Enzalutamide (MDV3100) is a second generation Androgen Receptor (AR) antagonist with proven efficacy in the treatment of castration resistant prostate cancer (CRPC). The majority of treated patients, however, develop resistance and disease progression and there is a critical need to identify novel targetable pathways mediating resistance. The purpose of this study was to develop and extensively characterize a series of enzalutamide-resistant prostate cancer cell lines. Four genetically distinct AR-positive and AR-pathway dependent prostate cancer cell lines (CWR-R1, LAPC-4, LNCaP, VCaP) were made resistant to enzalutamide by long-term culture (> 6 months) in enzalutamide. Extensive characterization of these lines documented divergent in vitro growth characteristics and AR pathway modulation. Enzalutamide-resistant LNCaP and CWR-R1 cells, but not LAPC-4 and VCAP cells, demonstrated increased castration-resistant and metastatic growth in vivo. Global gene expression analyses between short-term enzalutamide treated vs. enzalutamide-resistant cells identified both AR pathway and non-AR pathway associated changes that were restored upon acquisition of enzalutamide resistance. Further analyses revealed very few common gene expression changes between the four resistant cell lines. Thus, while AR-mediated pathways contribute in part to enzalutamide resistance, an unbiased approach across several cell lines demonstrates a greater contribution toward resistance via pleiotropic, non-AR mediated mechanisms.

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Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the synthesis of prostaglandins. Its overexpression induces numerous tumor-promoting phenotypes and is associated with cancer metastasis and poor clinical outcome. Although COX-2 inhibitors are promising chemotherapeutic and chemopreventative agents for cancer, the risk of significant cardiovascular and gastrointestinal complications currently outweighs their potential benefits. Systemic complications of COX-2 inhibition could be avoided by specifically decreasing COX-2 expression in epithelial cells. To that end, we have investigated the signal transduction pathway regulating the COX-2 expression in response to DNA damage in breast epithelial cells. In variant human mammary epithelial cells that have silenced p16 (vHMEC), double-strand DNA damage or telomere malfunction results in a p53-and activin A-dependent induction of COX-2 and continued proliferation. In contrast, telomere malfunction in HMEC with an intact p16/Rb pathway induces cell cycle arrest. Importantly, in ductal carcinoma in situ lesions, high COX-2 expression is associated with high γH2AX, TRF2, activin A, and telomere malfunction. These data show that DNA damage and telomere malfunction can have both cell-autonomous and cell-nonautonomous consequences and can provide a novel mechanism for the propagation of tumorigenesis. Cancer Prev Res; 3(2); 190-201. ©2010 AACR.

IntroductionTumors are characterized by alterations in the epithelial and stromal compartments, which both contribute to tumor promotion. However, where, when, and how the tumor stroma develops is still poorly understood. We previously demonstrated that DNA damage or telomere malfunction induces an activin A-dependent epithelial stress response that activates cell-intrinsic and cell-extrinsic consequences in mortal, nontumorigenic human mammary epithelial cells (HMECs and vHMECs). Here we show that this epithelial stress response also induces protumorigenic phenotypes in neighboring primary fibroblasts, recapitulating many of the characteristics associated with formation of the tumor stroma (for example, desmoplasia).MethodsThe contribution of extrinsic and intrinsic DNA damage to acquisition of desmoplastic phenotypes was investigated in primary human mammary fibroblasts (HMFs) co-cultured with vHMECs with telomere malfunction (TRF2-vHMEC) or in HMFs directly treated with DNA-damaging agents, respectively. Fibroblast reprogramming was assessed by monitoring increases in levels of selected protumorigenic molecules with quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and immunocytochemistry. Dependence of the induced phenotypes on activin A was evaluated by addition of exogenous activin A or activin A silencing. In vitro findings were validated in vivo, in preinvasive ductal carcinoma in situ (DCIS) lesions by using immunohistochemistry and telomere-specific fluorescent in situ hybridization.ResultsHMFs either cocultured with TRF2-vHMEC or directly exposed to exogenous activin A or PGE2 show increased expression of cytokines and growth factors, deposition of extracellular matrix (ECM) proteins, and a shift toward aerobic glycolysis. In turn, these "activated" fibroblasts secrete factors that promote epithelial cell motility. Interestingly, cell-intrinsic DNA damage in HMFs induces some, but not all, of the molecules induced as a consequence of cell-extrinsic DNA damage. The response to cell-extrinsic DNA damage characterized in vitro is recapitulated in vivo in DCIS lesions, which exhibit telomere loss, heightened DNA damage response, and increased activin A and cyclooxygenase-2 expression. These lesions are surrounded by a stroma characterized by increased expression of α smooth muscle actin and endothelial and immune cell infiltration.ConclusionsThus, synergy between stromal and epithelial interactions, even at the initiating stages of carcinogenesis, appears necessary for the acquisition of malignancy and provides novel insights into where, when, and how the tumor stroma develops, allowing new therapeutic strategies.

Effective immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex Class I (MHC-I). Recent developments in proteomics have improved the identification of peptides that are naturally presented by MHC-I, collectively known as the "immunopeptidome". Current approaches to profile tumor immunopeptidomes have been limited to in vitro investigation, which fails to capture the in vivo repertoire of MHC-I peptides, or bulk tumor lysates, which are obscured by the lack of tumor-specific MHC-I isolation. To overcome these limitations, we report here the engineering of a Cre recombinase-inducible affinity tag into the endogenous mouse MHC-I gene and targeting of this allele to the Kras LSL-G12D/+ ; p53 fl/fl (KP) mouse model (KP; K b Strep). This novel approach has allowed us to isolate tumor-specific MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD) in vivo. With this powerful analytical tool, we were able to profile the evolution of the LUAD immunopeptidome through tumor progression and show that in vivo MHC-I presentation is shaped by post-translational mechanisms. We also uncovered novel, putative LUAD tumor associated antigens (TAAs). Many peptides that were recurrently presented in vivo exhibited very low expression of the cognate mRNA, provoking reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance. Beyond cancer, the K b Strep allele is compatible with a broad range of Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease, and autoimmunity.