SEMA 3C drives cancer growth by transactivating multiple receptor tyrosine kinases via Plexin B1
2018; Springer Nature; Volume: 10; Issue: 2 Linguagem: Inglês
10.15252/emmm.201707689
ISSN1757-4684
AutoresJames W. Peacock, Ario Takeuchi, Norihiro Hayashi, Liangliang Liu, Kevin J. Tam, Nader Al Nakouzi, Nastaran Khazamipour, Tabitha Tombe, Takashi Dejima, Kevin C.K. Lee, Masaki Shiota, Daksh Thaper, Wilson CW Lee, Daniel H. Hui, Hidetoshi Kuruma, Larissa Ivanova, Parvin Yenki, Ivy Z. F. Jiao, Shahram Khosravi, Alice Mui, Ladan Fazli, Amina Zoubeidi, Mads Daugaard, Martin Gleave, Christopher J. Ong,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoResearch Article18 January 2018Open Access Source DataTransparent process SEMA3C drives cancer growth by transactivating multiple receptor tyrosine kinases via Plexin B1 James W Peacock James W Peacock Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Ario Takeuchi Ario Takeuchi Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urology, Graduate School of Medical Sciences, Kyushi University, Fukuoka, Japan Search for more papers by this author Norihiro Hayashi Norihiro Hayashi Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urology, The Jikei University School of Medicine, Tokyo, Japan Search for more papers by this author Liangliang Liu Liangliang Liu Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Kevin J Tam Kevin J Tam Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Nader Al Nakouzi Nader Al Nakouzi Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Nastaran Khazamipour Nastaran Khazamipour Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Tabitha Tombe Tabitha Tombe Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Takashi Dejima Takashi Dejima Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urology, Graduate School of Medical Sciences, Kyushi University, Fukuoka, Japan Search for more papers by this author Kevin CK Lee Kevin CK Lee Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Masaki Shiota Masaki Shiota Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urology, Graduate School of Medical Sciences, Kyushi University, Fukuoka, Japan Search for more papers by this author Daksh Thaper Daksh Thaper Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Wilson CW Lee Wilson CW Lee Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Daniel HF Hui Daniel HF Hui Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Hidetoshi Kuruma Hidetoshi Kuruma Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Larissa Ivanova Larissa Ivanova Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Parvin Yenki Parvin Yenki Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Ivy ZF Jiao Ivy ZF Jiao Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Shahram Khosravi Shahram Khosravi Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Alice L-F Mui Alice L-F Mui Department of Surgery, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Ladan Fazli Ladan Fazli Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Amina Zoubeidi Amina Zoubeidi Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Mads Daugaard Mads Daugaard Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Martin E Gleave Corresponding Author Martin E Gleave [email protected] orcid.org/0000-0003-4235-0167 Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Christopher J Ong Corresponding Author Christopher J Ong [email protected] [email protected] orcid.org/0000-0002-0175-8724 Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author James W Peacock James W Peacock Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Ario Takeuchi Ario Takeuchi Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urology, Graduate School of Medical Sciences, Kyushi University, Fukuoka, Japan Search for more papers by this author Norihiro Hayashi Norihiro Hayashi Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urology, The Jikei University School of Medicine, Tokyo, Japan Search for more papers by this author Liangliang Liu Liangliang Liu Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Kevin J Tam Kevin J Tam Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Nader Al Nakouzi Nader Al Nakouzi Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Nastaran Khazamipour Nastaran Khazamipour Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Tabitha Tombe Tabitha Tombe Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Takashi Dejima Takashi Dejima Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urology, Graduate School of Medical Sciences, Kyushi University, Fukuoka, Japan Search for more papers by this author Kevin CK Lee Kevin CK Lee Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Masaki Shiota Masaki Shiota Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urology, Graduate School of Medical Sciences, Kyushi University, Fukuoka, Japan Search for more papers by this author Daksh Thaper Daksh Thaper Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Wilson CW Lee Wilson CW Lee Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Daniel HF Hui Daniel HF Hui Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Hidetoshi Kuruma Hidetoshi Kuruma Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Larissa Ivanova Larissa Ivanova Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Parvin Yenki Parvin Yenki Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Ivy ZF Jiao Ivy ZF Jiao Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Shahram Khosravi Shahram Khosravi Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Alice L-F Mui Alice L-F Mui Department of Surgery, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Ladan Fazli Ladan Fazli Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Amina Zoubeidi Amina Zoubeidi Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Mads Daugaard Mads Daugaard Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Martin E Gleave Corresponding Author Martin E Gleave [email protected] orcid.org/0000-0003-4235-0167 Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Christopher J Ong Corresponding Author Christopher J Ong [email protected] [email protected] orcid.org/0000-0002-0175-8724 Vancouver Prostate Centre, Vancouver, BC, Canada Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Author Information James W Peacock1,2, Ario Takeuchi1,3, Norihiro Hayashi1,4, Liangliang Liu1, Kevin J Tam1,2, Nader Al Nakouzi1, Nastaran Khazamipour1, Tabitha Tombe1, Takashi Dejima1,3, Kevin CK Lee1, Masaki Shiota1,3, Daksh Thaper1,2, Wilson CW Lee1, Daniel HF Hui1, Hidetoshi Kuruma1, Larissa Ivanova1, Parvin Yenki1,2, Ivy ZF Jiao1, Shahram Khosravi1, Alice L-F Mui5, Ladan Fazli1, Amina Zoubeidi1,2, Mads Daugaard1,2, Martin E Gleave *,1,2 and Christopher J Ong *,*,1,2 1Vancouver Prostate Centre, Vancouver, BC, Canada 2Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada 3Department of Urology, Graduate School of Medical Sciences, Kyushi University, Fukuoka, Japan 4Department of Urology, The Jikei University School of Medicine, Tokyo, Japan 5Department of Surgery, University of British Columbia, Vancouver, BC, Canada *Corresponding author. Tel: +1 6046 752568; E-mail: [email protected] *Corresponding author. Tel: +1 6048 754111 ext. 63120; E-mail: [email protected] or [email protected] EMBO Mol Med (2018)10:219-238https://doi.org/10.15252/emmm.201707689 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand-independent manner via Plexin B1. SEMA3C expression levels increase in castration-resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide-resistant progression. Plexin B1 sema domain-containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post-castration in vivo. SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer. Synopsis SEMA3C is a secreted autocrine factor that drives cancer growth and treatment resistance by transactivating multiple receptor tyrosine kinases via Plexin B1 in a cognate ligand-independent manner. Antagonizing SEMA3C signaling inhibits prostate cancer growth in vitro and in vivo. SEMA3C drives EGFR, ErbB2, and MET signaling cascades. Plexin B1 (PLXNB1) is a receptor for SEMA3C. Autocrine SEMA3C promotes prostate cancer growth and treatment resistance. A recombinant PLEXIN B1 decoy protein attenuates SEMA3C signaling and prostate cancer growth. Introduction Androgen deprivation therapy (ADT) is first-line systemic therapy for men with metastatic prostate cancer (PCa). Unfortunately, the survival benefit from ADT is limited by emergence of lethal CRPC (Bruchovsky et al, 1989; Goldenberg et al, 1988). Development of CRPC is a complex process that has been attributed to a variety of mechanisms including reactivation of the androgen receptor (AR) axis and activation of growth factor signaling pathways (Yap et al, 2011). While many growth factor receptor pathways are activated in PCa progression (such as epidermal growth factor receptor (EGFR), ErbB2 (HER2/neu), and MET), ligands that drive activation of these pathways remain poorly defined. Our gene expression profiling data identified SEMA3C, a member of the secreted class 3 semaphorins, as a highly expressed gene in CRPC and AR pathway inhibitor-recurrent tumors. SEMA3C was prioritized as a putative CRPC driver as it was identified in two independent genomewide profiling studies. In search of novel targets associated with expression of PTEN, a gene that is frequently mutated in advanced PCa, DNA microarray profiling identified SEMA3C among the top three most differentially expressed genes between PTEN+ vs. PTEN−/− cancer cells (Peacock et al, 2009). In addition, SEMA3C was found to be an NFκB-regulated survival factor induced by CLU (Zoubeidi et al, 2010b), a molecular chaperone associated with treatment resistance, suggesting SEMA3C may be functionally relevant in CRPC (Zoubeidi et al, 2010a). As a whole, semaphorins are a large family of evolutionarily conserved secreted or cell surface signaling proteins originally discovered as important mediators of cell migration and axon guidance in the developing nervous system (Cagnoni & Tamagnone, 2014). While semaphorins have been best characterized in the nervous system, they have also been implicated in a variety of dynamic physiological processes including angiogenesis, tissue morphogenesis, immunity, and cancer (Cagnoni & Tamagnone, 2014). Semaphorins exhibit diverse activities in cancer. Depending on the tumor type and context, various class 3 semaphorins either promote (SEMA3C and 3E) or suppress (SEMA3A, 3B, 3D, 3E, and 3F) cancer growth (Rehman & Tamagnone, 2013). Among the class 3 semaphorins, SEMA3C is notable for its frequent association with tumor progression and poor prognosis across multiple tumor types including lung, breast, gastric, and ovarian cancers as well as glioblastoma (Rehman & Tamagnone, 2013). Increased expression of SEMA3C is associated with poor prognosis and tumor progression in a number of cancers (Yamada et al, 1997; Martin-Satue & Blanco, 1999; Konno, 2001; Galani et al, 2002; Herman & Meadows, 2007; Miyato et al, 2012; Xu et al, 2017). In PCa, SEMA3C promotes cell migration and invasion in vitro (Herman & Meadows, 2007) and drives EMT and stemness (Tam et al, 2017) and SEMA3C expression is a predictive marker for biochemical recurrence (BCR) (Li et al, 2013). Therefore, we investigated whether SEMA3C could be a key growth factor that drives CRPC progression and treatment resistance, and set out to develop a therapeutic protein inhibitor of SEMA3C signaling. Results Increased SEMA3C expression is associated with CRPC bone metastases To define SEMA3C expression levels in benign and cancerous prostate specimens and bone metastases, we assessed the levels of SEMA3C by tissue microarray (TMA) immunohistochemical (IHC) staining of a panel of 280 human PCa specimens representing benign prostatic hyperplasia (BPH, n = 12), untreated hormone naive (n = 114), neo-adjuvant hormone therapy (NHT)-treated (n = 87), NHT- and docetaxel-treated (n = 53) radical prostatectomy PCa specimens, as well as CRPC bone metastases (n = 30) collected immediately after death via University of Washington Rapid Autopsy program (Rocchi et al, 2005). Specimens were graded on a 0–3, intensity scale representing the range from no staining to high staining by visual scoring and automated quantitative image analysis. As shown in Fig 1A and B, increased SEMA3C expression was associated with advanced PCa that have been heavily treated with NHT and docetaxel (DTXL) (*P = 0.02), and CRPC bone metastases (**P = 0.0075; Fig 1B). We validated the specificity of SEMA3C (N20) antibodies to detect SEMA3C in DU145 cells transfected with scramble (siScr) or SEMA3C siRNA (siSEMA3C-1) using confocal immunofluorescent microscopy. The siSEMA3C treatment reduced SEMA3C staining compared to siScr control cells (Appendix Fig S1A). Figure 1. Increased SEMA3C expression correlates with CRPC and SEMA3C activates EGFR, ErbB2, MET, and SRC tyrosine kinase signaling Representative SEMA3C immunostaining of BPH, untreated hormone naïve, neo-adjuvant hormone therapy (NHT)-treated, NHT- and docetaxel-treated radical prostatectomy PCa specimens, and a bone CRPC metastasis specimen. Scale bar: 100 μm. IHC intensity scores of SEMA3C (N20) staining from BPH, untreated, NHT-treated, NHT- and docetaxel-treated, and bone metastasis. Mean ± SEM; *P = 0.02, **P = 0.0075. Statistical analysis was performed using the unpaired two-tailed Student's t-test. Immunoblot analyses of SEMA3C levels in conditioned media produced from androgen dependent (LNCaP), castration-resistant (C4-2, 22Rv1), enzalutamide-resistant (MR49F), and androgen receptor-negative (DU145) PCa cells seeded at equivalent cell density. Whole-cell lysates were immunoblotted with Ponceau staining as control. The data are representative of three independent experiments. Activation of EGFR, HER2/ErbB2, and downstream signaling in LNCaP cells treated with varying concentrations of SEMA3C:Fc (0–2 μM) for 10 min. Levels of indicated phosphoproteins and total proteins were assessed by immunoblot analyses. Vinculin is shown as loading control. Boxplots of RPPA measurements of indicated phosphoprotein levels in 498 patient tumor samples expressing SEMA3C high versus SEMA3C low mRNA levels from TCGA prostate adenocarcinoma provisional data set. Boxes span the interquartile range. Horizontal line within the box represents the median phosphoprotein levels. Error bars represent the range from the highest to the lowest observations. Statistical analysis was performed using the unpaired two-tailed Student's t-test. SEMA3C activates MET in a dose-dependent manner. DU145 cells were serum-starved and then stimulated with SEMA3C at the indicated doses for 10 min. Activation of MET was determined by immunoblotting with phospho-MET Abs. Source data are available online for this figure. Source Data for Figure 1 [emmm201707689-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Next, we examined SEMA3C expression in a panel of cell lines including benign prostate epithelial cells as well as androgen-sensitive, castrate-resistant, enzalutamide-resistant, and androgen receptor-negative cell lines. Elevated secreted SEMA3C levels were found in conditioned media of castrate-resistant C4-2 and 22Rv1, enzalutamide-resistant MR49F cells, and androgen-independent DU145 cells and androgen-sensitive LNCaP cells (Fig 1C and Appendix Fig S1B) compared to two benign immortalized prostatic cells, RWPE-1 and BPH-1 cells (Appendix Fig S1B). We have observed all forms of SEMA3C secreted from all of the prostate cancer cell lines. There is typically a doublet that runs at about 83 kDa that likely represents the full-length protein and the C-terminal cleavage product often referred to as the ∆13, respectively. We also typically see a band that runs below 70 kDa that represents the p65 cleavage product. As compared to prostate cancer cell lines, secreted SEMA3C expression was very low in immortalized benign prostate epithelial cell lines, RWPE-1 and BPH-1, respectively (Appendix Fig S1B). Mining of gene expression profiling data from Chen et al (2004) revealed an association between increased SEMA3C expression and resistance to anti-androgen therapy in six out of seven isogenic hormone-sensitive and castration-resistant xenograft pairs [NCBI GEO GDS535 (Barrett et al, 2007)] (Appendix Fig S1C). Consistent with these findings, we found a trend of higher levels of SEMA3C mRNA in castration-resistant LNCaP xenograft tumors as compared to tumors from non-castrate male mice (P = 0.075) (Appendix Fig S1D). We noted that a subgroup (approx. 50%) of the CRPC tumors had high SEMA3C, which suggests that SEMA3C overexpression may represent one mechanism to achieve castration resistance. SEMA3C activates signaling through EGFR, HER2, and MET receptor tyrosine kinases To identify potential signaling pathways regulated by SEMA3C in LNCaP cells, we performed an unbiased proteomic screen using the Kinex KAM-1.1 antibody microarray chip from Kinexus Biosciences Corp., containing over 650 antibodies, including > 270 phospho-site-specific antibodies as well as antibodies for detection of > 240 protein kinases, 28 phosphatases, and 90 other cell signaling proteins that regulate cell proliferation, stress, and apoptosis. The levels of signaling proteins were compared in SEMA3C-overexpressing LNCaP cells to empty vector-transduced LNCaP cells, and in parallel in SEMA3C antisense knockdown compared to scrambled oligonucleotide control-treated LNCaP cells. Intriguingly, the Kinex screen revealed a number of key phosphoproteins that were upregulated by SEMA3C overexpression and correspondingly, downregulated by SEMA3C silencing (Appendix Table S1), including Erb family RTKs (ErbB2 and EGFR), PI3K/PTEN cell survival pathway proteins (Akt1 and 4EBP1), and cell cycle regulatory proteins (Rb and CDK1/2), implicating the EGFR/ErbB2 pathway in SEMA3C signaling. To investigate whether naturally secreted SEMA3C could activate the EGFR/ErbB2 signaling pathway, we treated LNCaP cells with conditioned medium (CM) harvested from HEK 293T cells that overexpress full-length wild-type SEMA3C. We observed a dosage-dependent increase in EGFR, SHC, and MAPK phosphorylation with increasing concentration of SEMA3C containing CM and a corresponding decrease in EGFR, SHC, and MAPK phosphorylation in SEMA3C immuno-depleted CM, suggesting that SEMA3C is an autocrine growth factor that drives EGFR activation (Appendix Fig S1E). To validate whether SEMA3C activates the EGFR/ErbB2 pathway, LNCaP cells were treated with increasing concentrations of full-length recombinant SEMA3C-Fc fusion protein (SEMA3C:Fc) (0–2 μM) for 10 min and phosphorylation of EGFR, ErbB2 (HER2), and downstream signaling proteins (SRC, SHC, and MAPK) was analyzed by immunoblotting. SEMA3C treatment triggered dose-dependent increase in phosphorylation of EGFR, ErbB2, SRC, SHC, and p44/42 MAPK in LNCaP cells (Fig 1D and Appendix Fig S2A). SEMA3C stimulated EGFR and MAPK phosphorylation at levels as low as 1.6 nM SEMA3C in LNCaP cells (Appendix Fig S2B). Similarly, SEMA3C induced dose-dependent activation of EGFR signaling pathway in DU145 cells (Appendix Fig S2C). To investigate the relationship between SEMA3C expression and EGFR/ErbB2 signaling in clinical PCa samples, we analyzed phosphorylation status of EGFR, ErbB2, and downstream signaling proteins SRC and SHC in high SEMA3C versus low SEMA3C-expressing samples from The Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov/) prostate adenocarcinoma data set using cBioPortal tools (Cerami et al, 2012; Gao et al, 2013). As shown in Fig 1E, high SEMA3C mRNA levels were associated with increased levels of phospho-EGFR (Y1068), phospho-ErbB2 (Y1248), phospho-SRC (Y416), and phospho-SHC (Y317) as determined by reverse-phase protein array (RPPA) measurements of phosphoprotein levels in patient tumor samples expressing increased SEMA3C mRNA with z-score threshold >1.0 (SEMA3C high) versus unaltered SEMA3C (SEMA3C low) tumor samples from TCGA prostate adenocarcinoma provisional data set. Plot and P value were generated from cBioPortal. Semaphorin receptors such as Plexin B1 are also known to mediate signaling through MET receptor tyrosine kinase (RTK) (Giordano et al, 2002). To examine whether SEMA3C can trigger MET signaling, MET-expressing DU145 cells were stimulated with varying concentrations of SEMA3C:Fc and activation of MET signaling was examined by immunoblotting for phospho-MET. MET phosphorylation was induced by SEMA3C stimulation in DU145 in a dose-dependent manner (Fig 1F). To determine whether SEMA3C can drive activation of EGFR and MET signaling in other cell types, we first screened cBioPortal for Cancer Genomics data for tumor types that showed an association of high SEMA3C expression and poor prognosis. High SEMA3C expression was associated with poor overall survival in renal clear cell carcinoma and bladder carcinoma (Appendix Fig S2D). Furthermore, SEMA3C has recently been shown to promote tumorigenicity and survival of glioma stem cells (Man et al, 2014). Hence, we screened a panel of cells lines representing renal cancer (CAKI-1, CAKI-2, ACHN), bladder cancer (UC13, T24), and glioblastoma (U87MG), for expression of SEMA3C, EGFR, MET, Plexin B1, Plexin D1, NRP1, and NRP2 (Appendix Fig S2E). Treatment of CAKI-2, T24, and U87MG cells which express lower SEMA3C levels with recombinant SEMA3C:Fc showed activation of MET and EGFR signaling in a dose-dependent manner (Appendix Fig S2F). Conversely, siRNA-mediated silencing of SEMA3C reduced levels of phospho-EGFR proteins in high SEMA3C-expressing CAKI-1 and ACHN cell lines (Appendix Fig S2G). These data collectively suggest that SEMA3C may be a key driver of EGFR and MET signaling in a broad spectrum of cancers. SEMA3C drives signaling and growth via Plexin B1 and NRP1/2 Plexins and neuropilins are cell surface receptors responsible for secreted class 3 semaphorin signal transduction (Rehman & Tamagnone, 2013). Since Plexin B1 and D1 are known to mediate RTK activation such as HER2/ErbB2 (Swiercz et al, 2004, 2008; Casazza et al, 2010) and MET (Giordano et al, 2002), we therefore sought to examine whether NRP1, NRP2, Plexin B1, and Plexin D1 are receptors for SEMA3C in PCa cells. To this end, we first performed proliferation assays in DU145 cells treated with siRNA targeting Plexin D1 (siPLXND1), Plexin B1 (siPLXNB1), NRP1 (siNRP1), and NRP2 (siNRP2) (Fig 2A and Appendix Fig S2H). Knockdown of Plexin B1, NRP1, NRP2, and both NRP1 and NRP2 inhibited SEMA3C-induced growth of DU145 cells compared to siScramble (siScr) controls, whereas siRNA-mediated silencing of Plexin D1 did not (Fig 2A). Moreover, knockdown of Plexin B1 and of NRP1 and NRP2 individually and combined inhibited SEMA3C-activated phosphorylation of EGFR, HER2/ErbB2, and SHC compared to siScr control (Appendix Fig S2I–K). Taken together, these data suggest that Plexin B1 and coreceptors, NRP1 and NRP2, mediate SEMA3C signaling in prostate cancer cells. Figure 2. SEMA3C acts through Plexin B1 A. Proliferation assay of DU145 cells treated with scramble (siScr) or siRNAs specific for PLXND1, PLXNB1, NRP1, NRP2, and NRP1 and NRP2 together. SEMA3C-induced growth is expressed as percent of control (siScramble) using the crystal violet proliferation assay. Mean ± SEM; *P = 0.025 (NRP1), *P = 0.031 (NRP2), ***P = 0.0005, ****P < 0.0001. Statistical analysis was performed using the Holm–Sidak post hoc comparison test after one-way ANOVA (n = 6). B. Association of SEMA3C and Plexin B1 (Interactions/cell) as detected by the proximity ligation assay (PLA) in DU145 cells treated with either rhIgG1Fc or SEMA3C:Fc, ****P < 0.0001. Statistical analysis was performed using the Mann–Whitney test. C. PLA association between SEMA3C and Plexin B1 in DU145 KD cells with either siScrambled (siScr) or si-Plexin B1 siRNAs. Cells were treated with either SEMA3C or control (rhIgG1Fc), ****P < 0.0001. Statistical analysis was performed using the Mann–Whitney test. D. PLA interactions of SEMA3C and NRP1 in control (rhIgG1Fc) or SEMA3C:Fc-treated DU145 cells transfected with either siPLXNB1 or siScr, ****P < 0.0001. Statistical analysis was performed using the Mann–Whitney test. E. SEMA3C or rhIgG1Fc as control DU145 lysates were immunoprecipitated with antibodies against NRP-1, PLEXIN B1, HER2/ErbB2, and MET. The immunoblot was probed with C-terminal-specific Plexin B1 antibodies. Input levels of the corresponding proteins are shown. F–H. PLA association of Plexin B1 and MET (F), EGFR (G), and HER2 (H) in DU145 cells treated with either rhIgG1Fc or SEMA3C:Fc. Negative controls represent the background staining observed in the absence of primary antibody added in the PLA reaction. Representative corresponding fluorescence micrographic images of the PLA interactions of rIgG1Fc versus SEMA3C:Fc-treated DU145 cells are shown. DAPI staining in blue. **P = 0.005, ****P < 0.0001. Data were statistically tested using Kruskal–Wallis or Mann–Whitney test. Data information: Scale bars: 10 μm. The punctate red fluorescence staining represents the specific corresponding PLA interactions (B–D, F–H). Horizontal lines represent the Mean and the box range represents the minimum and maximum interactions /cell. Data is representative of PLA interactions from 5 fields of view. Source data are available online for this figure. Source Data for Figure 2 [emmm201707689-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint The proximity ligation assay (PLA) is a powerful technique used to visualize and quantitate protein–protein interactions (see Appendix Materials and Methods). The assay reports proximal association of two target proteins less than 40 nm apart (Fredriksson et al, 2002). Using PLA, we sought to determine whether SEMA3C treatment of DU145 cells could induce formation of a ligand–receptor complex comprised of SEMA3C with NRP1 and Plexin B1 on the cell surface and whether Plexin B1 associates with EGFR, HER2, and MET, thereby mediating transduction of downstream RTK signaling. As a first step, we used PLA to characterize the formation of ligand–receptor complexes between SEMA3C and its putative candidate receptors, Plexin B1 and NRP1 in siScr or siPLXNB1-treated DU145 cells stimulated with either recombinant full-length SEMA3C-Fc fusion (SEMA3C:Fc) or human IgGFc (rhIgGFc) as control. We found significant direct binding of SEMA3C:Fc with Plexin B1 (Fig 2B) that was significantly inhibited by siPLXNB1 treatment (Fig 2C). Furthermore, we found significant binding of SEMA3C with NRP1 (F
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