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Supplementary Figure S1. Generation of ENZR Xenografts and Cell Lines. Supplementary Figure S2. AR non-driven ENZR cells display a NE differentiation signature. Supplementary Figure S3. BRN2 expression is associated with PCa progression and a NE phenotype in human tumors. Supplementary Figure S4. BRN2 expression is inversely related to AR activity and is required for ENZ-induced NE marker expression in CRPC. Supplementary Figure S5. BRN2 overexpression induces NE marker expression and reciprocally regulates AR. Supplementary Figure S6. BRN2 activity and markers of NE differentiation are suppressed by androgens in CRPC and ENZR cells. Supplementary Figure S7. SOX2 expression is induced by ENZ and suppressed by androgens. Supplementary Figure S8. BRN2 is required for neuroendocrine marker expression and aggressive growth of CRPC cells in vitro.

Supplementary methods, figure legends, and primer lists.

RNAseq of ENZ-resistant cells.

NEPC signature for GSEA analysis.

Gene Profiling data of CRPC cells with BRN2 over-expression.

The acquisition of an invasive phenotype by epithelial cells occurs through a loss of cellular adhesion and polarity, heralding a multistep process that leads to metastatic dissemination. Since its characterization in 1995, epithelial-mesenchymal transition (EMT) has been closely linked to the metastatic process. As a defining aspect of EMT, loss of cell adhesion through downregulation of E-cadherin is carried out by several transcriptional repressors; key among them the SNAI family of transcription factors. Here we identify for the first time that Lyn kinase functions as a key modulator of SNAI family protein localization and stability through control of the Vav-Rac1-PAK1 (Vav-Rac1-p21-activated kinase) pathway. Accordingly, targeting Lyn in vitro reduces EMT and in vivo reduces metastasis of primary tumors. We also demonstrate the clinical relevance of targeting Lyn as a key player controlling EMT; patient samples across many cancers revealed a strong negative correlation between Lyn and E-cadherin, and high Lyn expression in metastatic tumors as well as metastasis-prone primary tumors. This work reveals a novel pancancer mechanism of Lyn-dependent control of EMT and further underscores the role of this kinase in tumor progression.

Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges. In particular, a subset of patients who relapse following ARPI therapy their dependence on AR signaling and emerge with neuroendocrine features. These tumors, termed treatment induced small-cell prostate cancer (t-SCPC) or neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapies. Previously, our group identified the neural transcription factor BRN2 as a major clinically relevant driver of SCPC and now report that targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC.Methods/Results: In silico screening of small molecules was conducted on a model of BRN2 which was later validated with the first-in-field crystal structure of BRN2 DNA binding domain. On the basis of the model, several small molecules were identified that showed direct binding to BRN2 and inhibited its activity. Pharmacokinetic studies measured stability and bioavailability of med-chem optimized lead compound (BRN2i) that significantly reduced tumor growth in multiple xenograft models with no measurable side-effects. In silico modeling predicted a 7Å “closing” in the DBD once it was bound to BRN2i, this shift translated to reduced interaction with DNA by chromatin fractionation and ChIP-seq, thus confirming the mode of action for BRN2i is through loss of DNA binding. Loss of BRN2 binding reduced cell proliferation in tSCPC cell line 42DENZR, de novo SCPC cell line NCI-H660 and NEPC organoids as well as downregulated several known targets like EZH2, ASCL1, SOX2 and PEG10. Down-regulation of these target genes was also measured in the xenograft tumors, confirming on target effect in vivo. Moreover, the specificity of BRN2i was validated with CRISPR/Cas9 mediated knockout of BRN2 with downstream mRNA expression and phenotypic changes. Conclusion: The described work aims to lay the pre-clinical foundation for the integration of BRN2 targeted therapies into the treatment landscape to improve survival for patients suffering from small-cell neuroendocrine prostate cancer. Citation Format: Daksh Thaper, Ravi Munuganti, Adeleke Aguda, Soojin Kim, Shungyu Ku, Sahil Kumar, Sepideh Vahid, Shaghayegh Nouruzi, Olena Sivak, Dwaiyapan Ganguli, Colm Morrissey, Eva Corey, Himisha Beltran, Amina Zoubeidi. Selective inhibition of transcription factor BRN2 as a treatment strategy for Small Cell Prostate Cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3083.

Most prostate cancer patients will progress to a castration-resistant state (CRPC) after androgen ablation therapy and despite the development of new potent anti-androgens, like enzalutamide (ENZ), which prolong survival in CRPC, ENZ-resistance (ENZR) rapidly occurs. Re-activation of the androgen receptor (AR) is a major mechanism of resistance. Interrogating our in vivo derived ENZR model, we discovered that transcription factor STAT3 not only displayed increased nuclear localization but also bound to and facilitated AR activity. We observed increased STAT3 S727 phosphorylation in ENZR cells, which has been previously reported to facilitate AR binding. Strikingly, ENZR cells were more sensitive to inhibition with STAT3 DNA-binding inhibitor galiellalactone (GPA500) compared to CRPC cells. Treatment with GPA500 suppressed AR activity and significantly reduced expression of Cyclin D1, thus reducing cell cycle progression into S phase and hindering cell proliferation. In vivo, GPA500 reduced tumor volume and serum PSA in ENZR xenografts. Lastly, the combination of ENZ and GPA500 was additive in the inhibition of AR activity and proliferation in LNCaP and CRPC cells, providing rationale for combination therapy. Overall, these results suggest that STAT3 inhibition is a rational therapeutic approach for ENZR prostate cancer, and could be valuable in CRPC in combination with ENZ.

Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges and patients generally die within two years. In particular, a subset of patients who relapse following ARPI therapy exhibit lineage switching whereby tumours shed their dependence on AR signaling and emerge with neuroendocrine features. These tumours, termed treatment induced neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapy which carries a short-lived response at the cost of significant toxicity. Therefore, targeted therapies for this deadly disease are desperately needed. Thus, the need to develop targeted treatments for this devastating disease is of paramount importance. Recently our group identified the neural transcription factor BRN2 as a major clinically relevant driver of NEPC and aggressive tumor growth, both in vitro and in vivo, suggesting targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC. Methods: Study the effects of BRN2 inhibition using siRNA, small molecule inhibitors and CRISPR K/O models. Results: Inhibition of BRN2 by siRNA and by CRISPR/Cas9 knockout drastically reduced cell proliferation in 42DENZR (NEPC) cell lines. This data was re-capitulated in human NEPC NCI-H660 cells. Loss of BRN2 initiated drastic epigenetic changes in NEPC cell lines as well as in G1 arrest through up-regulation of CDKN1A/1B. This was confirmed using our first in field BRN2 inhibitors. Targeting BRN2 also lead to downregulation several known targets in NEPC like EZH2, AURKA, SOX2 and Peg10. Treatment with BRN2i reduced recruitment of BRN2 to the chromatin by approximately 93% within 16 hours. Moreover, these BRN2 inhibitors displayed adequate pharmacokinetic properties and reduced NEPC proliferation in vivo. Conclusion: No therapies exist for highly lethal NEPC. Hence, the described work aims to verify BRN2, a central driver of NEPC, and lay the pre-clinical foundation for the integration of targeted therapies into the treatment landscape to improve survival and quality of life for patients suffering from deadly form of prostate cancer. Citation Format: Daksh Thaper, Ravi Munuganti, Shaghayegh Nouruzi, Sahil Kumar, Soojin Kim, Olena Sivak, Adeleke Aguda, Dwaipayan Ganguli, Sepideh Vahid, Loredana Puca, Himisha Beltran, Amina Zoubeidi. First-in-field small molecule inhibitors targeting BRN2 as a therapeutic strategy for small cell prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1295.

Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers.

Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges and patients generally die within two years. In particular, a subset of patients who relapse following ARPI therapy exhibit lineage switching whereby tumours shed their dependence on AR signaling and emerge with neuroendocrine features. These tumours, termed treatment induced neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapy which carries a short-lived response at the cost of significant toxicity. Therefore, targeted therapies for this deadly disease are desperately needed. Thus, the need to develop targeted treatments for this devastating disease is of paramount importance. Recently our group identified the neural transcription factor BRN2 as a major clinically relevant driver of NEPC and aggressive tumor growth, both in vitro and in vivo, suggesting targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC. Methods: Study the effects of BRN2 inhibition using siRNA and CRISPR K/O models. Results: Inhibition of BRN2 by siRNA and by CRISPR/Cas9 knockout drastically reduced cell proliferation in 42DENZR (NEPC) cell lines. This data was re-capitulated in human NEPC NCI-H660 cells. Loss of BRN2 initiated drastic epigenetic changes in NEPC cell lines as well as in G1 arrest through up-regulation of CDKN1A/1B. This was confirmed using our first in field BRN2 inhibitors. Targeting BRN2 also lead to downregulation several known targets in NEPC like EZH2, AURKA, SOX2 and Peg10. Conclusion: No therapies exist for highly lethal NEPC. Hence, the described work aims to verify BRN2 as a central driver of NEPC, and lay the pre-clinical foundation for the integration of targeted therapies into the treatment landscape to improve survival and quality of life for patients suffering from deadly form of prostate cancer. Citation Format: Daksh Thaper, Ravi Munuganti, Shaghayegh Norouzi, Sahil Kumar, Soojin Kim, Kriti Singh, Sepideh Vahid, Jennifer Bishop, Amina Zoubeidi. Targeting master neuronal transcription factor BRN2 in neuroendocrine prostate cancer [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 1931.

During the past several years, various species of Cousinia (Asteraceae) have been authenticated in Iran. However, data concerning their biological activities remain limited. The main purpose of this research was to assess potential cytotoxicity and matrix metalloproteinases (MMP) inhibitory effects of seven ethanol extracts of Cousinia using a cell line model (Fibrosarcoma-WEHI 164). We further investigated the antibacterial activity of these Cousinia ethanol extracts, using disk diffusion method. Among the ethanol extracts, the total extract of C. sulabadensis elicited significant inhibition of MMP activity in a dose-response fashion (49.2 ± 0.51, p < 0.05). However, this extract exhibited the lowest cytotoxicity effect at all tested concentrations. The concentration necessary to produce a 50% cell death rate (IC50) with C. shulabadensis was 304.5 ± 0.61 µg/mL. The calculated IC50 for cytotoxicity of the other Cousinia species extracts ranged between 18.4 ± 0.59 to 87.9 ± 0.58 µg/mL. The highest antibacterial activity was observed for the total extract of Cousinia phyllocephala. In conclusion, this study supports that Cousinia species display a remarkable inhibition of matrix metalloproteinases activity. The concomitant MMP-inhibitory and low cytotoxicity effects observed in C. sulabadensis might coin this extract for future potential anti-invasive herbal medicine studies.

Castrate-resistant prostate cancer (CRPC) progression is a complex process by which prostate cells acquire the ability to survive and proliferate in the absence or under very low levels of androgens. Most CRPC tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes owing to reactivation of AR. Protein tyrosine kinases have been implicated in supporting AR activation under castrate conditions. Here we report that Lyn tyrosine kinase expression is upregulated in CRPC human specimens compared with hormone naive or normal tissue. Lyn overexpression enhanced AR transcriptional activity both in vitro and in vivo and accelerated CRPC. Reciprocally, specific targeting of Lyn resulted in a decrease of AR transcriptional activity in vitro and in vivo and prolonged time to castration. Mechanistically, we found that targeting Lyn kinase induces AR dissociation from the molecular chaperone Hsp90, leading to its ubiquitination and proteasomal degradation. This work indicates a novel mechanism of regulation of AR stability and transcriptional activity by Lyn and justifies further investigation of the Lyn tyrosine kinase as a therapeutic target for the treatment of CRPC.Oncogenesis (2014) 3, e115; doi:10.1038/oncsis.2014.30; published online 18 August 2014.

Background: Androgen ablation remains the most effective therapy for patients with advanced disease. Unfortunately, most patients progress to castrate resistant prostate cancer (CRPC) characterized with hyper-activation of the androgen receptor (AR). Enzalutamide, a potent AR inhibitor showed efficacy by prolonging survival in CRPC patients. However, ENZ resistance (ENZR) rapidly occurs in patients and in our pre-clinical model targeting AR in ENZ resistant tumors with a 3rd generation AR inhibitor is short lived. The role of signal transducer and activator of transcription (STAT) 3 in the progression of prostate cancer is well established and has been repeatedly linked with maintenance of a stem cell phenotype across cancers. Additionally, drug resistance has been hypothesized to occur via enrichment of cancer stem-like cells (CSC), a phenotype associated with poor survival in patients. Therefore, we propose a shift of focus to target CSC phenotype using small molecule inhibitor of STAT3 called GPA500, instead of the AR axis to deal with ENZ resistance. Methods and Results: We developed a unique model of ENZ-resistance and found that cell lines derived from serially passaged ENZR tumors displayed broad genetic diversity and differential AR activity. Notably, the cell lines 42DENZR and 42FENZR are PSAlow, harbor an expanded CSC population and have STAT3 hyper-activation compared to CRPC controls measured by STAT3-luc reporter. Accordingly, Crystal Violet and MTT proliferation assays showed that 42DENZR and 42FENZR cell lines were more sensitive to the STAT3 inhibitor GPA500 compared to CRPC control. Targeting the ENZ-R cells with GPA500 reduced mRNA levels of CD133, CD44, SOX2, OCT4 and Nanog. The reduction of these CSC markers was accompanied by reduced self-renewal capacity measured by spheroid assay. Cytometry analysis revealed that treatment with GPA500 reduced α2β1+, CD44+ and CD133+ (CSC) population in the 42D and 42F cells. Conclusion: In this study, we provide pre-clinical proof that targeting the STAT3 pathway using GPA500 in ENZ resistance as well as CRPC reduces cell proliferation as well as expression of stem cell markers. Impact: Exploring mechanisms of ENZR resistance serves a critical unmet need in PCa oncology, as the number of men with ENZ resistant CRPC continues to rise. Targeting STAT3 with the small molecule inhibitor GPA500 may provide an effective method to treat ENZR patients or delay the emergence of ENZ resistance in CRPC by reducing the emergence of stem cells. Citation Format: Sepideh Vahid, Daksh Thaper, Alistair Davies, Micaela Janse Van Rensburg, Kate Gibson, Krisi Ketola, Martin Johansson, Jennifer L. Bishop, Amina Zoubeidi. Galiellalactone derivative targets stem cell population in ENZ-resistant prostate cancer through inhibition of STAT3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3342.

Introduction: Metastasis is the most common cause of death from cancer and occurs when malignant cells discard epithelial restraints and acquire invasive abilities, facilitating their dissemination to permissive micro-environments. This process is enhanced by tumor cell activation of Epithelial Mesenchymal Transition (EMT), a (normally embryonic) developmental program in which epithelial cells assume a mesenchymal phenotype during gastrulation and organogenesis, allowing single cell invasive movement away from the ectodermal layer. Recent evidence strongly implicates EMT induction in malignant progression and treatment resistance. For example, EMT regulatory transcription factors are required for breast cancer metastasis. Several oncogenic pathways (growth factors, Src family, MAPK, AKT) induce EMT. Lyn tyrosine kinase, a member of Src family tyrosine kinase is up-regulated in advanced prostate cancer and has been reported to correlate with aggressive breast cancer. Our objective is to determine the role of Lyn tyrosine kinase in EMT. Methods: LNCaP (Lymph Node Metastasis of Prostate Cancer), BT-549 (Triple Negative Breast Cancer) and UM-UC-13 (Bladder Cancer) cells were transfected with Lyn siRNA; EMT markers were monitored by western blot and qRT-PCR and immunofluorescence, migration by scratch assay and invasion by Boyden chamber. Sub cellular localization of proteins was examined by IF and nuclear/cyto extraction. In vivo experiments were performed in UC13-luc cells with shRNA of Lyn. Results: Here we report that Lyn expression is low in epithelial cells and is up-regulated in mesenchymal cells. Targeting Lyn using siRNA decreases EMT markers (Fibronectin, Vimentin and Zeb-1) at both mRNA and protein levels while increasing the epithelial marker (E-cadherin). Moreover, we found that Lyn siRNA decreases cell migration and invasion. Thisis decrease in mesenchymal phenotype can be attributed to the decrease in the amount of Slug and Snail, transcriptional repressors of E-Cadherin and activator of Vimentin and Fibronectin. Interestingly, we found that Lyn knockout induces a decrease of SLUG only at protein levels and not at mRNA levels. We discovered that Lyn triggers a signaling cascade through Vav-Rac-Pak1 pathway to alter sub cellular localization of the SNAI proteins leading to their proteasomal degradation. This effect results in decreased invasion and migration in vitro as well as decreased metastasis in vivo. Conclusion: Expression of Lyn kinase can be correlated to low prognosis and aggressive/metastatic phenotype. We show that knocking down Lyn by siRNA initiates a switch to a more epithelial phenotype reducing cell migration and invasion. Citation Format: Daksh Thaper, Sepideh Vahid, Ka Mun Nip, Igor Moskalev, Sebastian Frees, Morgan E. Roberts, Krisi Ketola, Kenneth W. Harder, Jennifer L. Bishop, Amina Zoubeidi. Lyn drives cancer metastasis via post-translational regulation of SNAI proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1682.

Introduction: Heat shock protein 27 (Hsp27) is a molecular chaperone highly and uniformly expressed in treatment resistant cancers like castrate resistant prostate cancer (CRPC). Hsp27 regulates activity of several oncogenic pathways and its levels correlate with aggressive tumor behaviour, drug resistance and tumor growth. Similarly, dysregulation of the Hippo tumor suppressor pathway, which restricts organ size and cell proliferation, occurs in many types of cancers. In healthy cells, activation of the Hippo pathway results in phosphorylation and cytoplasmic retention of two transcriptional co-activators YAP and TAZ, whereas in cancer YAP/TAZ are free to translocate to the nucleus and increase cell proliferation by promoting the activities of certain transcriptional factors including TEAD1. Inactivation of the Hippo pathway correlates with poor patient outcome and progression of tumors as well as an increase in migration, invasion and metastatic potential of cancer cells. Therefore it is of great importance to establish the correlation between Hsp27 and the Hippo pathway to further discover suitable targets in the treatment of metastatic malignancies. Methods: Hsp27 gain and loss of function experiments were done on 3 different cancer cell lines and the functional effects on every step of the pathway were monitored via Western blots and Immunofluorescence. Activity of YAP/TAZ after Hsp27 gain and loss of function was monitored by conducting qRT-PCR on TEAD target genes. Transcriptional activity of TEAD1 was also examined using a TEAD-dependent Luciferase reporter construct. Co-immunoprecipitation assay was conducted to analyze protein interactions in the absence/presence of Hsp27. Pathway activity in prostate cancer will be assessed by immunohistochemistry staining of core components of the pathway in patients’ tissue samples. Results: Our preliminary findings indicate that Hsp27 negatively affects the Hippo pathway. We found that targeting Hsp27 using siRNA in the PC3 (prostate cancer), A549 (lung cancer) and MDA-MB-453 (triple negative breast cancer) leads to increased cytoplasmic retention of p-YAP compared to control siRNA treated cells. Moreover, using immunofluorescence, we observed reduced nuclear translocation of the YAP/TAZ as well as sequestration of these components with cytoplasmic 14-3-3 proteins in siRNA treated PC3 cells. Furthermore inhibition of Hsp27 in prostate and lung tumour cells resulted in suppression of TEAD transcriptional activity analyzed by qRT-PCR and luciferase assay. Hsp27 over-expression experiments yielded opposite results compared to knockdown. Conclusion: Hsp27 overexpression contributes to inactivation of Hippo pathway. Targeting Hsp27 leads to inactivation of YAP and TAZ onco-proteins affecting cancer cell survival. Impact: Our data further supports the significance of targeting Hsp27 as a treatment option in cancers, especially metastatic malignancies like CRPC. Citation Format: Sepideh Vahid, Daksh Thaper, Amina Zoubeidi. Hsp27 negatively affects Hippo tumor suppressor pathway to regulate cell survival in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4956. doi:10.1158/1538-7445.AM2015-4956

Introduction: Metastasis is the most common cause of death from cancer and occurs when malignant cells discard epithelial restraints and acquire invasive abilities, facilitating their dissemination to permissive micro-environments. This process is enhanced by tumor cell activation of Epithelial Mesenchymal Transition (EMT), a (normally embryonic) developmental program in which epithelial cells assume a mesenchymal phenotype during gastrulation and organogenesis, allowing single cell invasive movement away from the ectodermal layer. Recent evidence strongly implicates EMT induction in malignant progression and treatment resistance. For example, EMT regulatory transcription factors are required for breast cancer metastasis. Several oncogenic pathways (growth factors, Src family, MAPK, AKT) induce EMT. Lyn tyrosine kinase, a member of Src family tyrosine kinase is up-regulated in advanced prostate cancer and has been reported to correlate with aggressive breast cancer. Our objective is to determine the role of Lyn tyrosine kinase in EMT. Methods: LNCaP, BT-549, UC-13 cells were transfected with Lyn siRNA; EMT markers were monitored by western blot and qRT-PCR and immunofluorescence, migration by scratch assay, invasion by Boyden chamber and matrix metalloproteinase (MMP) activity by Zymography. Lyn specific Kinase inhibitor Bafetinib will also be used along with over-expression experiments with Lyn Kinase Dead and Constitutively active mutants. Results: Here we report that Lyn expression is low in epithelial cells and is up-regulated in mesenchymal cells. Targeting Lyn using siRNA decreases EMT markers (Fibronectin, Vimentin) at both mRNA and protein levels while increasing the epithelial marker (E-cadherin). Moreover, we found that targeting Lyn by siRNA/shRNA and small molecule inhibitor Bafetinib decreases cell migration, invasion and the activity of MMPs. This decrease in mesenchymal phenotype can be attributed to the decrease in the amount of Slug and Snail, transcriptional repressors of E-Cadherin and activators of Vimentin. Consequently, we also found that over-expressing Lyn in both as WT or constitutively active (CA) mutant rescues expression of Slug and Snail, inducing EMT, increased cell migration and invasion while Kinase Dead (KD) mutant has no effect. Conclusion: Expression of Lyn kinase can be correlated to low prognosis and aggressive/metastatic phenotype. We show that targeting Lyn activity initiates a switch to a more epithelial phenotype reducing cell migration and invasion. Impact: The data suggests that Lyn tyrosine kinase plays a role in Epithelial Mesenchymal Transition and could be considered as a target for metastatic disease; especially in the more aggressive forms of cancer like Triple Negative Breast Cancer or Castration Resistant Prostate Cancer. This could be a realistic therapeutic option, as the Lyn small molecule inhibitor Bafetinib, is currently in clinical trials for treatment of several cancers. Note: This abstract was not presented at the meeting. Citation Format: Daksh Thaper, Sepideh Vahid, Ka Mun Nip, Kirsi Ketola, Jennifer Bishop, Amina Zoubeidi. Lyn kinase promotes metastasis through EMT in cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4106. doi:10.1158/1538-7445.AM2015-4106

Background: The role of signal transducer and activator of transcription (STAT) 3 in the progression of Prostate Cancer (PCa), the most common male cancer in North America, is well established. Although androgen ablation remains the most effective therapy for patients with advanced disease, most progress to castrate resistant prostate cancer (CRPC). Despite the development of new potent anti-androgens, like Enzalutamide (ENZ), which prolongs survival in CRPC patients, ENZ resistance rapidly occurs. Re-activation of the androgen receptor (AR) is a hallmark of CRPC and can occur via different mechanisms, including STAT3 activation. Furthermore, STAT3 can promote CRPC independently of AR activity, however whether STAT3 promotes resistance to ENZ in CRPC remains unknown. We have found that our LNCaP xenograft derived ENZR cell lines exhibit higher activity of the STAT3 pathway compared to CRPC. Additionally, in this model further targeting the AR using novel anti-androgens is short lived or ineffective. Therefore, we propose that targeting STAT3 using the small molecule inhibitor GPA500, alone or in combination with anti-androgens may delay or treat ENZR CRPC. Methods and Results: We developed a unique model of ENZ-resistance and found that cell lines derived from serially passaged ENZR tumors displayed broad genetic diversity and differential AR activity but consistent STAT3 hyper-activation compared to CRPC controls. WB analysis showed that all ENZR cell lines had increased pSTAT3Y705 compared to CRPC. Accordingly, Crystal Violet and MTT proliferation assays showed that ENZR cell lines were more sensitive to the STAT3 inhibitor GPA500 compared to CRPC control. The reduction in growth of GPA500 treated ENZR cells was independent of PARP cleavage, whereas GPA500 induced PARP cleavage in CRPC cells. Moreover, in ENZR cell lines with high AR activity and CRPC control cells, inhibition of STAT3 reduced AR activity as shown with WB and qRT-PCR for AR downstream targets like PSA and an AR-luciferase reporter assay. In ENZR cells with low AR activity and a stem cell phenotype, GPA500 treatment reduced surface expression of α2β1, CD44 and CD133. Inhibition of STAT3 activity was confirmed by WB as well as qRT-PCR analysis of STAT3 regulated genes like c-Myc, MCL1, BCL-XL. Conclusion: In this study, we provide pre-clinical proof that targeting the STAT3 pathway using GPA500 in ENZ resistance as well as CRPC reduces cell proliferation and AR activity as well as expression of stem cell markers. Impact: Exploring mechanisms of ENZR resistance serves a critical unmet need in PCa oncology, as the number of men with ENZ resistant CRPC continues to rise. Targeting STAT3 with the small molecule inhibitor GPA500 may provide an effective method to treat ENZR patients or delay the emergence of ENZ resistance in CRPC via reduction of AR activity and/or by suppressing aggressive tumor cell phenotypes such as cancer stem cells. Citation Format: Daksh Thaper, Sepideh Vahid, Jennifer L. Bishop, Martin Johansson, Amina Zoubeidi. New derivative of galiellalactone inhibits the STAT3 activity and suppresses ENZ-resistant Prostate Cancer in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2015-728