PDF file - 60K, Breeding strategy to produce the experimental animals. To produce the required F1 experimental animals, heterozygous TRAMP C57BL/6 females were crossed with Cd151 KO C57BL/6 males. The resultant Cd151 heterozygous C57BL/6 and TRAMP heterozygous females were then crossed with Cd151 heterozygous FVB/N males. Genetic frequencies shown are specific for the male experimental animals. This breeding program ensured the absence of kidney disease that occurs in FVB/N Cd151 homozygous KO mice. It also ensured a homogeneous genetic background for test and control mice which, in addition, were littermates

PDF file - 112K, Characterisation for expression of the epithelial cell marker cell marker, E-Cadherin. 5 m FFPE sections of normal prostate glands from 20 week old TRAMP negative animals along with sections from poorly differentiated primary prostate tumours from TRAMP positive animals were characterised by indirect peroxidase IHC with the anti E-Cadherin antibody (clone 36) that labels epithelial cells, as the primary antibody. The epithelial cells in the normal prostate glands in Panel A stained highly and specifically for E-Cadherin both on the membrane and also through the cytoplasm. In Panel B, the prostate gland that displays PIN has reduced labelling of E-cadherin and the surrounding poorly differentiated prostate tumour tissue was void of specific labelling for E-cadherin

PDF file - 297K, Characterisation of tumour lesions for expression of the neuroendocrine cell marker, synaptophysin. 5 m FFPE sections of primary prostate tumours along with metastatic lesions in the lung and liver were characterised by indirect peroxidase IHC with the anti synaptophysin antibody (clone SY38) that labels neuroendocrine cells, as the primary antibody. Brain sections stained for neuroendocrine cells (panel A, 50 x and B 200x) show positive stained layers of the dendate gyrus indicated by the star in panel B while the arrow shows specificity as the layer is negative for synaptophysin labelling. PIN and well differentiated primary prostate tumours represented in panel C, moderately differentiated prostate tumour, represented in panel D along with liver metastatic lesion represented in panel E were negative for the labelling of synaptophysin

Background Prostate cancer (PC) remains a leading cause of cancer mortality and the most successful chemopreventative and treatment strategies for PC come from targeting the androgen receptor (AR). Although AR plays a key role, it is likely that other molecular pathways also contribute to PC, making it essential to identify and develop drugs against novel targets. Recent studies have identified peroxisome proliferator-activated receptor gamma (PPAR gamma), a nuclear receptor that regulates fatty acid (FA) metabolism, as a novel target in PC, and suggest that inhibitors of PPAR gamma could be used to treat existing disease. We hypothesized that PPAR gamma acts through AR-dependent and independent mechanisms to control PC development and growth and that PPAR gamma inhibition is a viable PC treatment strategy. Methods Immunohistochemistry was used to determine expression of PPAR & x4af; in a cohort of patients with PC. Standard molecular techniques were used to investigate the PPAR & x4af; signaling in PC cells as well a xenograft mouse model to test PPAR & x4af; inhibition in vivo. Kaplan-Meier curves were created using cBioportal. Results We confirmed the expression of PPAR & x4af; in human PC. We then showed that small molecule inhibition of PPAR gamma decreases the growth of AR-positive and -negative PC cells in vitro and that T0070907, a potent PPAR gamma antagonist, significantly decreased the growth of human PC xenografts in nude mice. We found that PPAR gamma antagonists or small interfering RNA (siRNA) do not affect mitochondrial activity nor do they cause apoptosis; instead, they arrest the cell cycle. In AR-positive PC cells, antagonists and siRNAs reduce AR transcript and protein levels, which could contribute to growth inhibition. AR-independent effects on growth appear to be mediated by effects on FA metabolism as the specific FASN inhibitor, Fasnall, inhibited PC cell growth but did not have an additive effect when combined with PPAR gamma antagonists. Patients with increased PPAR & x4af; target gene expression, but not alterations in PPAR & x4af; itself, were found to have significantly worse overall survival. Conclusions Having elucidated the direct cancer cell effects of PPAR gamma inhibition, our studies have helped to determine the role of PPAR gamma in PC growth, and support the hypothesis that PPAR gamma inhibition is an effective strategy for PC treatment.

Purpose Prostate carcinoma consists of tumor epithelium and malignant stroma. Until recently, diagnostic and therapeutic efforts have focused exclusively on targeting characteristics of the tumor epithelium, ignoring opportunities to target inflammatory infiltrate and extracellular matrix components. Prostate tumors are rich in tumor-associated macrophages (TAMs), which can be either of the cytotoxic M1 or protumorigenic M2 phenotype. We have quantified the proportion of each in seven common human prostate tumor lines grown subcutaneously in athymic nude mice and have imaged macrophage densities in vivo in xenografts derived from these lines. Procedures A panel of seven human prostate cancer xenografts was generated in intact male athymic nude mice reflecting variable expression of the androgen receptor (AR) and prostate-specific membrane antigen (PSMA). Mice were imaged ex vivo using near-infrared fluorescence (NIRF) imaging for PSMA expression and total macrophage densities to enable direct comparison between the two. Tumors were harvested for sectioning and additional staining to delineate M1 and M2 phenotype along with vascular density. Results Macrophage polarization analysis of sections revealed that all xenografts were > 94% M2 phenotype, and the few M1-polarized macrophages present were confined to the periphery. Xenografts displaying the fastest growth were associated with the highest densities of macrophages while the slowest growing tumors were characterized by focal, tumor-infiltrating macrophage densities. Xenograft sections displayed a strong positive spatial relationship between macrophages, vasculature, and PSMA expression. Conclusions Prostate TAM disposition can be imaged ex vivo and is associated with growth characteristics of a variety of tumor subtypes regardless of PSMA or AR expression.

Abstract Prostate cancer (PC) is the second most diagnosed cancer and sixth leading cause of cancer mortality in men in the world. The androgen receptor (AR) plays key roles in the development of prostate tissue as well as in the development and progression of PC. Following male development, AR guides cytodifferentiation and homeostasis in benign luminal epithelial cells. However, in PC, AR instead drives the uncontrolled proliferation of these cells; this "AR malignancy shift" is a central event in tumorigenesis, as the AR becomes a primary driver of growth in malignant neoplastic cells. Despite this change in AR behavior having been recognized for decades, the details of the AR malignancy shift remain unclear. Using a ChIP-seq approach in primary human tissues, cell lines, and mouse models, we demonstrate that the AR malignancy shift occurs in every sample analyzed, suggesting that it is necessary for PC development. Using molecular and genetic techniques, we demonstrate that FOXA1, HOXB13, GATA2, JUN, and CEBPA are involved in the regulation of the AR malignancy shift. AR binding sites (ARBS) are enriched for FOX, HOX, and GATA motifs in PC cells but for JUN and CEBP motifs in benign cells. We show that mutations commonly found in localized PCs, including ERG, SPOP and FOXA1 alterations, can CAUSE the AR malignancy shift. Furthermore, these alterations are not transformative on their own but must be coupled to other mutations to transform cells, suggesting that the AR malignancy shift is not sufficient for tumorigenesis. We show that the AR malignancy shift occurs in mouse models of PC as well and that chronic low T, which is associated with increased PC risk and aggressiveness in humans, also causes the AR malignancy shift in mouse prostate tissue without the development of frank carcinoma. In conclusion, we have discovered a previously-unrecognized, fundamental tenet of PC, one which explains how and why AR signaling is different in cancer and benign cells. Our work has the potential to create genomic classifiers that accurately predict clinical outcomes and could be used to stratify patients with localized PC for specific treatments. Furthermore, our work suggests that the AR malignancy shift is a novel target for the treatment and/or prevention of PC.

The androgen receptor (AR) plays key roles in the development of prostate tissue and the development and progression of prostate cancer (PC). AR guides cytodifferentiation and homeostasis in benign luminal epithelial cells; however, in PC, AR instead drives the uncontrolled proliferation of these cells. This ‘AR malignancy shift’ (AMS) is a central event in tumorigenesis. Using a ChIP‐seq approach in primary human tissues, cell lines, and mouse models, we demonstrate that the AMS occurs in every sample analyzed, suggesting that it is necessary for PC development. Using molecular and genetic techniques, we demonstrate that forkhead box (FOX)A1, HOXB13, GATA2, and c‐JUN are involved in the regulation of the AMS. AR‐binding sites (ARBS) are enriched for FOX, HOX, and GATA motifs in PC cells but not for c‐JUN motifs in benign cells. We show that the SPOP mutation commonly found in localized PCs can cause the AMS but is not transformative on its own and must be coupled to another mutation to transform cells. We show that the AMS occurs in mouse models of PC as well and that chronic low T, which is associated with increased PC risk and aggressiveness in humans, also causes the AMS in mice. We have discovered a previously unrecognized, fundamental tenet of PC, one which explains how and why AR signaling is different in cancer and benign cells. Our work has the potential to be used to stratify patients with localized PC for specific treatments. Furthermore, our work suggests that the AMS is a novel target for the treatment and/or prevention of PC.

BACKGROUND:Androgens and the androgen receptor (AR) are necessary for the development, function, and homeostatic growth regulation of the prostate gland. However, once prostate cells are transformed, the AR is necessary for the proliferation and survival of the malignant cells. This change in AR function appears to occur in nearly every prostate cancer. We have termed this the AR malignancy shift. METHODS:In this review, we summarize the current knowledge of the AR malignancy shift, including the DNA-binding patterns that define the shift, the transcriptome changes associated with the shift, the putative drivers of the shift, and its clinical implications. RESULTS:In benign prostate epithelial cells, the AR primarily binds consensus AR binding sites. In carcinoma cells, the AR cistrome is dramatically altered, as the AR associates with FOXA1 and HOXB13 motifs, among others. This shift leads to the transcription of genes associated with a malignant phenotype. In model systems, some mutations commonly found in localized prostate cancer can alter the AR cistrome, consistent with the AR malignancy shift. Current evidence suggests that the AR malignancy shift is necessary but not sufficient for transformation of prostate epithelial cells. CONCLUSIONS:Reinterpretation of prostate cancer genomic classification systems in light of the AR malignancy shift may improve our ability to predict clinical outcomes and treat patients appropriately. Identifying and targeting the molecular factors that contribute to the AR malignancy shift is not trivial but by doing so, we may be able to develop new strategies for the treatment or prevention of prostate cancer.

Protein phosphatase 2A (PP2A) is a family of serine/threonine phosphatases that regulate multiple cellular signalling pathways involved in proliferation, survival and apoptosis. PP2A inhibition occurs in many cancers and is considered a tumour suppressor. Deletion/downregulation of PP2A genes has been observed in breast tumours, but the functional role of PP2A subunit loss in breast cancer has not been investigated.

Breast cancer is the most common cancer in women and a leading cause of death. Dysregulation of cellular signalling pathways controlling proliferation, survival and migration, such as the PI3K/Akt and Ras/MAPK pathways, are key features of breast cancer. Protein phosphatase 2A (PP2A) negatively regulates many components of these pathways. PP2A is a family of trimeric serine/threonine phosphatases, each consisting of a structural, a catalytic and a regulatory subunit of which there are multiple isoforms. The addition of specific regulatory subunits provides subcellular targeting and substrate specificity to the enzyme. While PP2A is generally considered a tumor suppressor, a specific role for individual PP2A subunits in breast cancer has not been described. To address this, we first examined PP2A subunit expression in human breast tumors. Immunohistochemical analysis revealed significantly lower expression of the structural subunit, PP2A-A, and regulatory subunits PP2A-B55α and PP2A-B56α, in primary tumors and metastases, compared to adjacent normal mammary tissue. We further found an association of low PP2A-B55α with aggressive breast cancer subtypes, and with worse disease-free and overall survival. Functionally, shRNA-mediated knockdown of PP2A-B55α in normal mammary epithelial 3D cultures induced a tumorigenic phenotype, characterised by increased proliferation and enlarged multi-lobular acini. In contrast, overexpression of PP2A-B55α in breast cancer cells inhibited proliferation. Thus PP2A inactivation, in particular loss of B55α, is functionally important in breast tumorigenesis. PP2A-B55α complexes play an important role in DNA damage repair, and we found B55α knockdown impaired DNA damage repair. Thus low PP2A-B55α may contribute to genomic instability. To examine the functional role of PP2A-B55α in vivo, we have generated the first PP2A-B55α (Ppp2r2a) knockout mouse. Constitutive knockout of Ppp2r2a is embryonic lethal, with embryos dying during late development, post 14.5 days p.c. Heterozygous Pppr2ra mice (Ppp2r2a+/-) are viable, despite expressing only ~10% of PP2A-B55α protein levels compared to WT mice. Interestingly, Ppp2r2a+/- mice have significantly reduced branching in the developing mammary gland, similar to that observed in mice with mammary-specific loss of the breast tumor susceptibility gene, Brca1. Analysis of breast tumor formation in these mice, either alone or when crossed with MMTV-Neu animals, is underway. Finally, we show that pharmacological activation of PP2A, using FTY720, or the non-phosphorylatable analogue, AAL(S), inhibits tumor growth and metastases in an orthotopic xenograft model of aggressive, triple negative breast cancer (MDA-MB-231). Together this work demonstrates the importance of PP2A as a tumor suppressor in breast cancer, and suggests that targeting PP2A is a potential therapeutic strategy for poor outcome patients. Citation Format: Nikita Panicker, Abdul Mannan, Lauren F. Watt, Ben Copeland, Matt D. Dun, Simon King, Megan Clarke, Kathryn Skelding, Severine Roselli, Nicole M. Verrills. Functional role of the tumor suppressor protein phosphatase, PP2A-B55α, in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2375. doi:10.1158/1538-7445.AM2017-2375

BACKGROUND Low serum testosterone (low T) has been repeatedly linked to worse outcomes in men with newly diagnosed prostate cancer (PC). How low T contributes to these outcomes is unknown. Here we demonstrate that exposure to low T causes significant changes in the mouse prostate and prostate stem cells. METHODS Mice were castrated and implanted with capsules to achieve castrate, normal, or sub‐physiological levels of T. After 6 weeks of treatment, LC‐MS/MS was used to quantify the levels of T and dihydrotestosterone (DHT) in serum and prostate tissue. FACS was used to quantify the percentages of purported prostate stem and transit amplifying (TA) cells in mouse prostates. Prostate tissues were also stained for the presence of CD68+ cells and RNA was extracted from prostate tissue or specific cell populations to measure changes in transcript levels with low T treatment. RESULTS Despite having significantly different levels of T and DHT in the serum, T and DHT concentrations in prostate tissue from different T treatment groups were similar. Low T treatment resulted in significant alterations in the expression of androgen biosynthesis genes, which may be related to maintaining prostate androgen levels. Furthermore, the expression of androgen‐regulated genes in the prostate was similar among all T treatment groups, demonstrating that the mouse prostate can maintain functional levels of androgens despite low serum T levels. Low T increased the frequency of prostate stem and TA cells in adult prostate tissue and caused major transcriptional changes in those cells. Gene ontology analysis suggested that low T caused inflammatory responses and immunofluorescent staining indicated that low T treatment led to the increased presence of CD68+ macrophages in prostate tissue. CONCLUSIONS Low T alters the AR signaling axis which likely leads to maintenance of functional levels of prostate androgens. Low T also induces quantitative and qualitative changes in prostate stem cells which appear to lead to inflammatory macrophage infiltration. These changes are proposed to lead to an aggressive phenotype once cancers develop and may contribute to the poor outcomes in men with low T. Prostate 77:530–541, 2017 . © 2016 Wiley Periodicals, Inc.

BACKGROUND. The LNCaP cell line was originally isolated from the lymph node of a patient with metastatic prostate cancer. Many cell lines have been derived from LNCaP by selective pressures to study different aspects of prostate cancer progression. When injected subcutaneously into male athymic nude mice, LNCaP and its derivatives rarely metastasize.METHODS. Here, we describe the characteristics of a new LNCaP derivative, JHU-LNCaP-SM, which was generated by long term passage in normal cell culture conditions.RESULTS. Short tandem repeat (STR) analysis and genomic sequencing verified JHU-LNCaP-SM derivation from parental LNCaP cells. JHU-LNCaP-SM cells express the same mutated androgen receptor (AR) but unlike LNCaP, are no longer androgen dependent for growth. The cells demonstrate an attenuated androgen responsiveness in transcriptional assays and retain androgen sensitive expression of PSA, AR, and PSMA. Unlike parental LNCaP, JHU-LNCaP-SM cells quickly form subcutaneous tumors in male athymic nude mice, reliably metastasize to the lymph nodes and display a striking intra-tumoral and spreading hemorrhagic phenotype as tumor xenografts.CONCLUSIONS. The JHU-LNCaP-SM cell line is a new isolate of LNCaP, which facilitates practical, preclinical studies of spontaneous metastasis of prostate cancer through lymphatic tissues. (C) 2015 Wiley Periodicals, Inc.

Tetraspanins are transmembrane proteins that serve as scaffolds for multiprotein complexes containing, for example, integrins, growth factor receptors and matrix metalloproteases, and modify their functions in cell adhesion, migration and transmembrane signaling. CD151 is part of the tetraspanin family and it forms tight complexes with β1 and β4 integrins, both of which have been shown to be required for tumorigenesis and/or metastasis in transgenic mouse models of breast cancer. High levels of the tetraspanin CD151 have been linked to poor patient outcome in several human cancers including breast cancer. In addition, CD151 has been implicated as a promoter of tumor angiogenesis and metastasis in various model systems.

Prostate cancer is an extremely heterogeneous disease; patients that do progress to late-stage metastatic prostate cancer have limited treatment options, mostly palliative. Molecules involved in the metastatic cascade may prove beneficial in stratifying patients to assign appropriate treatment modalities and may also prove to be therapeutic antimetastatic targets. The tetraspanin group of molecules are integral membrane proteins that associate with motility-related proteins such as integrins. Clinical studies have mostly shown that reduced expression levels of the tetraspanin CD9 are correlated with tumour progression in a range of cancers. Furthermore, functional studies have shown CD9 to be involved in cell motility and adhesion and that it may influence metastasis. The effects of endogenous CD9 on prostate cancer initiation and progression were analysed by crossing a Cd9(-/-) (KO) murine model with a model of de novo developing and spontaneously metastasising prostate cancer, namely the transgenic adenocarcinoma of mouse prostate model. Our study demonstrates for the first time that ablation of Cd9 had no detectable effect on de novo primary tumour onset, but did significantly increase metastasis to the liver but not the lungs.What's new? The ability to distinguish between prostate tumors that are more or less likely to spread could facilitate decisions regarding treatment options for patients. A promising marker for such distinction is the tetraspanin protein CD9, which in experimental studies has been linked to metastasis. Here, Cd9 genetic ablation in the well-characterized transgenic adenocarcinoma of mouse prostate (TRAMP) model reveals an association between loss of Cd9 expression and increased incidence of metastases to the liver but not the lung. In contrast, Cd9 ablation did not affect primary prostate tumor initiation. The findings suggest that Cd9 acts as a suppressor of metastasis.

Abstract Tetraspanins are integral membrane proteins that associate with motility-related molecules such as integrins. Experimental studies have indicated that they may be important regulators of tumor invasion and metastasis, and high expression of the tetraspanin CD151 has been linked to poor prognosis in a number of cancers. Here, we show for the first time that genetic ablation of CD151 inhibits spontaneous metastasis in a transgenic mouse model of de novo tumorigenesis. To evaluate the effects of CD151 on de novo prostate cancer initiation and metastasis, a Cd151−/− (KO) murine model was crossed with the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model. Mice were analyzed for initiation of prostate tumor by palpation and primary tumors were analyzed by immunohistochemistry. Liver and lungs were examined for incidence and size of spontaneous metastatic lesions by histopathology. Knocking-out Cd151 had no significant effect on prostate cancer initiation or on expression of markers of proliferation, apoptosis, or angiogenesis in primary tumors. However, it did significantly decrease metastasis in a site-specific fashion, notably to the lungs but not the liver. Thus, CD151 acts principally as promoter of metastasis in this model. Prostate cancer is the second highest cause of cancer-related deaths in men in most Western countries, with the majority of deaths attributed to late-stage metastatic disease. CD151 may prove to be a valuable prognostic marker for treatment stratification and is a possible antimetastatic target. Mol Cancer Res; 11(1); 95–105. ©2012 AACR.

Previous studies showed that CD151-null mice have a skin wound healing deficit. To gain an understanding of the role of CD151 in re-epithelialisation and dermal contraction, keratinocyte and fibroblast functions were assayed. Primary CD151-null keratinocytes displayed defective migration on Matrigel (a basement membrane equivalent) and laminin-332, the primary adhesion component of basement membranes, but not on collagen-I. Adhesion, spreading and proliferation were also deficient on laminin-332, but not collagen-I. The data suggest that loss of CD151 impairs the function of its primary interaction partners, integrin alpha3beta1- and/or alpha6beta4 which bind to laminin-332. Skin fibroblasts also produce CD151 mRNA. CD151-null fibroblasts migrated significantly faster on collagen I than wild type fibroblasts, confirming that they possess functional collagen receptors. However, no significant decrease in the ability of CD151-null fibroblasts to cause contraction in floating collagen gel assays in response to transforming growth factor beta-1 (TGF-beta1) or platelet derived growth factor (PDGF-BB) was observed, nor was there an effect on fibroblast adhesion or proliferation on collagen-I. The data implicate CD151 as a facilitator of laminin-332-mediated keratinocyte functions that impact on the re-epithelialisation process intrinsic to wound healing and further suggest a potential novel role for CD151 in fibroblast migration.