and EphB4 phosphorylation, immunoblotting of tumor lysates were performed. Immunoblot of tumors treated with daily vehicle, imatinib, and both high- or low-dose dasatinib showed that Pdgfrβ and EphB4 phosphorylation was decreased in dasatinibtreated mice, whereas only Pdgfrβ phosphorylation was decreased in mice treated with imatinib (Fig. 7 B and C). Taken together, these data suggest that blockade of only PDGFRβ activity (by imatinib) can impart some inhibition of tumor growth, whereas direct blockade of both Pdgfrβ and EphB4 activity (by dasatinib) imparts the greatest effect on inhibition of tumor growth. Discussion Through an unbiased RNAi screen targeted to the tyrosine kinome, we identified EphB4 as a novel target critical to tumor cell viability. We also found EphB4 to be a poor prognostic indicator in PAX3:FOXO1 fusion-positive aRMS. EphB4 has
that the phosphorylation of EphB4 is mediated by an imatinib target, such as PDGFRβ (one of only a few imatinib targets). Finally, we observed that PDGF-BB ligand increased cell growth by two- to threefold in a dose-dependent manner, affirming a mitogenic phenotype in murine and human aRMS (Fig. 6 C and D, respectively). In addition, the demonstrated ability of kinase inhibitors imatinib or dasatinib to ablate this mitogenic phenotype in murine primary cell culture of aRMS (U23674) (Fig. S8D) suggests that PDGF-BB ligand-mediated growth is a kinasedependent process. EphrinB2 Treatment Induces Apoptosis in Murine and Human aRMS.
Although PDGF-BB activated PDGFRβ, EphB4, Akt, and Erk 1/2, resulting in stimulating mitogenesis of tumor cells, EphrinB2 was not capable of stimulating these downstream signaling pathways, despite inducing phosphorylation of EphB4, suggesting that canonical EphB4 signaling may serve a different role. In prior reports, the Eph RTK family has been shown to function as tumor suppressors (5, 11) or as oncogenes (4, 27) in a wide variety of malignancies. An EphrinB2 induced EphB4 signaling cascade mediated by the Abl-Crkl pathway resulting in apoptosis has been shown in mammary carcinoma (6). Based on our previous observations in these murine and human aRMS, we hypothesized Aslam et al.
Fig. 7. Dasatinib significantly slows aggressive murine aRMS tumor growth versus imatinib in vivo. (A) Kaplan–Meier survival curve of SCID/hairless/ outbred mice treated with vehicle daily, dasatinib 15 mg·kg·d, dasatinib 50 mg·kg·d or imatinib 100 mg·kg·d, with endpoint measurement being days for tumor volume to reach 2.0 cm3. Mantel–Cox analysis of survival curves revealed P < 0.05 between comparison of vehicle and dasatinib 15 mg/kg and imatinib 100 mg/kg and P < 0.05 between 50 mg/kg dasatinib and all other treatment groups. (B) Immunoblot confirming inhibition of PDGFRβ phosphorylation by imatinib and dasatinib in vivo and (C) inhibition of EphB4 phosphorylation by dasatinib only (imatinib unable to inhibit EphB4 activation) from randomly selected tumor-bearing mice. White bar separates lanes cropped from the same gel.
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Dasatinib-Mediated Inhibition of both PDGFRβ and EphB4 Improves Survival More than Imatinib-Mediated Inhibition of PDGFRβ Alone in an Orthotopic Allograft Model of aRMS. To determine whether
in Eph kinase-ephrin interactions (9). We show dasatinib is efficacious at physiologically achievable serum concentrations (31) but acknowledge that while dasatinib inhibits both PDGFRβ and EphB4, it also inhibits many other tyrosine (and serine/threonine) kinases. Therefore, its antagonistic effects on aRMS cells in culture and in the xenograft studies could be due to inhibition of not only PDGFRβ and EphB4, but could also be a result of its inhibition of other kinases as well. It might also stand to reason that clinical trials targeting EphB4 in aRMS would benefit well from a biomarker-driven strategy to identify which patients are best suited for EphB4-targeted therapy.
previously been identified to contribute toward tumor suppression, apoptosis, or an increased malignant phenotype in a variety of other cancers (4–6, 9, 11, 27, 28). In our studies, we further identified a previously undescribed mechanism of cross-talk between EphB4 with another RTK, PDGFRβ, which is known to be activated and expressed in aRMS (29, 30). Our studies demonstrated that both cell survival (Akt) and cell proliferation (Erk) signaling pathways are downstream of both RTKs, suggesting that both RTKs converge on similar pathways. We show EphB4 stimulation by its cognate ligand, EphrinB2, was unable to stimulate Akt or Erk; however, PDGFRβ stimulation with PDGF-BB ligand was able to phosphorylate EphB4 as well as activate these downstream pathways in both murine and human aRMS. Signaling interrogation revealed p-FAK was abrogated with EphB4 knockdown, but not by Pdgfrβ knockdown. However, PDGFRβ stimulation via PDGF-BB was able to stimulate FAK phosphorylation, presumably through cross-talk with EphB4. These results allude to EphB4 having an ability to activate oncogenic signaling pathways independent of PDGFRβ or its cognate ligand, EphrinB2, as we also describe. Based on our results (summarized in Fig. S9), as well as previous literature corroborating the phenomenon of ephrin kinase signaling initiated by noncanonical pathways, we deduce that EphB4 in aRMS may serve as a scaffold to activate other signaling pathways or associate with other proteins that are able to activate the receptor, similar to its cross-talk with PDGFRβ. Such a ligand-independent function of Eph receptors has been described in other model systems, such as prostate cancer and glioma cells (9). To translate our results to the clinic, we chose to test the Food and Drug Administration-approved, dual PDGFRβ and EphB4 inhibitor, dasatinib. A promising secondary attribute of dasatinib is its ability to inhibit the Src family kinases, which is relevant because the Src family kinases are responsible for reverse signaling
ACKNOWLEDGMENTS. We thank Stephanie Willis, Elaine Huang, and Chris Eide for technical assistance, as well as other B.J.D. laboratory members for their invaluable support. M.I.A. is a Howard Hughes Medical Institute Medical Research Fellow. B.J.D. is an investigator of the Howard Hughes Medical Institute. This work was supported by Grant 5R01CA133229 and in part by a grant from the Joanna McAfee Childhood Cancer Foundation (to C.K.); a grant from the William Lawrence and Blanche Hughes Foundation (to J.W.T.); a grant from the Leukemia and Lymphoma Society (to J.W.T.); a grant from the V Foundation for Cancer Research (to J.W.T.); and National Cancer Institute Grant 4R00CA151457 (to J.W.T.). The Cooperative Human Tissue Network and the Children’s Oncology Group Biorepository are funded by the National Cancer Institute.
1. Arndt CA, Crist WM (1999) Common musculoskeletal tumors of childhood and adolescence. N Engl J Med 341(5):342–352. 2. Sultan I, Qaddoumi I, Yaser S, Rodriguez-Galindo C, Ferrari A (2009) Comparing adult and pediatric rhabdomyosarcoma in the surveillance, epidemiology and end results program, 1973 to 2005: An analysis of 2,600 patients. J Clin Oncol 27(20):3391–3397. 3. Blandford MC, et al. (2006) Rhabdomyosarcomas utilize developmental, myogenic growth factors for disease advantage: A report from the Children’s Oncology Group. Pediatr Blood Cancer 46(3):329–338. 4. Xia G, et al. (2006) EphB4 receptor tyrosine kinase is expressed in bladder cancer and provides signals for cell survival. Oncogene 25(5):769–780. 5. Oricchio E, et al. (2011) The Eph-receptor A7 is a soluble tumor suppressor for follicular lymphoma. Cell 147(3):554–564. 6. Noren NK, Foos G, Hauser CA, Pasquale EB (2006) The EphB4 receptor suppresses breast cancer cell tumorigenicity through an Abl-Crk pathway. Nat Cell Biol 8(8):815–825. 7. Kumar SR, et al. (2009) Preferential induction of EphB4 over EphB2 and its implication in colorectal cancer progression. Cancer Res 69(9):3736–3745. 8. Kertesz N, et al. (2006) The soluble extracellular domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth. Blood 107(6):2330–2338. 9. Miao H, et al. (2009) EphA2 mediates ligand-dependent inhibition and ligandindependent promotion of cell migration and invasion via a reciprocal regulatory loop with Akt. Cancer Cell 16(1):9–20. 10. Leroy C, et al. (2009) Quantitative phosphoproteomics reveals a cluster of tyrosine kinases that mediates SRC invasive activity in advanced colon carcinoma cells. Cancer Res 69(6):2279–2286. 11. Batlle E, et al. (2005) EphB receptor activity suppresses colorectal cancer progression. Nature 435(7045):1126–1130. 12. Andrae J, Gallini R, Betsholtz C (2008) Role of platelet-derived growth factors in physiology and medicine. Genes Dev 22(10):1276–1312. 13. Ostman A, Heldin CH (2007) PDGF receptors as targets in tumor treatment. Adv Cancer Res 97:247–274. 14. Steller EJ, et al. (2013) PDGFRB promotes liver metastasis formation of mesenchymallike colorectal tumor cells. Neoplasia 15(2):204–217. 15. Noel P, Mesa RA (2013) Eosinophilic myeloid neoplasms. Curr Opin Hematol 20(2): 157–162. 16. Heinrich MC, et al. (2003) PDGFRA activating mutations in gastrointestinal stromal tumors. Science 299(5607):708–710.
17. Taniguchi E, et al. (2008) PDGFR-A is a therapeutic target in alveolar rhabdomyosarcoma. Oncogene 27(51):6550–6560. 18. He S, et al. (2010) Soluble EphB4 inhibition of PDGF-induced RPE migration in vitro. Invest Ophthalmol Vis Sci 51(1):543–552. 19. Hawkins DS, Spunt SL, Skapek SX; COG Soft Tissue Sarcoma Committee (2013) Children’s Oncology Group’s 2013 blueprint for research: Soft tissue sarcomas. Pediatr Blood Cancer 60(6):1001–1008. 20. Nishijo K, et al. (2009) Credentialing a preclinical mouse model of alveolar rhabdomyosarcoma. Cancer Res 69(7):2902–2911. 21. Keller C, et al. (2004) Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: Cooperativity of Ink4a/ARF and Trp53 loss of function. Genes Dev 18(21):2614– 2626. 22. Tyner JW, et al. (2008) RNAi screening of the tyrosine kinome identifies therapeutic targets in acute myeloid leukemia. Blood 111(4):2238–2245. 23. Tyner JW, et al. (2009) RNAi screen for rapid therapeutic target identification in leukemia patients. Proc Natl Acad Sci USA 106(21):8695–8700. 24. Ginsberg JP, Davis RJ, Bennicelli JL, Nauta LE, Barr FG (1998) Up-regulation of MET but not neural cell adhesion molecule expression by the PAX3-FKHR fusion protein in alveolar rhabdomyosarcoma. Cancer Res 58(16):3542–3546. 25. Wang Q, Kumar S, Mitsios N, Slevin M, Kumar P (2007) Investigation of downstream target genes of PAX3c, PAX3e and PAX3g isoforms in melanocytes by microarray analysis. Int J Cancer 120(6):1223–1231. 26. Cao L, et al. (2010) Genome-wide identification of PAX3-FKHR binding sites in rhabdomyosarcoma reveals candidate target genes important for development and cancer. Cancer Res 70(16):6497–6508. 27. Masood R, et al. (2006) EphB4 provides survival advantage to squamous cell carcinoma of the head and neck. Int J Cancer 119(6):1236–1248. 28. Noren NK, Lu M, Freeman AL, Koolpe M, Pasquale EB (2004) Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth. Proc Natl Acad Sci USA 101(15):5583–5588. 29. Cen L, et al. (2007) Phosphorylation profiles of protein kinases in alveolar and embryonal rhabdomyosarcoma. Mod Pathol 20(9):936–946. 30. Armistead PM, et al. (2007) Expression of receptor tyrosine kinases and apoptotic molecules in rhabdomyosarcoma: Correlation with overall survival in 105 patients. Cancer 110(10):2293–2303. 31. Johnson FM, et al. (2010) Phase 1 pharmacokinetic and drug-interaction study of dasatinib in patients with advanced solid tumors. Cancer 116(6):1582–1591.
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Methods Human Samples. Human samples (normal skeletal muscle and tumor RNA, and human tissue microarrays) were provided by the Cooperative Human Tissue Network or Children’s Oncology Group Biorepository. Studies were performed under an Institutional Review Board-approved protocol. Other Methods. Detailed descriptions of cell culture, RT-qPCR, immunohistochemistry, immunoblotting and immunoprecipitation, RNA-interference studies, in vitro growth inhibition and proliferation assays, cell viability and apoptosis assays, in vivo studies, and statistical considerations are given in SI Methods.
Aslam et al.
Alveolar rhabdomyosarcoma (aRMS) is an aggressive myogenic childhood malignancy, not infrequently presenting as incurable metastatic disease. To identify therapeutic targets, we performed an unbiased tyrosine kinome RNA interference screen in primary cell cultures from a genetically engineered, conditional mouse model of aRMS. We identified ephrin receptor B4 (EphB4) as a target that is widely expressed in human aRMS and that portends a poor clinical outcome in an expression level-dependent manner. We also uncovered cross-talk of this ephrin receptor with another receptor tyrosine kinase, PDGFRβ, which facilitates PDGF ligand-dependent, ephrin ligand-independent activation of EphB4 converging on the Akt and Erk1/2 pathways. Conversely, EphB4 activation by its cognate ligand, EphrinB2, did not stimulate PDGFRβ; instead, apoptosis was paradoxically induced. Finally, we showed that smallmolecule inhibition of both PDGFRβ and EphB4 by dasatinib resulted in a significant decrease in tumor cell viability in vitro, as well as decreased tumor growth rate and significantly prolonged survival in vivo. To our knowledge, these results are the first to identify EphB4 and its cross-talk with PDGFRβ as unexpected vital determinants of tumor cell survival in aRMS, with EphB4 at the crux of a bivalent signaling node that is either mitogenic or proapoptotic. sarcoma
progression in the malignancy. Particularly in colorectal cancer, loss of EphB4 expression was shown to be critical at the adenoma-carcinoma transition (11). The platelet derived growth factor receptors (PDGFRs) α and β have been shown to be expressed both on tumor cells and stromal cells of mesenchymal origin in the tumor microenvironment (12, 13). In turn, the PDGFRs are involved in multiple malignant processes through activating mutations or driving oncogenic signaling pathways in both epithelial and hematologic malignancies (14–16). In aRMS, PDGFRα is a transcriptional target of a translocation-mediated fusion gene of PAX3 and FOXO1 (PAX3:FOXO1) and a therapeutic target in preclinical studies using the small molecule inhibitor imatinib or antibodymediated receptor blockade (17). Although cross-talk between the PDGFRs and the Eph receptors has not been directly investigated in tumorigenesis, evidence of an existing functional interface between the receptors has been implicated in select nonmalignant biological systems (18). Furthermore, targeting RTKs Significance Effective targeted therapies to complement already intensive chemotherapy are much needed for the childhood muscle cancer rhabdomyosarcoma, yet few targeted agents have been identified that improve long-term survival. In particular, the alveolar subtype of rhabdomyosarcoma accounts for disproportionate mortality. Herein, the receptor tyrosine kinase EphB4 is identified as a potential two-way switch for alveolar rhabdomyosarcoma. Whereas the typical EphB4 ligand, EphrinB2, drives tumor cells toward apoptosis, the interaction between EphB4 and another receptor tyrosine kinase, PDGFRβ, drives tumors to proliferate in the presence of the PDGFRβ ligand, PDGF-BB. The Food and Drug Administration-approved dual EphB4-PDGFRβ inhibitor, dasatinib, is found to have significant preclinical activity, which is clinically relevant because EphB4 and PDGFRβ are independent poor prognostic factors in this childhood disease.
| pediatric | muscle
T
he childhood cancer alveolar rhabdomyosarcoma (aRMS) is a malignancy for which molecularly targeted therapies are in great need. More than half of aRMS patients present with unresectable or metastatic disease, yet despite maximally intensive chemotherapy, survival of these patients has remained unchanged for four decades (1). For adults with aRMS, survival probability is even more dismal (2). Thus, viable targeted therapies to incorporate into clinical trials for aRMS are greatly anticipated. We have previously identified receptor tyrosine kinases (RTKs) to be high-value target candidates in aRMS based on clinical survival data (3), which has led to the present studies performing an unbiased examination of the tyrosine kinome. Ephrin receptors (Eph proteins) are the largest family of RTKs and bind to Eph receptor-interacting (ephrin) ligands, which are glycosylphosphatidylinositol-linked or transmembrane ligands at sites of cell–cell contact. This receptor-ligand axis is atypical for RTKs in that it generates a bidirectional signaling cascade in both the cell expressing the receptor and the cell presenting the ligand. Although both Eph receptors and ephrins are demonstrated to be widely expressed in a variety of malignancies, their contribution to tumorigenesis has been controversial. This disparity has been because of the ability of Eph receptors, in particular, to either contribute to tumorigenesis or to perform a role in tumor suppression, depending upon the cellular context (4–8). In cancer, these signaling proteins may support progression, not necessarily through association with their respective ligands, but through cross-talk using other oncogenic signaling pathways, such as Akt, the Src family kinases, or EGFR (4, 9, 10). Another interesting and important aspect of the Eph receptors’ function in cancer is the regulation of their expression with respect to the stage of www.pnas.org/cgi/doi/10.1073/pnas.1403608111
Author contributions: M.I.A., J.A., J.W.T., and C.K. designed research; M.I.A., J.A., and C.K. performed research; M.I.A., J.W.T., and C.K. contributed new reagents/analytic tools; M.I.A., J.A., A.M., B.J.D., J.W.T., and C.K. analyzed data; and M.I.A., B.J.D., J.W.T., and C.K. wrote the paper. Conflict of interest statement: B.J.D. is principal investigator or coinvestigator on Novartis and Bristol-Myers Squibb (BMS) clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. B.J.D. does not derive salary, nor does his laboratory receive funds from these contracts. Oregon Health and Science University (OHSU) and B.J.D. have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. C.K. previously employed a technician who is a family member of a BMS employee. Freely available online through the PNAS open access option. 1
To whom correspondence may be addressed. E-mail: [email protected], tynerj@ohsu. edu, or [email protected].
2
J.W.T. and C.K. contributed equally to this work.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1403608111/-/DCSupplemental.
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Contributed by Brian J. Druker, March 21, 2014 (sent for review January 28, 2013)
with small-molecule inhibitors is a major clinical trial interest area of the Children’s Oncology Group (19). Thus far, the importance of EphB4 in tumorigenesis has been interrogated by methods involving inhibition of extracellular domain interaction with its ligand, as well as in vivo silencing of the receptor in murine tumor xenograft models (4, 8); however, the potential benefit of clinical small-molecule inhibitors targeting EphB4 kinase activity in cancer is yet to be explored. In this report, we have identified EphB4 as a druggable target in aRMS, a result obtained after investigating a surprising dichotomy for the signaling role of EphB4 through EphrinB2 versus through crosstalk with PDGFRβ and PDGF ligand. Results RNA-Interference–Assisted Protein Identification Screen. With the aim of identifying novel tyrosine kinase gene targets in aRMS, we first established primary tumor cell cultures from our genetically engineered, conditional mouse model of aRMS, as previously described (20, 21). Primary cell culture U23674 was derived from a primary limb tumor of a mouse comparable to Intergroup Rhabdomyosarcoma Study Group (IRSG) surgical stage T2b/N1/M0, and culture U48484 was derived from a pulmonary metastasis of a different tumor-bearing animal comparable to IRSG surgical stage T2b/N0/M1. We performed an RNA-interference assisted protein target identification (RAPID) screen of validated siRNAs to individually silence each member of the mouse tyrosine kinome (22, 23). Cell viability was assessed by MTS assay to determine the siRNAs that have deleterious effects on cell viability. The statistical threshold used to classify a significant decrease in tumor cell viability after siRNA gene silencing was as previously described (22, 23). This method identified Ntrk4, Pdgfrβ, and EphB4 in U23674, and Pdgfrβ, EphB4, and EphB6 in U48484. Interestingly, Pdgfrβ and EphB4 were both identified as targets in both cell lines (Fig. 1A). Expression of these RTKs at the protein level was validated by immunoblotting (Fig. 1B), and knockdown confirmed at the protein level (Fig. 1C). The average knockdown of Pdgfrβ and EphB4 in U23674 cells was ∼98% and 78%, respectively, and ∼95% and ∼98% in U48484, respectively (Fig. S1 A and B). We further validated the RAPID screen results using pooled EphB4 and Pdgfrβ siRNA in aRMS compared with a control mouse myoblast cell line (C2C12) (Fig. 1D). To determine if decrease in tumor cell viability from EphB4 silencing was a ligand-dependent or -independent phenotype, we silenced EphrinB2 and saw no appreciable decrease in tumor cell viability (Fig. 1D), suggesting EphB4’s oncogenic effect is independent of its cognate ligand, EphrinB2. There was no cooperative decrease in tumor cell viability when both receptors were silenced concurrently (Fig. 1D). Results of all MTS assays were confirmed by flow cytometry (Fig. S1C). To control for potential off-target effects causing the observed decrease in cell viability, we used the individual duplex siRNAs that comprise the Pdgfrβ and EphB4 siRNA pools used in the aforementioned studies to transfect U23674 and U48484 cells. The decrease in relative tumor cell viability we observed correlated with the individual duplex siRNA that most efficiently silenced Pdgfrβ or EphB4 (Fig. S1 D–G). EphB4 and EphrinB2 Is Expressed in Human and Mouse aRMS and EphB4 Is a Poor Prognostic Indicator in Fusion-Positive Human aRMS. No previous studies have examined expression or func-
tional significance of EphB4 or its ligand in aRMS. We examined expression of EphB4 and EphrinB2 in human and mouse aRMS. By quantitative RT-PCR (RT-qPCR), EPHB4 and EPHRINB2 expression were elevated in human aRMS compared with normal skeletal muscle, whereas in mouse aRMS only EphB4 levels were elevated (Fig. S2A). In both human skeletal muscle and aRMS, we saw a direct correlation between EphB4 and PDGFRβ expression level, with PAX3:FOXO1+ aRMS demonstrating a higher Pearson’s correlation coefficient (r = 0.91) compared 6384 | www.pnas.org/cgi/doi/10.1073/pnas.1403608111
Fig. 1. RNA-interference screen identifying EphB4 and PDGFRβ are required for tumor cell survival. (A) Cell viability result for the RNAi-assisted protein identification screen of primary (U23674) and metastatic (U48484) mouse aRMS tumor cultures. Dashed lines are ±2 SD with respect to median viability of cells. (B) Expression of EphB4, PDGFRβ, and EphrinB2 in U23674 and U48484 in addition to C2C12, an immortalized murine myoblast cell line. (C) Representative immunoblot of U23674 cells showing expression and knockdown efficiency of EphB4 and PDGFRβ in conditions identical to RNAi screen in A. White bar separates lane cropped from a lane on same gel. (D) Cell viability assessed by MTS assay on day 4 of murine aRMS cells and C2C12 transfected with nonspecific targeting siRNA, in addition to siRNA targeting EphB4 or Pdgfrß alone or in combination, EphrinB2 and Plk1 (+ control). *P < 0.05 compared with nonspecific siRNA by Student t test. For all experiments we included an siRNA targeted to Plk1 to serve as a positive control.
with PAX3:FOXO1− aRMS (r = 0.82) or human skeletal muscle (r = 0.80) (Fig. 2A). Immunoblot of PDGFRβ and EphB4 in four previously described human RMS cell lines, Rh5, Rh30, Rh3 (a subclone of Rh28), and Rh18 was also performed (Fig. 2B). Rh5, Rh30, and Rh3 are PAX3:FOXO1 fusion-positive aRMS, whereas Rh18 is a fusion-negative rhabdomyosarcoma. Both RTKs were present at the protein level in Rh5, Rh30, and Rh18, although the cell line Rh3 did not express EphB4. Immunohistochemistry on a human aRMS tissue microarray consisting of 19 unique cases with 31 sections confirmed expression of EphB4 (scores of 0–3) (Fig. S2B) and PDGFRβ (scores of 0–3) (Fig. S3A) at the protein level. The majority of tumor sections demonstrated strong staining of both EphB4 and PDGFRβ, although there were sections with weak expression of either receptor. Scoring by staining intensity is summarized in SI Methods. Fig. 2C includes representative images of human aRMS tissue stained for EphB4 and PDGFRβ, as well as their ligands EphrinB2 and PDGF-BB, respectively. In addition, wildtype human skeletal muscle was stained for EphB4 and PDGFRβ (Fig. S3 B and C). Additional images stained for the receptors and their corresponding ligands can be found in Figs. S4 and S5. To control for antibody specificity, antibodies were preincubated with blocking peptides before sections were stained (Figs. S4 and S5). PDGFRβ expression has been shown to be a strongly negative prognostic factor in rhabdomyosarcoma, reducing 5-y survival probability by >35% when adjusting for known clinical covariates (3). A similar analysis of this IRSG-IV microarray dataset (3) demonstrated that EPHB4 expression showed a >70% decrease in 5-y survival between high and low EPHB4 levels in fusion positive aRMS (Fig. 2D). Taken together, these results are consistent with a role of potential clinical significance for EphB4 in aRMS. Furthermore, Aslam et al.
unidentified target of PAX3:FOXO1. An indirect relationship is likely because previous ChIP-Seq performed on global/genomewide PAX3:FOXO1 binding sites in human aRMS, as well as various PAX3 isoform binding sites, did not identify EphB4 or PDGFRβ as direct targets of these transcription factors (25, 26). In our murine aRMS primary cell culture U23674, we observed that decreased Pax3:Foxo1 levels further decreases cell viability when silencing PDGFRβ or EphB4 individually (Fig. S6E). To confirm that EphB4 is a target of Pax3:Foxo1, we introduced a pCDNA3 vector expressing the Pax3:Foxo1 fusion transcript, or an empty vector, into murine myoblast (C2C12) cells. Accordingly, we observed increased expression of EphB4 at both the mRNA and protein level in C2C12 cells transfected with the fusion transcript compared with control C2C12 cells (Fig. S7). Pax3:Foxo1 expression was confirmed at the protein level in these cells (Fig. S7B). These data are consistent with EphB4 being either a direct or indirect target of Pax3:Foxo1a.
significant correlating expression of PDGFRβ and EPHB4 suggested potential interactions for further investigation.
status of EphB4 and Pdgfrβ in U23674 and U48484 cells by immunoprecipitating both RTKs from cell lysates and probing with antiphospho-tyrosine (p-Tyr) antibody, revealing that both receptors are phosphorylated at baseline (Fig. 4A). The primary cell cultures (U23674 and U48484) were treated with imatinib (targets PDGFRβ) and dasatinib (targets PDGFRβ and EphB4) for 72 h and cell viability assessed. These inhibitors decreased tumor cell viability and phosphorylation of their targeted receptors (Fig. 4 A and B). Dasatinib proved more potent; IC50s were achieved in the nanomolar range, whereas the imatinib IC50 was in the micromolar range. The IC50 for dasatinib in Rh5 and Rh30 (human aRMS which express PDGFRβ and EphB4) (Fig. 2B) were 35 and 200 nM, respectively (Fig. S8A).
Knockdown of PAX3:FOXO1 Regulates EphB4 Levels in Murine and Human aRMS. Previously, other RTKs such as PDGFRα and
siRNA-Mediated Knockdown of Pdgfrβ or EphB4 Induces Apoptosis and Abrogates Anchorage-Independent Colony Formation. To determine
c-Met have been established as downstream transcriptional targets of PAX3:FOXO1 (17, 24). To investigate whether EphB4 and PDGFRβ are directly or indirectly regulated by the PAX3: FOXO1 fusion protein, we silenced the fusion gene in both U23674 and U48484 using siRNA targeted to eYFP. This approach can be taken in our conditional mouse model because eYFP is expressed by means of an internal ribosomal entry site on a bicistronic transcript with Pax3:Foxo1 (Pax3:Foxo1-IRESeYFP). We are thus able to knock down Pax3:Foxo1 in tumor cells by targeting eYFP. Silencing of Pax3:Foxo1 led to decreased levels of EphB4 protein (Fig. 3A), but Pdgfrβ expression surprisingly increased. This result for Pdgfrβ is the opposite of what we have observed for Pdgfrα (17). The same phenomenon was observed in Rh5 (human aRMS) when PAX3:FOXO1 was silenced (Fig. 3A). Furthermore, silencing of PAX3:FOXO1 in murine and human aRMS resulted in a decrease and increase in EphB4 and PDGFRβ levels, respectively (Fig. S6 A and B). We hypothesized that the increase observed in PDGFRβ protein may represent a compensatory response to loss of EphB4 after silencing of PAX3:FOXO1 or a tumor cell response to stress. To test the former hypothesis, we studied the effect on PDGFRβ protein level after direct silencing of EphB4 in murine and human aRMS cells. Indeed, we observed that EphB4 silencing resulted in a significant increase in PDGFRβ protein (Fig. 3B), but had no effect on PDGFRβ mRNA levels in murine and human aRMS (Fig. S6 C and D); this suggests a posttranslational mechanism may be responsible for increased PDGFRβ protein. Silencing of PDGFRβ in both murine and human aRMS resulted in a significant decrease of EphB4 at the protein (Fig. 3B) and mRNA level (Fig. S6 C and D). Decreased EphB4 expression from PAX3:FOXO1 knockdown suggests EphB4 is a previously
whether the decrease in cell viability observed after knockdown
Aslam et al.
Fig. 3. Effect of PAX3:FOXO1 on EphB4 and PDGFRβ expression and sensitivity to siRNA mediated silencing. (A) Effect of Pax3:Foxo1 silencing by eYFP-targeted siRNA on expression of EphB4 and PDGFRβ by immunoblot in U23674 and U48484 murine aRMS cells, and PAX3:FOXO1 silencing in human aRMS cell line Rh5 demonstrates an identical phenomenon. (B) Effect on PDGFRβ expression after EphB4 silencing in U23674, U48484, and Rh5 cells.
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Fig. 2. EphB4 and EphrinB2 levels in mouse aRMS are similar to human aRMS and tumor microarray of human aRMS shows strong expression of EphB4 and PDGFRβ by immunohistochemistry. (A) Correlation of PDGFRβ and EphB4 between human normal skeletal muscle (SKM) and aRMS tumors. r indicates calculated Pearson’s correlation coefficient. (B) Immunoblot of previously established human aRMS cell lines positive for PAX3:FOXO1: Rh5, Rh30, Rh3, and fusion negative Rh18 show expression of PDGFRβ and EphB4. (C) Immunohistochemistry of human aRMS tumors stained for EphB4, EphrinB2, PDGFRβ and PDGF-BB. (Scale bar, 50 μm.) (D) EPHB4 expression portends a poor overall survival for patients in PAX3:FOXO1 fusion positive aRMS (P = 0.032). Histogram signifies frequency of varying EPHB4 expression level in fusion positive aRMS patients.
Activation of PDGFRβ and EphB4 in Murine aRMS Can Be Blocked by Tyrosine Kinase Inhibitors. We next determined the activation
of each receptor individually, we observed a reduction in phosphorylation of Erk 1/2 and Akt. FAK phosphorylation was reduced after silencing of EphB4, but not Pdgfrβ in both U23674 and U48484 cells (Fig. 4E). To further interrogate signaling from Pdgfrβ and EphB4 receptor stimulation, cells were serumstarved then stimulated with their respective ligands. PDGF-BB stimulated phosphorylation of Pdgfrβ, Akt, FAK, and Erk 1/2 (Fig. 5A). EphrinB2 stimulation of either cell line did not yield an increase in FAK, Akt, or Erk 1/2 phosphorylation (Fig. 5A). These results were also observed in the human aRMS cell lines Rh5 and Rh30 (Fig. 5B). Because EphrinB2 stimulation did not result in activation of Akt, Erk, or FAK, we immunoprecipitated EphB4 and probed with an anti–p-Tyr antibody to confirm EphB4 phosphorylation upon EphrinB2 stimulation (Fig. 6A). Taken together, these data suggest that with PDGF-BB ligand stimulation, PDGFRβ activates the common downstream effectors Akt and Erk1/2, whereas EphB4 signaling through Akt and Erk 1/2 appears to be independent of its cognate ligand, EphrinB2, in human and murine aRMS. Therefore, to test if Pdgfrβ could activate EphB4 (and thus, hypothetically, downstream mediators Akt and Erk 1/2), we looked for evidence of the ability of Pdgfrβ to phosphorylate EphB4. We immunoprecipitated EphB4 with and without stimulation of Pdgfrβ with PDGF-BB and showed that phosphorylation of EphB4 increases after PDGF-BB treatment (Fig. 6B). Imatinib was able to inhibit this effect despite EphB4 not being a direct target of imatinib. This result suggested
Fig. 4. PDGFRβ and EphB4 inhibition abrogates growth of mouse tumor primary cell cultures. (A) Small molecule inhibition of Pdgfrβ phosphorylation by imatinib or dasatinib and EphB4 phosphorylation by dasatinib. DMSO was used as a vehicle control. (B) A 72 h MTS assay on cells treated with increasing concentrations of imatinib and dasatinib. IC50 for each drug and cell line are shown. (C) Knockdown of Pdgfrβ and EphB4 results in tumor cells undergoing apoptosis as measured by Annexin V. Early apoptotic cells are Annexin V+ only, late apoptotic cells are Annexin V and 7-AAD+. (D) Anchorage-independent colony formation is decreased significantly in murine aRMS U23674 and U48484 cells when Pdgfrβ and EphB4 are silenced by siRNA. Relative number of colonies normalized to nonspecific siRNA. (E) Immunoblot showing signaling downstream EphB4 and PDGFRβ shown after siRNA mediated silencing of each kinase in U23674 and U48484 cells. For PDGFRβ and EphB4 levels after corresponding siRNAs, see Fig. 3B. *P < 0.05 compared with nonspecific siRNA by Student t test.
of Pdgfrβ and EphB4 was from either cell cycle redistribution and/or apoptosis, we performed cell cycle analysis of U23674 and U48484 cells by FACS after transfection with nonspecific Pdgfrβor EphB4-targeted siRNA, revealing cell cycle distribution was not significantly altered (Fig. S8B). Silencing of EphB4 or Pdgfrβ individually or simultaneously resulted in significant apoptosis as determined by FACS (Fig. 4C). Silencing of Pax3:Foxo1 was used as a comparator of tumor cell apoptosis (Fig. 4C). The ability of murine aRMS cells to form colonies in an anchorage-independent colony formation assay was significantly inhibited upon Pdgfrβ and EphB4 silencing (Fig. 4D and Fig. S8C). Taken together, these data indicate a vital role of Pdgfrβ and EphB4 in tumor cell survival and transformation. Akt, Erk 1/2 Are Downstream of PDGFRβ and EphB4, Whereas FAK Is Downstream of EphB4 and EphB4 Is Phosphorylated upon PDGF-BB Stimulation. We next assessed the downstream signaling pathways
affected by silencing of Pdgfrβ and EphB4. Upon knockdown 6386 | www.pnas.org/cgi/doi/10.1073/pnas.1403608111
Fig. 5. PDGFRβ but not EphB4 stimulation by their respective ligands activates downstream cell survival and proliferative pathways in murine and human aRMS. (A) Immunoblots showing U23674 and U48484 cells stimulated (after overnight serum starvation) with PDGF-BB (10 ng/mL, 15 min) alone or in the presence of imatinib (100 nM, 1 h) or with EphrinB2 (2 μg/mL, 15 min) alone or in the presence of dasatinib (100 nM, 1 h). Fc only (Fc portion lacking clustered ligand, 2 μg/mL for 15 min) lane represents control for EphrinB2. (B) Immunoblots showing Rh5 and Rh30 cells stimulated (after overnight serum starvation) with PDGF-BB (10 ng/mL, 15 min) alone or in the presence of imatinib (100 nM, 1 h) or with EphrinB2 (2 μg/mL, 15 min) alone or in the presence of dasatinib (100 nM, 1 h).
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that EphrinB2 was similarly stimulating a distinct signaling cascade. Because Crkl is a monogamous substrate for phosphorylation by Abl (6), we examined Crkl phosphorylation upon EphrinB2 treatment. p-Crkl was stimulated by EphrinB2 treatment in both murine and human aRMS cells, U48484 and Rh5, respectively (Fig. 6E). The murine aRMS primary cell cultures U23674 and U48484 along with human aRMS cell lines Rh5 and Rh30 in the presence of Fc only control or EphrinB2 for a 3 d period showed that all cell lines underwent apoptosis upon EphrinB2 treatment (Fig. 6 F and G, respectively). These results indicate that although EphrinB2 does stimulate EphB4, the signaling cascade initiated by EphrinB2 is distinct from that stimulated through cross-talk with PDGFRβ.
Fig. 6. PDGFRβ and EphB4 cross-talk; EphrinB2 signals through EphB4 to induce apoptosis in murine and human aRMS. (A) Representative immunoblot indicating EphrinB2 treatment (2 μg/mL, 15 min) results in phosphorylation of EphB4 and (B) PDGF-BB treatment (10 ng/mL, 15 min) results in EphB4 phosphorylation in murine aRMS (U48484) and human aRMS (Rh30). PDGF-BB mediated phosphorylation is inhibited by imatinib. (C) A graded increase in PDGF-BB concentrations of 0, 100, 200, 500, and 1,000 ng/mL serves as a mitogen in U23674 and U48484 cells. (D) A graded increase in PDGF-BB concentrations of 0, 100, 200, 500, and 1,000 ng/mL serves as a mitogen in Rh5 and Rh30 cells. (E) In murine aRMS U48484 and human aRMS Rh5, EphrinB2 (2 μg/mL, 15 min) treatment stimulates p-Crkl, which dasatinib inhibits. (F) EphrinB2 (20 μg/mL) treatment versus Fc only for 3 d of mouse aRMS and (G) human aRMS cells results in tumor cells undergoing apoptosis as measured by Annexin V. Early apoptotic cells are Annexin V+ only, late apoptotic cells are Annexin V and 7-AAD+. *P < 0.01, **P < 0.05 by Student t test compared with respective control.
dasatinib’s increased efficacy relative to imatinib was conserved in vivo, we orthotopically engrafted (SCID/hairless/outbred) mice with U23674 cells and treated these mice with dasatinib (15 or 50 mg/kg) or imatinib (100 mg/kg). Once tumors were 0.2 cc3, we commenced daily treatment with drug or vehicle control. Survival was significantly extended with dasatinib at a dose of 50 mg/kg compared with all other treatment groups (P < 0.05) (Fig. 7A). To confirm drug administration in vivo inhibited Pdgfrβ and EphB4 phosphorylation, immunoblotting of tumor lysates were performed. Immunoblot of tumors treated with daily vehicle, imatinib, and both high- or low-dose dasatinib showed that Pdgfrβ and EphB4 phosphorylation was decreased in dasatinibtreated mice, whereas only Pdgfrβ phosphorylation was decreased in mice treated with imatinib (Fig. 7 B and C). Taken together, these data suggest that blockade of only PDGFRβ activity (by imatinib) can impart some inhibition of tumor growth, whereas direct blockade of both Pdgfrβ and EphB4 activity (by dasatinib) imparts the greatest effect on inhibition of tumor growth. Discussion Through an unbiased RNAi screen targeted to the tyrosine kinome, we identified EphB4 as a novel target critical to tumor cell viability. We also found EphB4 to be a poor prognostic indicator in PAX3:FOXO1 fusion-positive aRMS. EphB4 has
that the phosphorylation of EphB4 is mediated by an imatinib target, such as PDGFRβ (one of only a few imatinib targets). Finally, we observed that PDGF-BB ligand increased cell growth by two- to threefold in a dose-dependent manner, affirming a mitogenic phenotype in murine and human aRMS (Fig. 6 C and D, respectively). In addition, the demonstrated ability of kinase inhibitors imatinib or dasatinib to ablate this mitogenic phenotype in murine primary cell culture of aRMS (U23674) (Fig. S8D) suggests that PDGF-BB ligand-mediated growth is a kinasedependent process. EphrinB2 Treatment Induces Apoptosis in Murine and Human aRMS.
Although PDGF-BB activated PDGFRβ, EphB4, Akt, and Erk 1/2, resulting in stimulating mitogenesis of tumor cells, EphrinB2 was not capable of stimulating these downstream signaling pathways, despite inducing phosphorylation of EphB4, suggesting that canonical EphB4 signaling may serve a different role. In prior reports, the Eph RTK family has been shown to function as tumor suppressors (5, 11) or as oncogenes (4, 27) in a wide variety of malignancies. An EphrinB2 induced EphB4 signaling cascade mediated by the Abl-Crkl pathway resulting in apoptosis has been shown in mammary carcinoma (6). Based on our previous observations in these murine and human aRMS, we hypothesized Aslam et al.
Fig. 7. Dasatinib significantly slows aggressive murine aRMS tumor growth versus imatinib in vivo. (A) Kaplan–Meier survival curve of SCID/hairless/ outbred mice treated with vehicle daily, dasatinib 15 mg·kg·d, dasatinib 50 mg·kg·d or imatinib 100 mg·kg·d, with endpoint measurement being days for tumor volume to reach 2.0 cm3. Mantel–Cox analysis of survival curves revealed P < 0.05 between comparison of vehicle and dasatinib 15 mg/kg and imatinib 100 mg/kg and P < 0.05 between 50 mg/kg dasatinib and all other treatment groups. (B) Immunoblot confirming inhibition of PDGFRβ phosphorylation by imatinib and dasatinib in vivo and (C) inhibition of EphB4 phosphorylation by dasatinib only (imatinib unable to inhibit EphB4 activation) from randomly selected tumor-bearing mice. White bar separates lanes cropped from the same gel.
PNAS | April 29, 2014 | vol. 111 | no. 17 | 6387
MEDICAL SCIENCES
Dasatinib-Mediated Inhibition of both PDGFRβ and EphB4 Improves Survival More than Imatinib-Mediated Inhibition of PDGFRβ Alone in an Orthotopic Allograft Model of aRMS. To determine whether
in Eph kinase-ephrin interactions (9). We show dasatinib is efficacious at physiologically achievable serum concentrations (31) but acknowledge that while dasatinib inhibits both PDGFRβ and EphB4, it also inhibits many other tyrosine (and serine/threonine) kinases. Therefore, its antagonistic effects on aRMS cells in culture and in the xenograft studies could be due to inhibition of not only PDGFRβ and EphB4, but could also be a result of its inhibition of other kinases as well. It might also stand to reason that clinical trials targeting EphB4 in aRMS would benefit well from a biomarker-driven strategy to identify which patients are best suited for EphB4-targeted therapy.
previously been identified to contribute toward tumor suppression, apoptosis, or an increased malignant phenotype in a variety of other cancers (4–6, 9, 11, 27, 28). In our studies, we further identified a previously undescribed mechanism of cross-talk between EphB4 with another RTK, PDGFRβ, which is known to be activated and expressed in aRMS (29, 30). Our studies demonstrated that both cell survival (Akt) and cell proliferation (Erk) signaling pathways are downstream of both RTKs, suggesting that both RTKs converge on similar pathways. We show EphB4 stimulation by its cognate ligand, EphrinB2, was unable to stimulate Akt or Erk; however, PDGFRβ stimulation with PDGF-BB ligand was able to phosphorylate EphB4 as well as activate these downstream pathways in both murine and human aRMS. Signaling interrogation revealed p-FAK was abrogated with EphB4 knockdown, but not by Pdgfrβ knockdown. However, PDGFRβ stimulation via PDGF-BB was able to stimulate FAK phosphorylation, presumably through cross-talk with EphB4. These results allude to EphB4 having an ability to activate oncogenic signaling pathways independent of PDGFRβ or its cognate ligand, EphrinB2, as we also describe. Based on our results (summarized in Fig. S9), as well as previous literature corroborating the phenomenon of ephrin kinase signaling initiated by noncanonical pathways, we deduce that EphB4 in aRMS may serve as a scaffold to activate other signaling pathways or associate with other proteins that are able to activate the receptor, similar to its cross-talk with PDGFRβ. Such a ligand-independent function of Eph receptors has been described in other model systems, such as prostate cancer and glioma cells (9). To translate our results to the clinic, we chose to test the Food and Drug Administration-approved, dual PDGFRβ and EphB4 inhibitor, dasatinib. A promising secondary attribute of dasatinib is its ability to inhibit the Src family kinases, which is relevant because the Src family kinases are responsible for reverse signaling
ACKNOWLEDGMENTS. We thank Stephanie Willis, Elaine Huang, and Chris Eide for technical assistance, as well as other B.J.D. laboratory members for their invaluable support. M.I.A. is a Howard Hughes Medical Institute Medical Research Fellow. B.J.D. is an investigator of the Howard Hughes Medical Institute. This work was supported by Grant 5R01CA133229 and in part by a grant from the Joanna McAfee Childhood Cancer Foundation (to C.K.); a grant from the William Lawrence and Blanche Hughes Foundation (to J.W.T.); a grant from the Leukemia and Lymphoma Society (to J.W.T.); a grant from the V Foundation for Cancer Research (to J.W.T.); and National Cancer Institute Grant 4R00CA151457 (to J.W.T.). The Cooperative Human Tissue Network and the Children’s Oncology Group Biorepository are funded by the National Cancer Institute.
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Methods Human Samples. Human samples (normal skeletal muscle and tumor RNA, and human tissue microarrays) were provided by the Cooperative Human Tissue Network or Children’s Oncology Group Biorepository. Studies were performed under an Institutional Review Board-approved protocol. Other Methods. Detailed descriptions of cell culture, RT-qPCR, immunohistochemistry, immunoblotting and immunoprecipitation, RNA-interference studies, in vitro growth inhibition and proliferation assays, cell viability and apoptosis assays, in vivo studies, and statistical considerations are given in SI Methods.
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