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Background Genomic analyses of hundreds of prostate tumors have defined a diverse landscape of mutations and genome rearrangements, but the transcriptomic effect of this complexity is less well understood, particularly at the individual tumor level. We selected a cohort of 25 high-risk prostate tumors, representing the lethal phenotype, and applied deep RNA-sequencing and matched whole genome sequencing, followed by detailed molecular characterization. Results Ten tumors were exposed to neo-adjuvant hormone therapy and expressed marked evidence of therapy response in all except one extreme case, which demonstrated early resistance via apparent neuroendocrine transdifferentiation. We observe high inter-tumor heterogeneity, including unique sets of outlier transcripts in each tumor. Interestingly, outlier expression converged on druggable cellular pathways associated with cell cycle progression, translational control or immune regulation, suggesting distinct contemporary pathway affinity and a mechanism of tumor stratification. We characterize hundreds of novel fusion transcripts, including a high frequency of ETS fusions associated with complex genome rearrangements and the disruption of tumor suppressors. Remarkably, several tumors express unique but potentially-oncogenic non-ETS fusions, which may contribute to the phenotype of individual tumors, and have significance for disease progression. Finally, one ETS-negative tumor has a striking tandem duplication genotype which appears to be highly aggressive and present at low recurrence in ETS-negative prostate cancer, suggestive of a novel molecular subtype. Conclusions The multitude of rare genomic and transcriptomic events detected in a high-risk tumor cohort offer novel opportunities for personalized oncology and their convergence on key pathways and functions has broad implications for precision medicine. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0426-y) contains supplementary material, which is available to authorized users.

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.

The signal transducer and activator of transcription (STAT)3 governs essential functions of epithelial and hematopoietic cells that are often dysregulated in cancer. While the role for STAT3 in promoting the progression of many solid and hematopoietic malignancies is well established, this review will focus on the importance of STAT3 in prostate cancer progression to the incurable metastatic castration-resistant prostate cancer (mCRPC). Indeed, STAT3 integrates different signaling pathways involved in the reactivation of androgen receptor pathway, stem like cells and the epithelial to mesenchymal transition that drive progression to mCRPC. As equally important, STAT3 regulates interactions between tumor cells and the microenvironment as well as immune cell activation. This makes it a major factor in facilitating prostate cancer escape from detection of the immune response, promoting an immunosuppressive environment that allows growth and metastasis. Based on the multifaceted nature of STAT3 signaling in the progression to mCRPC, the promise of STAT3 as a therapeutic target to prevent prostate cancer progression and the variety of STAT3 inhibitors used in cancer therapies is discussed.

Increased expression of the molecular chaperone Hsp27 is associated with the progression of prostate cancer (PCa) to castration-resistant disease, which is lethal due to metastatic spread of the prostate tumor. Metastasis requires epithelial to mesenchymal transition (EMT), which endows cancer cells with the ability to disseminate from the primary tumor and colonize new tissue sites. A wide variety of secreted factors promote EMT, and while overexpression and constitutive activation of epidermal growth factor (EGF) signaling is associated with poor prognosis of PCa, a precise role of EGF in PCa progression to metastasis remains unclear. Here, we show that Hsp27 is required for EGF-induced cell migration, invasion and MMPs activity as well as the expression of EMT markers including Fibronectin, Vimentin and Slug with concomitant decrease of E-cadherin. Mechanistically, we found that Hsp27 is required for EGF-induced AKT and GSK3β phosphorylation and β-catenin nuclear translocation. Moreover, silencing Hsp27 decreases EGF dependent phosphorylation of β-catenin on tyrosine 142 and 654, enhances β-catenin ubiquitination and degradation, prevents β-catenin nuclear translocation and binding to the Slug promoter. These data suggest that Hsp27 is required for EGF-mediated EMT via modulation of the β-catenin/Slug signaling pathway. Together, our findings underscore the importance of Hsp27 in EGF induced EMT in PCa and highlight the use of Hsp27 knockdown as a useful strategy for patients with advanced disease.

Treatment with Enzalutamide (ENZ) prolongs survival in patients with incurable castration-resistant prostate cancer (CRPC). However, the rapid recurrence of ENZ resistant tumors and the increased incidence of lethal neuroendocrine cancers in CRPC patients underscore the need for understanding how aggressive cell types emerge in resistant disease in order to design more effective therapies. As an unbiased approach to uncovering novel mechanisms of ENZ resistance, ENZ resistant cells were established from castration-resistant LNCaP xenografts that recurred under ENZ treatment in vivo and were genetically profiled using Illumina Platform gene expression analysis as well as RNA Seq. Genes involved in myeloid cell differentiation and immune checkpoint responses, as well as neuroendocrine differentiation, were strongly upregulated in ENZ resistant cell lines compared to CRPC controls. To investigate whether ENZ resistant cells may pass through a “dedifferentiated” state allowing progression to a neuroendocrine phenotype with immune-evasive properties, markers of cancer stem cells (CD133, CD44, CD117, CD49F, TROP2) neuroendocrine cells (neuron specific enolase, snaptophysin, chromagranin A, aurora kinase A, NCAM-1) and immune-evasion (programmed death ligands-1 and 2, B7H3, ICOSL) were assessed by quantitative PCR and flow cytometry. Indeed, all cancer stem cell markers were expressed at a higher frequency on ENZ resistant cells than CRPC controls and neuroendocrine markers were highly upregulated. Consistent with gene profiling results suggestive of a myeloid differentiation process, neuroendocrine-like resistant cells displayed an “immune” phenotype, expressing cell surface markers and transcription factors characteristic of dendritic cells (CD11c, HLA-DR, IRF8, ID2, AHR, BATF) that were absent from CRPC cells. Moreover, a greater frequency of neuroendocrine-like ENZ resistant cells expressed PD-L1, PD-L2 and ICOSL, and they showed intense staining for B7H3. Each of these molecules is well known to limit T cell effector responses and have been implicated in immune evasion strategies of solid tumors. Taken together, our results suggest that acquisition of resistance to Enzalutamide promotes the emergence of neuroendocrine-like cells that may be able to subvert anti-tumor T cell responses via dysregulation of antigen presentation or by enhancing immune checkpoint signaling. These results support anecdotal findings of increased frequency of neuroendocrine prostate cancer in patients undergoing long-term androgen withdrawal therapy and suggest investigating the utility of immunotherapies, such as anti-CTLA-4 or PD-1, in this subset of patients.

Defining the mechanisms underlying metastatic progression of prostate cancer may lead to insights into how to decrease morbidity and mortality in this disease. An important determinant of metastasis is epithelial-to-mesenchymal transition (EMT), and the mechanisms that control the process of EMT in cancer cells are still emerging. Here, we report that the molecular chaperone Hsp27 (HSPB1) drives EMT in prostate cancer, whereas its attenuation reverses EMT and decreases cell migration, invasion, and matrix metalloproteinase activity. Mechanistically, silencing Hsp27 decreased IL-6–dependent STAT3 phosphorylation, nuclear translocation, and STAT3 binding to the Twist promoter, suggesting that Hsp27 is required for IL-6–mediated EMT via modulation of STAT3/Twist signaling. We observed a correlation between Hsp27 and Twist in patients with prostate cancer, with Hsp27 and Twist expression each elevated in high-grade prostate cancer tumors. Hsp27 inhibition by OGX-427, an antisense therapy currently in phase II trials, reduced tumor metastasis in a murine model of prostate cancer. More importantly, OGX-427 treatment decreased the number of circulating tumor cells in patients with metastatic castration-resistant prostate cancer in a phase I clinical trial. Overall, this study defines Hsp27 as a critical regulator of IL-6–dependent and IL-6–independent EMT, validating this chaperone as a therapeutic target to treat metastatic prostate cancer. Cancer Res; 73(10); 3109–19. ©2013 AACR.

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.

Intestinal homeostasis requires a complex balance of interactions between diverse resident microbial communities, the intestinal epithelium, and the underlying immune system. We show that the Lyn tyrosine kinase, a critical regulator of immune cell function and pattern-recognition receptor (PRR) responses, has a key role in controlling gastrointestinal inflammation. Lyn⁻/⁻ mice were highly susceptible to dextran sulfate sodium (DSS)-induced colitis, whereas Lyn gain-of-function (Lyn(up)) mice exhibited attenuated colitis during acute and chronic models of disease. Lyn(up) mice were hypersensitive to lipopolysaccharide (LPS), driving enhanced production of cytokines and factors associated with intestinal barrier function, including interleukin (IL)-22. Oral administration of LPS was sufficient to protect antibiotic-treated Lyn(up) but not wild-type mice from DSS, highlighting how Lyn-dependent changes in the nature/magnitude of PRR responses can impact intestinal health. Furthermore, protection from DSS-induced colitis and increased IL-22 production in response to LPS did not depend on the adaptive immune system, with increased innate lymphoid cell-derived IL-22 correlating with Lyn activity in dendritic cells. These data reveal a key role for Lyn in the regulation of innate immune responses and control of intestinal inflammation.

Foot morphology and gait patterns of children have been explored in order to understand the developmental stages [1–3]. However, these studies have mostly focused on static and walking conditions. The purpose of this study was to investigate the development of running in children between the ages of 3 and 11 years and to investigate gender differences. This was done as a multi-lab, multi-country study; but the foot pressure results from the Japanese children are presented here.Copyright © 2009 by ASME

The SH2 domain-containing inositol 5′-phosphatase, SHIP, negatively regulates various hematopoietic cell functions and is critical for maintaining immune homeostasis. However, whether SHIP plays a role in controlling bacterial infections in vivo remains unknown. Salmonella enterica causes human salmonellosis, a disease that ranges in severity from mild gastroenteritis to severe systemic illness, resulting in significant morbidity and mortality worldwide. The susceptibility of ship +/+ and ship −/− mice and bone marrow-derived macrophages to S. enterica serovar Typhimurium infection was compared. ship −/− mice displayed an increased susceptibility to both oral and intraperitoneal serovar Typhimurium infection and had significantly higher bacterial loads in intestinal and systemic sites than ship +/+ mice, indicating a role for SHIP in the gut-associated and systemic pathogenesis of serovar Typhimurium in vivo. Cytokine analysis of serum from orally infected mice showed that ship −/− mice produce lower levels of Th1 cytokines than do ship +/+ animals at 2 days postinfection, and in vitro analysis of supernatants taken from infected bone marrow-derived macrophages derived to mimic the in vivo ship −/− alternatively activated (M2) macrophage phenotype correlated with these data. M2 macrophages were the predominant population in vivo in both oral and intraperitoneal infections, since tissue macrophages within the small intestine and peritoneal macrophages from ship −/− mice showed elevated levels of the M2 macrophage markers Ym1 and Arginase 1 compared to ship +/+ cells. Based on these data, we propose that M2 macrophage skewing in ship −/− mice contributes to ineffective clearance of Salmonella in vivo.

Anterior cruciate ligament (ACL) injuries are very common, causing pain, loss of function, and medical expense. Women sustain ACL injuries more often than men. Most of these injuries (70%) are non-contact for women [1], occurring during pivoting or sudden deceleration [2]. Training programs have been developed to try to prevent these injuries in women. These programs are designed to improve strength, muscle balance, and knee control. Evaluating the effectiveness of training programs is difficult. To date, these evaluations have only assessed factors such as muscle activation and joint angles that are not directly related to ACL strain and ACL injury risk.Copyright © 2007 by ASME

The ability to evade host immune surveillance is a critical virulence determinant for any pathogenic microorganism. The intracellular pathogen Listeria monocytogenes is highly adept at reaching privileged niches within the host without alerting immune responses. Upon infection, Listeria penetrates epithelial cells in the gastrointestinal tract and spreads silently from cell to cell. Once this barrier is breached, it has the ability to escape the phagosome and replicate within phagocytic cells, using them as vehicles to disseminate throughout the host (reviewed in ref. 1). The capacity to defend against the onslaught of immune defenses is another key aspect to being a successful pathogen. In this issue of PNAS, Boneca et al. (2) demonstrate that Listeria modifies peptidoglycan (PG) in its cell wall, thereby conferring resistance to the host bacteriolytic product, lysozyme. By preventing bacterial degradation and subsequent release of immunostimulants, PG modification is therefore also a means by which Listeria evades immune detection by host pattern–recognition receptors (PRRs). Importantly, although many pathogens are known to modify their PG, this report by Boneca et al. (2) is the first to demonstrate that PG N-deacetylation has an important role in virulence and evasion of host defense.

Androgen receptor- (AR-) expressing prostate cancer (PCa) cell lines can transdifferentiate to neuro-ectodermal cell lineages under androgen deprivation. We have proposed that this transdifferentiation is mediated by initial regression to an unstable cancer stem cell-like (CSCL) intermediate state and we have developed a serum- and androgen-free custom cell culture medium that now allows us to efficiently reprogram PCa cells to this CSCL state and expand them for characterization. Exposure of adherent cultures of parental LNCaP, VCaP, LAPC4 or CWR22rv1 cells to the reprogramming medium induced a rapid and efficient (>67%) morphological change of plated cells to smaller, rounded cells with large nuclei and prominent nucleoli that grow in 3-dimensions. Following transfer of the reprogrammed cells they, thereafter, grow as cell rosettes that will form larger spheroids. Analysis of changes in gene expression by microarray gene expression profiling or immunohistochemistry (IHC) showed a remarkable overexpression of stem cell genes in the reprogrammed cells and a change in cell surface CD protein expression that resembles neural stem cells. Reprogramming drastically reduced expression of AR RNA and protein in all cell lines. Once reprogrammed, the CSCLs have significantly higher tumor-initiating capacity based upon tumors formed from cell serial dilutions xenografted into intact or castrated male nude mice. In vitro, reprogrammed cells were significantly more resistant to the anti-androgen enzalutamide and to hypoxia compared to parentals. By placing CSCLs into specific differentiation mediums, we could differentiate them back into neural-, oligodendrocyte-, glia- or osteoblast-like cells based upon induced expression of lineage-specific RNAs/proteins or return them to prostate-like cells (that re-express AR) by supplementing them with serum and androgen. Reprogrammed cells have an EMT phenotype based on increased expression of EMT-related genes and significantly increased invasiveness in in vitro and in vivo models. Comparison of gene expression changes acquired by reprogrammed CSCLs amongst the different cell models revealed a shared set of 65 over-/under-expressed genes (≥1.8-fold) between them. Finally, we have found that growth of parental LNCaP cells in androgen-deprived or enzalutamide-supplemented (serum-containing) growth medium increased the population of cells bearing CSCL surface CD stem markers. In summary, our studies show that commonly used PCa cell lines have the ability to efficiently transition between a differentiated and a CSCL state in an appropriate microenvironment that is depleted of androgens. While in the CSCL state, these cells show increased invasiveness, increased resistance to hormonal therapeutics and a propensity to transdifferentiate to alternate cell lineages under appropriate stimulation. Citation Format: Josselin Caradec, Amy A. Lubik, Mannan Nouri, Na Li, Manuel Altimarano-Dimas, Jennifer Bishop, Mani Moniri, Down Cochrane, Martin Gleave, Ralph Buttyan. Efficient conditional reprogramming of differentiated prostate cancer cells back to a stem cell-like state with increased aggressive properties. [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 4073. doi:10.1158/1538-7445.AM2015-4073

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

e16075 Background: Treatment benefits of the anti-androgen Enzalutamide (ENZ) in patients with castration-resistant prostate cancer (CRPC) are limited, underscoring the need for alternative therapi...

Although Salmonella enterica serovars are some of the best-studied bacterial pathogens, the field still has a long way to go, especially when one considers that (i) they cause significant morbidity and mortality worldwide; (ii) they have broad host ranges, but for unknown reasons, infections result in different diseases in different hosts; (iii) they are able to establish persistent infections, which serve as reservoirs for transmission/shedding; and (iv) they are increasingly resistant to many antibiotics. The Second Conference on Salmonella, sponsored by the American Society for Microbiology, was held in Victoria, Canada, from 9 to 13 September 2006. Over 200 participants from around the world gathered at the historic Empress Hotel to discuss their latest findings on Salmonella population biology, animal models, pathogenic mechanisms, clinical disease, and vaccination. The first highlight of the conference was the keynote address by Roy Curtiss III (Arizona State University, Temple). He embodied the spirit of the conference “from pathogenesis to therapeutics,” as his life with Salmonella has taken him from investigating bacterial genetics and pathogenic mechanisms to translating this research into therapeutic approaches and vaccine development. This review will highlight some of the central themes that arose during the course of the conference; however, as it is so brief, it will describe only a fraction of the many excellent talks and posters presented.

Retroviruses evolve rapidly to avoid the immune response of the infected host. We show here that the wild-type mouse mammary tumor virus MMTV(C3H) persisted indefinitely in C3H/HeN mice. However, it was rapidly lost in mice of the closely related C3H/HeJ strain and was replaced by a virus recombinant with an endogenous Mtv provirus. Maintenance of the wild-type virus was dependent on Toll-like receptor-4 (TLR4) signaling, which triggered production of the immunosuppressive cytokine interleukin-10. In the presence of mutant TLR4 in C3H/HeJ mice, wild-type virus was eliminated by the cytotoxic immune response, promoting selection of the immune escape recombinant MMTV variants. Thus, subversion of the innate immune system is yet another survival strategy used by retroviruses.

Advances in castrate-resistant prostate cancer (CRPC) treatment using androgen receptor (AR) targeted therapies have produced striking survival advantages in clinical studies; recently approved AR inhibitor Enzalutamide (ENZ) prolongs survival of patients with incurable CRPC. Although enthusiasm for this approach remains high, prostate tumor heterogeneity and the inevitable development of resistance as seen currently in the clinic and in our pre-clinical model, dictates a critical need for a better understanding of the mechanisms of resistance. We have recently developed a unique model of LNCaP-CRPC-ENZ-resistance and found that cell lines derived from serially passaged ENZ resistant tumors express different levels of prostate specific antigen (PSA). Especially low PSA expressing cells are believed to be highly enriched with cancer stem cells with high self-renewal and these cells have been considered difficult to target with current therapies. To define molecular mechanisms contributing to treatment resistance in an unbiased manner, we performed gene profiling on LNCaP, CRPC and ENZ resistant cell lines expressing low and high levels of PSA. Using systems biological approaches, we found that several genes and pathways involved in stem cell maintenance were altered in Enzalutamide resistant cells. Interestingly, forkhead box M1 (FOXM1) was one of the key pathways potentially maintaining cancer stem cells and driving dedifferentiation during ENZ resistance. FOXM1 is a transcription factor that confers treatment resistance and is required for maintenance of pluripotency in stem cells and PCa CSC-like spheroid formation as well as induction of epithelial-to-mesenchymal transition and invasion. Interestingly, FOXM1 and its downstream targets were upregulated in both PSA high and low expressing ENZ resistant cells compared to CRPC cells and correlated with poor survival and Gleason score in CRPC. In addition, mining public data, we found that FOXM1 is highly expressed in prostate cancer cells with low PSA expression, indicating that also low PSA expressing prostate cancer stem cells could be targeted by inhibiting FOXM1. Using systems biological approaches, we further found out that the antibiotic ionophore and oxidative stress inducer Monensin, reduces FOXM1 pathway activity in ENZ resistant cells. The validation results indicated that Monensin reduced cell viability, FOXM1 transcriptional activity as well as FOXM1 protein, gene and its target gene expressions in both PSA high and low expressing ENZ resistant prostate cancer cells. Taken together, the results suggest that inhibition of FOXM1 by Monensin is a potent novel mean to target ENZ resistant cells including low PSA expressing prostate cancer stem cell-like cells. Citation Format: Kirsi Ketola, Jennifer Bishop, Ka Mun Nip, Soojin Kim, Fazli Ladan, Martin Gleave, Amina Zoubeidi. Inhibition of FOXM1 targets both high and low PSA expressing prostate cancer cells resistant to Enzalutamide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 676. doi:10.1158/1538-7445.AM2014-676

Androgen-dependent prostate cancer (PCa) cells can acquire features of neuroendocrine- (NE-) cells under acute androgen deprivation (AD). Gene expression studies show that other neuroectodermal/mesenchymal (NE/M) cell phenotypes develop under chronic AD. We propose that a transient PCa stem-like cell mediates these transdifferentiations and we sought to reveal and maintain this stem-like state using a special growth medium that is compatible with stem cells that could give rise to these complex lineages. LNCaP or CWR22r cells were plated at low density on coated plates then switched to an androgen-free “stem-transition medium” compatible with NE/M-derived stem cells. Eight days later, cells were transferred in the same medium and were characterized for morphology and 3-dimensional (3D) growth properties. Growth in the medium converted these cells, en masse, to small, rounded proliferating cells that grew as 3D rosettes then to spheroids. Gene expression and cell surface CD antigen profiling of converted LNCaP cells were most consistent with a neural/neural crest stem-like (N/NCSL) identity. N/NCSL PCa cells fail to express androgen receptor (AR) or PSA protein and they are significantly more resistant to hypoxia and enzalutamide than parental LNCaPs. LNCaP-N/NCSL cells have significantly increased tumor-initiating capability and formed tumors in nude mice from only 100 xenografted cells vs 1 X 106 parental LNCaP cells needed to form tumors. When LNCaP N/NCSL cells were placed into differentiation mediums without androgen, they acquired morphological features and expressed biomarkers of neuron-, glia-, oligodendrocyte or osteocyte-like cells. When they were redifferentiated in androgen-containing medium, they reverted to an epithelial cell that resembles parental LNCaPs but they overexpressed AR and retained expression of nestin, a neural stem marker. Our outcomes show that differentiated (AR+) PCa cells have a previously unsuspected developmental plasticity that allows them to return back to a neural/neural crest stem-like state with drastically increased tumor initiating capability under certain microenvironmental conditions. Our new model cell culture system allows us to maintain them in this state or to differentiate them to a variety of NE/M-derived cell lineages. Reactivation of androgen signaling, however, returns them to a modified prostate epithelial-like state that overexpresses AR and retains expression of some stem genes. We believe that our model culture system reveals a novel pathway associated with progression of PCa to therapy-resistance. *Authors contributed equally Citation Format: Josselin Caradec*, Amy Lubik*, Mannan Nouri, Na Li, Jennifer Bishop, Martin Gleave, Ralph Buttyan. A spontaneous developmental lineage plasticity that underlies the response of prostate cancer cells to androgen deprivation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3017. doi:10.1158/1538-7445.AM2014-3017

149 Background: ASCO recommends CSFs such as pegfilgrastim for dose dense doxorubicin cyclophosphamide paclitaxel (ddACT) in breast cancer. It is unclear whether dose dense paclitaxel requires pegfilgrastim, and some community oncologists use pegfilgrastim selectively with the dose dense paclitaxel portion of ddACT. We reviewed the results of pegfilgrastim use based on physician preference within a single breast oncology community-based practice. Methods: This is a single-center, single-practice retrospective cohort study of adjuvant ddACT for breast cancer patients identified from the pharmacy database between 1/2010 and 6/2014. Patient demographics, stage, chemotherapy and pegfilgrastim use, chemotherapy delays, febrile neutropenic (FN) events, and pain records including narcotic use were collected from patient charts. Patients receiving pegfilgrastim routinely at cycle 1 were compared to those within the practice given pegfilgrastim selectively for ANC < 1.5 on day of treatment or nadir ANC < 1.0 during the preceding cycle. Differences between groups were analyzed using the Mann-Whitney test, with significance at the p < 0.05 level. Results: The two groups were balanced with regard to age and number of cycles. There were no significant differences in cycle delay, and FN. Routine pegfilgrastim resulted in more bone pain and higher cost. Conclusions: The selective use of pegfilgrastim during the paclitaxel portion of ddACT did not result in chemotherapy delay, more infections or FN. It did decrease painful myalgia and arthralgias, and dramatically lowered treatment cost. These retrospective data, although limited by participant number, question the need for the routine use of pegfilgrastim for the paclitaxel portion of ddACT and require prospective validation. [Table: see text]

Prostate cancer initially responses to androgen ablation or castration, but progression to incurable castrate-resistant prostate cancer often occurs within three years. Androgen receptor pathway inhibitor Enzalutamide (ENZ) has produced promising survival gains of patients with CRPC. However, resistance to this novel therapy also rapidly occurs and novel treatment strategies are needed. We have recently developed a unique ENZ and castrate-resistant LNCaP xenograft model. To identify key regulators and active pathways in ENZ resistant cells, genome-wide gene expression analysis was performed and analyzed using systems biological approaches. We identified that forkhead box M1 (FOXM1) is one of the key upstream regulators activated in ENZ resistant prostate cancer cells. FOXM1 is overexpressed in several cancers and it is an important regulator of cancer characteristics such as cell cycle, proliferation, invasion, metastases and cancer stem cells. FOXM1 is also a critical regulator of oxidative stress and its depletion sensitises cells to oxidative stress induced apoptosis. Interestingly, we further found out that the antibiotic ionophore and oxidative stress inducer monensin that was previously identified as a cancer-specific prostate cancer inhibitor in high-throughput screening, reduces FOXM1 pathway activity and viability of ENZ resistant cells. Thus, the results suggest that inhibition of FOXM1 is a potent novel mean to impair redox control and target ENZ resistant prostate cancer cells. Taken together, our results indicated that FOMX1 activity is increased in ENZ resistant prostate cancer cells and that targeting FOXM1 is a potential means to overcome ENZ resistance in prostate cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C91. Citation Format: Kirsi Ketola, Jennifer Bishop, Martin Gleave, Amina Zoubeidi. Targeting enzalutamide resistance in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C91.

Introduction: Prostate cancer (PCa) is the most common cancer and the second leading cause cancer death in North American men. An important determinant for metastasis is epithelial to mesenchymal transition (EMT) which is known to be correlated to castrate resistant prostate cancer (CRCP). Previous report demonstrated the overexpression of Hsp27 and its phosphorylated form in poor diagnosis prostate cancer. In addition, both epidermal growth factor (EGF) and its receptor (EGFR) are up–regulated in PCa during progression to CRPC. However, the role of EGF and EGFR in PCa progression remains unclear. Our objective is to establish that Hsp27 is a central “node” for several signaling pathways activated by EGF and known to modulate EMT. Methods: To assess the role of Hsp27 in EMT, gain and loss of Hsp27 function has been conducted in LNCaP cells with or without EGF stimulation. EMT markers expression has been evaluated at the protein (WB) and mRNA level (qRT–PCR). Furthermore, invasion, migration assays in addition with MMP–9 activity have been done to evaluate the migratory capacity of those cells. Also, to better understand the molecular mechanism involved in EMT induction, transactivation and Chip assays have been performed. The in vitro results have been confirmed with an in vivo study using an Hsp27 antisense oligonucleotide (OGX-427) in a murine model. Results: We found that Heat shock protein 27 (Hsp27) is up–regulated with CRPC progression and drives EMT in PCa cells by decreasing epithelial cell markers, up–regulating mesenchymal markers and enhancing cell migration and MMP–9 activity. Hsp27 knockdown induces sequestration of beta–catenin in the cytoplasm by increasing its binding to GSK–3beta and abrogating EGF induced beta–catenin nuclear translocation. Additionally, by decreasing the nuclear translocation of beta-catenin, Hsp27 knockdown decreases the binding of beta–catenin to slug promoter thereby lessening its transcriptional activity. As Slug is known to be a critical key in EMT induction, we confirmed the role of EGF and Hsp27 in EMT induction and our capacity to abrogate the EGF-induced EMT effect by Hsp27 knockdown. Finally, targeting Hsp27 using antisense oligonucleotide OGX-427 in vivo, significantly reduced tumor cell metastasis in a PCa murine model. Conclusion: This study shows that EGF through Hsp27 induces modifications in gene expression and morphology toward a mesenchymal phenotype. The mechanism involving Hsp27, β-catenin, nuclear β-catenin/TCF and EGF signaling cascades described in the present study contribute to the comprehension of mechanisms driving metastasis in aggressive castration-resistant prostate cancer. This study reveals that Hsp27 is a crucial effector of EGF–dependent and independent EMT and suggests that targeting Hsp27 holds promises for a new strategy in metastatic PCa. Citation Format: Thomas D. Cordonnier, Jennifer L. Bishop, Masaki Shiota, Ario Takeuchi, Ka Mun Nip, Martin Gleave, Amina Zoubeidi. Hsp27 is an essential effector of EGF-induced epithelial to mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 281. doi:10.1158/1538-7445.AM2013-281

Prostate cancer (PCa) is the most common cancer and the second leading cause cancer death in North American men. Understanding the molecular mechanisms underlying metastatic PCa development is essential to decrease morbidity and mortality. An important determinant for metastasis is epithelial to mesenchymal transition (EMT). We show that the molecular chaperone Hsp27 drives EMT in PCa cells by decreasing epithelial cell markers, upregulating mesenchymal markers and enhancing cell migration and MMP-9 activity. Mechanistically, Hsp27 is required for EMT induced by epidermal growh factor (EGF) via modulation of the beta-catenin/Slug signaling pathway. Hsp27 knockdown induces sequestration of beta-catenin in the cytoplasm by increasing its binding to GSK3beta and abrogating EGF induced beta-catenin nuclear translocation. Additionally, by decreasing the nuclear translocation of beta-catenin, Hsp27 knockdown decreases the binding of beta-catenin to the Slug promoter, thereby abrogating its transcriptional activity. As Slug is known to be a critical factor in EMT induction, we confirmed the role of Hsp27 in EGF mediated EMT using Hsp27 knockdown. To address a role for Hsp27 in promoting aggressive PCa in vivo, Hsp27 was suppressed in a murine model of PCa using the antisense drug OGX-427 (currently in phase II clinical trials). We found OGX-427 treatment significantly reduces tumor cell metastasis in mice, and moreover, decreases circulating tumor cell counts in patients with castration resistant prostate cancer. This study defines a role for Hsp27 as a critical regulator of EGF dependent EMT and highlights Hsp27 is an attractive anti-cancer target for metastatic PCa. Citation Format: Thomas Cordonnier, Jennifer L. Bishop, Masaki Shiota, Ario Takeuchi, Ka Mun Nip, Martin Gleave, Amina Zoubeidi. Hsp27 is required for EGF-induced epithelial to mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C25.