Androgen refractory cancer of the prostate (ARCaP) cells contain androgen receptor (AR) and synthesize and secrete prostate specific antigen (PSA). We isolated epithelia-like ARCaP(E) from parental ARCaP cells and induced them to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors including TGFbeta1 plus EGF, IGF-1, beta2-microglobulin (beta2-m), or a bone microenvironment. The molecular and behavioral characteristics of the resultant ARCaP(M) were characterized extensively in comparison to the parental ARCaP(E) cells. In addition to expressing mesenchymal biomarkers, ARCaP(M) gained 100% incidence of bone metastasis. ARCaP(M) cells express receptor activator of NF-kappaB ligand (RANKL), which was shown to increase tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in culture, and when metastatic to bone in vivo. We provide evidence that RANKL expression was promoted by increased cell signaling mediated by the activation of Stat3-Snail-LIV-1. RANKL expressed by ARCaP(M) cells is functional both in vitro and in vivo. The lesson we learned from the ARCaP model of EMT is that activation of a specific cell signaling pathway by soluble factors can lead to increased bone turnover, mediated by enhanced RANKL expression by tumor cells, which is implicated in the high incidence of prostate cancer bone colonization. The ARCaP EMT model is highly attractive for developing new therapeutic agents to treat prostate cancer bone metastasis.

Purpose: h2-Microglobulin (h2M) has been shown to promote osteomimicry and the proliferation of human prostate cancer cells. The objective of this study is to determine the mechanism by which targeting h2M using anti-h2M antibody inhibited growth and induced apoptosis in prostate cancer cells. Experimental Design: Polyclonal and monoclonal h2M antibodies were used to interrupt h2M signaling in human prostate cancer cell lines and the growth of prostate tumors in mice. The effects of the h2M antibody on a survival factor, androgen receptor (AR), and its target gene, prostate-specific antigen (PSA) expression, were investigated in cultured cells and in tumor xenografts. Results: The h2M antibody inhibited growth and promoted apoptosis in both AR-positive and PSA-positive, and AR-negative and PSA-negative, prostate cancer cells via the down-regulation of the AR in AR-positive prostate cancer cells and directly caused apoptosis in AR-negative prostate cancer cells in vitro and in tumor xenografts. The h2M antibody had no effect on AR expression or the growth of normal prostate cells. Conclusions: h2M downstream signaling regulates AR and PSA expression directly in ARpositive prostate cancer cells. In both AR-positive and AR-negative prostate cancer cells, interrupting h2M signaling with the h2M antibody inhibited cancer cell growth and induced its apoptosis. The h2M antibody is a novel and promising therapeutic agent for the treatment of human prostate cancers.h2-Microglobulin (h2M) is produced by all nucleated cells as a 119-amino-acid residue protein and, after processing, is secreted in a 99-amino-acid form (11,800 Da; refs. 1,2). The most common known function of h2M, a light-chain antigenpresenting molecule, is to serve as a coreceptor for the presentation of the MHC class I in nucleated cells for cytotoxic T-cell recognition (3). However, cancer cells frequently downregulate the expression of MHC class I to evade recognition by the immune system (4 -7), presumably allowing the secretion of free h2M into circulation or in the tumor microenvironment. Our laboratory first identified h2M, an active component secreted by prostate cancer, and prostate and bone stromal cells, as a major growth factor and signaling molecule (8). h2M conferred osteomimicry, the ability of cancer cells to mimic gene expression by bone cells, in prostate cancer cells through the activation of a cyclic AMP (cAMP) -dependent protein kinase A (PKA) and cAMP-responsive element binding (CREB) protein signaling pathway (9). The use of a sequence-specific small interfering RNA (siRNA) targeting h2M and its signaling resulted in extensive prostate cancer cell death in vitro and greatly promoted prostate tumor regression in immunocompromised mice (8). We also showed that interrupting h2M signaling similarly blocked human renal cell carcinoma growth (10). h2M has recently been shown to be a useful biomarker for advanced human prostate cancer (11). h2M seems to be a downstream androgen target gene, more specific than prostatespecific antig...

Increased AR activity has been shown to be preserved in spatially distinct metastatic tumors from the same patient suggesting the requirement for lineage-specific dependencies for metastatic castration resistant prostate cancer (mCRPC). Amplification of the AR gene is a common mechanism by which mCRPC increase AR activity. To determine whether AR amplification in circulating tumor cells (CTC) could complement metastatic tissue biopsies in men undergoing treatment for mCRPC, we developed a novel two-step assay to isolate CTCs and subsequently analyzed AR amplification status in CTCs and matched biopsy tissue from the same patient by fluorescence in situ hybridization (FISH). AR gene status in CTCs showed strong concordance with AR gene status in matched tissue samples in 24 of 25 patients (Correlation: 96%; Kappa: 0.83; Sensitivity: 100%, Specificity: 83%). Our work demonstrates that AR amplification is conserved between CTCs and biopsies and that CTCs can serve as non-invasive surrogate to document AR amplification in mCRPC.

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