Phenotypic switching is an emerging driver of cancer treatment resistance, yet early signals regulating this process remain unclear. Here, using longitudinal single-cell RNA sequencing, we mapped differentiation trajectories in the LTL331 prostate adenocarcinoma patient-derived xenograft (PDX) model undergoing neuroendocrine prostate cancer (NEPC) transformation post castration. Our analyses identified a key differentiation node marked by epithelial-mesenchymal transition (EMT) and repressor element-1 silencing transcription factor (REST) downregulation driven by the CXCR4-LASP1-G9a-SNAIL axis. Mechanistically, CXCR4 activation promotes nuclear translocation of LASP1 that links G9a and SNAIL via SH3/proline-rich motif and LIM/SNAG domain interactions, enabling SNAIL-mediated REST repression via promoter E-box motifs. Inhibition of CXCR4 or G9a reversed LTL331R NEPC cells toward a luminal androgen receptor-active phenotype. CXCR4-targeted radioligands enabled both imaging and inhibition of NEPC tumors in vivo. These findings highlight the CXCR4-LASP1-G9a-SNAIL axis as a key regulator of epigenetic and transcriptional reprogramming in NEPC transdifferentiation and support its therapeutic targeting in aggressive NEPC.

Abstract Our work sets out to show that semaphorin 3C is a driver of epithelial-to-mesenchymal transition and stemness in prostate cancer. Prostate cancer (PCa) is among the most commonly-occurring non-cutaneous cancers in men. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted chemotactic proteins whose normal functions reside in embryogenesis and development where they are responsible for directing cell movement. Semaphorins act through autocrine, paracrine, and juxtacrine signaling and have been implicated in a broad range of biological functions ranging from tissue morphogenesis to immunity; altered semaphorin expression has also been observed in numerous cancers. One member of the class 3 semaphorins, semaphorin 3C (SEMA3C), has been implicated in several forms of cancer and its increased expression is correlated with prostate cancer severity. Additionally, SEMA3C has been shown to be upregulated in response to chemotherapy and radiation treatment, promote metastasis to the lung, and promote tumourigenicity of glioma cells. SEMA3C has also been documented to increase cell proliferation and migration, decrease apoptosis, and promote integrin signaling and VEGF secretion in endothelial cells. SEMA3C was shown to drive migration of breast cancer cells and recent studies have highlighted the importance and prognostic value of SEMA3C in prostate cancer. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes cancer progression by driving mesenchymal and stem-like phenotypes. Other class 3 semaphorins have been shown to drive EMT and the link between SEMA3C and stemness has been established in glioma cells. In the present study, using gain of function studies coupled to gene expression (qPCR, Western blot, FACS, immunofluorescence microscopy) and functional studies (migration, invasion, and sphere-forming assays) we show that ectopic expression of SEMA3C in RWPE-1, a normal prostate epithelial cell line, promotes epithelial-to-mesenchymal transition and stemness. Specifically, we find that overexpression of SEMA3C leads to an upregulation of EMT markers and migratory and invasive phenotypes. SEMA3C overexpression was also associated with an upregulation of the cancer stem cell marker, CD44, and heightened sphere-forming capabilities. Additionally, using ultrasound-guided intracardiac injection of SEMA3C-overexpressing cells, we show that SEMA3C drives cell dissemination in vivo. We conclude from our studies that SEMA3C is a driver of prostate cancer by promoting epithelial-to-mesenchymal transition and stemness. Citation Format: Kevin J. Tam, Daniel H. Hui, Wilson C. Lee, Mingshu Dong, Tabitha Tombe, Ivy Z. Jiao, Shahram Khosravi, Ario Takeuchi, James W. Peacock, Larissa Ivanova, Igor Moskalev, Martin E. Gleave, Ralph Buttyan, Michael E. Cox, Christopher J. Ong. Semaphorin 3C drives invasiveness in prostate cells through epithelial-to-mesenchymal transition and stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1087.

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 invivo. SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer.

Despite the amenability of early-stage prostate cancer to surgery and radiation therapy, locally advanced and metastatic prostate cancer is clinically problematic. Chemical castration is often used as a first-line therapy for advanced disease, but progression to the castration-resistant prostate cancer phase occurs with dependable frequency, largely through mutations to the androgen receptor (AR), aberrant AR signaling, and AR-independent mechanisms, among other causes. Semaphorin 3C (SEMA3C) is a secreted signaling protein that is essential for cardiac and neuronal development and has been shown to be regulated by the AR, to drive epithelial-to-mesenchymal transition and stem features in prostate cells, to activate receptor tyrosine kinases, and to promote cancer progression. Given that SEMA3C is linked to several key aspects of prostate cancer progression, we set out to explore SEMA3C inhibition by small molecules as a prospective cancer therapy. A homology-based SEMA3C protein structure was created, and its interaction with the neuropilin (NRP)-1 receptor was modeled to guide the development of the corresponding disrupting compounds. Experimental screening of 146 in silico‒identified molecules from the National Cancer Institute library led to the discovery of four promising candidates that effectively bind to SEMA3C, inhibit its association with NRP1, and attenuate prostate cancer growth. These findings provide proof of concept for the feasibility of inhibiting SEMA3C with small molecules as a therapeutic approach for prostate cancer.

Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in cancer-related deaths. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted signalling proteins whose normal roles reside in embryogenesis and neuronal development. In this context, semaphorins help establish chemotactic gradients and direct cell movement. Various semaphorin family members have been found to be up- and down-regulated in a number of cancers. One family member, Semaphorin 3 C (SEMA3C), has been implicated in prostate, breast, ovarian, gastric, lung, and pancreatic cancer as well as glioblastoma. Given SEMA3C’s roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes epithelial-to-mesenchymal transition (EMT) and stem-like phenotypes in prostate cells. In the present study we show that ectopic expression of SEMA3C in RWPE-1 promotes the upregulation of EMT and stem markers, heightened sphere-formation, and cell plasticity. In addition, we show that SEMA3C promotes migration and invasion in vitro and cell dissemination in vivo.

BACKGROUND LIM and SH3 domain protein 1 (LASP1) has been implicated in several human malignancies and has been shown to predict PSA recurrence in prostate cancer. However, the anti‐tumor effect of LASP1 knockdown and the association between LASP1 and the androgen receptor (AR) remains unclear. The aim of this study is to clarify the significance of LASP1 as a target for prostate cancer, and to test the effect of silencing LASP1 in vivo using antisense oligonucleotides (ASO). METHODS A tissue microarray (TMA) was performed to characterize the differences in LASP1 expression in prostate cancer treated after hormone deprivation therapy. Flow cytometry was used to analyze cell cycle. We designed LASP1 ASO for knockdown of LASP1 in vivo studies. RESULTS The expression of LASP1 in TMA was increased after androgen ablation and persisted in castration resistant prostate cancer (CRPC). Also in TMA, compared with LNCaP cell, LASP1 expression is elevated in CRPC cell lines (C4‐2 and VehA cells). Interestingly, suppression of AR elevated LASP1 expression conversely, AR activation decreased LASP1 expression. Silencing of LASP1 reduced cell growth through G1 arrest which was accompanied by a decrease of cyclin D1. Forced overexpression of LASP1 promoted cell cycle and induced cell growth which was accompanied by an increase of cyclin D1. Systemic administration of LASP1 ASO with athymic mice significantly inhibited tumor growth in CRPC xenografts. CONCLUSIONS These results indicate that LASP1 is negatively regulated by AR at the transcriptional level and promotes tumor growth through induction of cell cycle, ultimately suggesting that LASP1 may be a potential target in prostate cancer treatment. Prostate 77:309–320, 2017 . © 2016 Wiley Periodicals, Inc.

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2015MP66-02 INHIBITION OF LIM-SH3 DOMAIN PROTEIN1 AUGMENTS THE ANTI-CANCER EFFECT OF ENZALUTAMIDE IN PROSTATE CANCER. Takashi Dejima, Ario Takeuchi, Jeffrey Leong, Tabitha Tombe, Kevin Tam, Seiji Naito, Martin Gleave, and Christopher Ong Takashi DejimaTakashi Dejima More articles by this author , Ario TakeuchiArio Takeuchi More articles by this author , Jeffrey LeongJeffrey Leong More articles by this author , Tabitha TombeTabitha Tombe More articles by this author , Kevin TamKevin Tam More articles by this author , Seiji NaitoSeiji Naito More articles by this author , Martin GleaveMartin Gleave More articles by this author , and Christopher OngChristopher Ong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2355AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES LIM-SH3 domain protein 1 (LASP1) has been shown to promote cancer progression and invasion in several malignancies. In prostate cancer, LASP1 is a strong predictor of early metastasis and correlates with invasion. However, the anti-tumor effects of LASP1 knockdown and the association between LASP1 and androgen receptor (AR) remain unclear. In this study, we investigated the anti-cancer activity of LASP1 knockdown in prostate cancer. METHODS The LASP1 expression in several prostate cancer cell lines was determined by Western blot analysis and quantitative reverse transcription-PCR. Silencing of LASP1 was achieved using siRNA. The tissue microarray (TMA) consisted of 164 patients' radical prostatectomies (Vancouver General Hospital). The effect of LASP1 knockdown in vivo was assessed by treating LASP1 antisense oligonucleotide (ASO) with mice in PC-3 xenograft model. RESULTS LASP1 is higher in PC-3 and DU145 cells than in LNCaP cells. Knockdown of LASP1 by siRNA reduced cell growth in PC-3 and DU145 cells. Flow cytometric analysis revealed that silencing of LASP1 induced G1 arrest, and was accompanied by an increase in p21 and a decrease in cyclinD1. Stable knockdown of LASP1 also reduced cell growth. Conversely, transient LASP1 overexpression in LNCaP promoted transition from G0/G1 to S phase and increased cyclinD1 and decreased p21. Additionally, stable overexpression of LASP1 in LNCaP, PC-3, and DU145 cells promoted cell growth and increased cyclinD1. In order to investigate the association between LASP1 and AR, we analyzed LASP1 expression in patients after radical prostatectomy. TMA data shows that LASP1 expression correlates with PSA recurrence after radical prostatectomy. Interestingly, LASP1 expression in TMA is increased after androgen-deprivation therapy (ADT). Consistent with TMA, LASP1 expression is higher in CRPC cancer cell lines (C4-2, VehA16 and VehD16) than in LNCaP cells. In addition, AR inhibition using Enzalutamide or siRNA induced LASP1 expression. Conversely, AR stimulation using R1881 suppressed LASP1 expression. LASP1 knockdown in combination with Enzalutamide showed synergistic effects in vitro. Systemic administration of LASP1 ASO in athymic mice injected with PC-3 cells significantly delays tumor progression. CONCLUSIONS These results suggest that inhibiting LASP1 through silencing technology holds promise as a novel therapeutic approach in the treatment of prostate cancer, and that this approach may have synergy with conventional anti-androgens. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e816 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Dejima More articles by this author Ario Takeuchi More articles by this author Jeffrey Leong More articles by this author Tabitha Tombe More articles by this author Kevin Tam More articles by this author Seiji Naito More articles by this author Martin Gleave More articles by this author Christopher Ong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-13 LIM-SH3 DOMAIN PROTEIN 1 KNOCKDOWN INHIBITS CELL GROWTH AND ENHANCES ACTIVITY OF CISPLATIN IN BLADDER CANCER. Takashi Dejima, Ario Takeuchi, Tetsutaro Hayashi, Jeffrey Leong, Tabitha Tombe, Kevin Tam, Htoo Oo, Peter Black, Seiji Naito, Martin Gleave, and Christopher Ong Takashi DejimaTakashi Dejima More articles by this author , Ario TakeuchiArio Takeuchi More articles by this author , Tetsutaro HayashiTetsutaro Hayashi More articles by this author , Jeffrey LeongJeffrey Leong More articles by this author , Tabitha TombeTabitha Tombe More articles by this author , Kevin TamKevin Tam More articles by this author , Htoo OoHtoo Oo More articles by this author , Peter BlackPeter Black More articles by this author , Seiji NaitoSeiji Naito More articles by this author , Martin GleaveMartin Gleave More articles by this author , and Christopher OngChristopher Ong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.517AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer is the 6th most common cancer in the USA. The current standard therapy for the first line of metastatic or local advanced bladder cancer is combination therapy with cisplatin (CDDP) and gemcitabine (GEM). However 5-years survival is still below 50%; therefore additional therapy is needed. LIM-SH3 domain protein 1 (LASP1), identified from cDNA library of metastatic axillary lymph nodes of breast cancer, has been shown to promote cancer progression and invasion in several malignancies. In bladder cancer, LASP1 expression associates with cell invasion and can be used for detecting bladder cancer. However, the anti-tumor effects of LASP1 knockdown in vivo as well as combination therapy with chemotherapy remain unclear. In this study, we investigated the anti-cancer activity of LASP1 knockdown in bladder cancer. METHODS LASP1 gene expression of tumor samples is analyzed using the Affymetrix Exon Array. The LASP1 expression in several bladder cancer cell lines was assessed by Western blot analysis and quantitative reverse transcription-PCR. Silencing of LASP1 in vitro was achieved using siRNA. The In vivo effect of LASP1 antisense oligonucleotide (ASO) treatment was assessed in the T24 CDDP-R (cisplatin-resistant) orthotopic bladder cancer model. RESULTS High LASP1 expression correlated with recurrence rate between patients receiving adjuvant chemotherapy or not. This difference is increased in patients after adjuvant chemotherapy. The LASP1 expression is higher in UC1 and UC15 cells than in UC13 and UC6 cells. Knockdown of LASP1 using siRNA inhibited cell growth, and was accompanied by an increase in p21 and p27. Conversely, stable LASP1 overexpression drove cell growth with increase of cyclinD1 in UC13 and UC6 cells. The treatment of CDDP and GEM induced LASP1 expression in vitro. Combination treatment with LASP1 knockdown using siRNA and CDDP inhibited cell growth in UC1 cells. Furthermore, compared with parental cell line, LASP1 is higher in T24 CDDP-R and RT112 CDDP-R cells than in parental cells. In the orthotopic bladder cancer model, systemic LASP1 ASO administration to athymic nude mice delayed tumor progression in T24 CDDP-R cells. CONCLUSIONS These data revealed that LASP1 inhibition might be as a promising novel therapeutics modality in the treatment of bladder cancer, as well as a possible synergy with chemotherapy. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e607-e608 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Dejima More articles by this author Ario Takeuchi More articles by this author Tetsutaro Hayashi More articles by this author Jeffrey Leong More articles by this author Tabitha Tombe More articles by this author Kevin Tam More articles by this author Htoo Oo More articles by this author Peter Black More articles by this author Seiji Naito More articles by this author Martin Gleave More articles by this author Christopher Ong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2010681 REDUCED ANDROGEN DEPENDENT AND INDEPENDENT GROWTH OF HUMAN PROSTATE CANCER XENOGRAFTS IN GROWTH HORMONE RELEASING HORMONE RECEPTOR MUTANT HOSTS Kiyoshi Takahara, Howard Tearle, Tabitha Tombe, Martin E. Gleave, and Michael E. Cox Kiyoshi TakaharaKiyoshi Takahara More articles by this author , Howard TearleHoward Tearle More articles by this author , Tabitha TombeTabitha Tombe More articles by this author , Martin E. GleaveMartin E. Gleave More articles by this author , and Michael E. CoxMichael E. Cox More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1080AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Signaling through the growth hormone/insulin-like growth factor (GH/IGF) axis has been linked to prostate cancer (PCa) risk. Previous studies have indicated that human breast cancer xenografts and murine PCa models develop more slowly in little murine hosts. The little (lit/lit) phenotype is due to a D60G missense mutation in the GH-releasing hormone receptor (GHRHR) resulting in loss of pituitary GHRHR function and suppression of GH and IGF-1 expression. We hypothesized that the GH/IGF axis influences PCa growth and castration-resistant (CR) progression of human PCa xenografts. METHODS To determine whether PCa growth in vivo models is influenced by the host GH/IGF axis, we performed in vivo growth studies using the androgen-responsive human PCa cell line, LNCaP, and CR human PCa cell line, PC3, in Nod/SCID lit/lit mice and compared them to growth and CR progression in mice heterozygous for the lit allele (lit/+). To assess whether suppressed GH/IGF levels affected androgen-responsive growth of PCa xenografts, we monitored tumor size and serum PSA levels before and after castration of LNCaP tumor-bearing lit/lit and lit/+ mice. We also monitored growth rate of PC3 xenografts in intact mice to assess the impact of GH/IGF axis deficiencies on CR PCa. Growth of LNCaP and PC3 cells in vitro using serum from lit/lit mice or lit/+ mice serum was examined to complement the in vivo experiments. RESULTS In intact mice, androgen-responsive growth rate of LNCaP xenografts was significantly reduced in lit/lit mice as compared to lit/+ mice [mm3/day (mean±SD) 56.0±8.5 vs 93.2±16.3, p < 0.05]. CR progression of LNCaP xenografts was also significantly delayed in lit/lit mice as compared to lit/+ mice [mm3/day (mean±SD) 65.2±12.8 vs 147.2±47.3, p < 0.1]. After castration, serum PSA levels in lit/lit mice was significantly lower than in lit/+ mice [ng/ml/day (mean±SD) 18.0±6.5 vs 60.0±33.0, p < 0.1]. Growth of CR PC3 xenografts was significantly delayed in lit/lit mice (n=19) as compared to lit/+ mice (n=17) [mm3/day (mean±SD) 234.5±24.1 vs 441.6±42.4, p < 0.001]. LNCaP and PC3 cells grown in vitro with serum from lit/lit mice showed decreased proliferation as compared with serum from lit/+ mice. CONCLUSIONS The GH/IGF axis appears to be an important stimulator of both androgen-responsive growth and CR progression of these PCa xenograft models. The results motivate clinical trials of novel hormonal treatment strategies that target the GH/IGF axis for PCa patients. Vancouver, Canada© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e266 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kiyoshi Takahara More articles by this author Howard Tearle More articles by this author Tabitha Tombe More articles by this author Martin E. Gleave More articles by this author Michael E. Cox More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The use of the green fluorescent protein (GFP) to label specific cell types and track gene expression in animal models, such as mice, has evolved to become an essential tool in biological research. Transgenic animals expressing genes of interest linked to GFP, either as a fusion protein or transcribed from an internal ribosomal entry site (IRES) are widely used. Enhanced GFP (eGFP) is the most common form of GFP used for such applications. However, a red fluorescent protein (RFP) would be highly desirable for use in dual-labeling applications with GFP derived fluorescent proteins, and for deep in vivo imaging of tissues. Recently, a new generation of monomeric (m)RFPs, such as monomeric (m)Cherry, has been developed that are potentially useful experimentally. mCherry exhibits brighter fluorescence, matures more rapidly, has a higher tolerance for N-terminal fusion proteins, and is more photostable compared with its predecessor mRFP1. mRFP1 itself was the first true monomer derived from its ancestor DsRed, an obligate tetramer in vivo. Here, we report the successful generation of a transgenic mouse line expressing mCherry as a fluorescent marker, driven by the ubiquitin-C promoter. mCherry is expressed in almost all tissues analyzed including pre- and post-implantation stage embryos, and white blood cells. No expression was detected in erythrocytes and thrombocytes. Importantly, we did not encounter any changes in normal development, general physiology, or reproduction. mCherry is spectrally and genetically distinct from eGFP and, therefore, serves as an excellent red fluorescent marker alone or in combination with eGFP for labelling transgenic animals.