Mutations in the androgen receptor (AR) that enable activation by antiandrogens occur in hormone-refractory prostate cancer, suggesting that mutant ARs are selected by treatment. To validate this hypothesis, we compared AR variants in metastases obtained by rapid autopsy of patients treated with flutamide or bicalutamide, or by excision of lymph node metastases from hormone-naïve patients. AR mutations occurred at low levels in all specimens, reflecting genetic heterogeneity of prostate cancer. Base changes recurring in multiple samples or multiple times per sample were considered putative selected mutations. Of 26 recurring missense mutations, most in the NH 2 -terminal domain (NTD) occurred in multiple tumors, whereas those in the ligand binding domain (LBD) were case specific. Hormone-naïve tumors had few recurring mutations and none in the LBD. Several AR variants were assessed for mechanisms that might underlie treatment resistance. Selection was evident for the promiscuous receptor AR-V716M, which dominated three metastases from one flutamide-treated patient. For the inactive cytoplasmically restricted splice variant AR23, coexpression with AR enhanced ligand response, supporting a decoy function. A novel NTD mutation, W435L, in a motif involved in intramolecular interaction influenced promoter-selective, cell-dependent transactivation. AR-E255K, mutated in a domain that interacts with an E3 ubiquitin ligase, led to increased protein stability and nuclear localization in the absence of ligand. Thus, treatment with antiandrogens selects for gain-of-function AR mutations with altered stability, promoter preference, or ligand specificity. These processes reveal multiple targets for effective therapies regardless of AR mutation. [Cancer Res 2009;69(10):4434-42]

Previous work in this laboratory has shown that endoderm cells in the heart forming region (HFR endoderm) of the chicken embryo induce terminal cardiac differentiation in explanted precardiac mesoderm cells. Immunostaining patterns indicating that HFR endoderm cells express Drosophila decapentaplegic (dpp)-like antigens prompted a degenerate polymerase chain reaction (PCR) screen to identify cDNAs in the dpp subgroup of the transforming growth factor-beta (TGF-beta) family. Among 50 clones of PCR products that have been sequenced, over half have identity with bone morphogenetic protein-2 (BMP-2). No other TGF-beta cDNAs have been detected, suggesting that BMP-2 is the major dpp subgroup protein synthesized by HFR endoderm cells. However, BMP-2 protein did not promote survival of either precardiac or non-precardiac mesoderm cells in culture. Whereas FGF-4 supports cardiogenesis in precardiac mesoderm, it did not induce cardiogenesis in nonprecardiac mesoderm, although explant viability was maintained. In contrast to the isolated effects of these growth factors, treatment of non-precardiac mesoderm with combined BMP-2 and FGF-4 induced cardiogenesis in the majority of explants, as revealed by the formation of a rhythmically contractile multicellular vesicle that expresses sarcomeric alpha-actin. These findings suggest that BMP-2 and FGF-4 possess respective differentiative and proliferative activities, the combination of which specifies cells to the cardiac lineage.

Highlights d Slx and Slxl1 are mouse lineage-specific copies of their autosomal progenitor Sycp3 d Slx and Slxl1 gene families are essential for male fertility d Changes in Slxl1 gene copy number distorts the ratio of male to female offspring d Slxl1 versus Sly competition for spindlin proteins may mediate sex-ratio distortion

Polymorphism in the length of the N-terminal glutamine (Q) tract in the human androgen receptor (AR) has been implicated in affecting aspects of male health ranging from fertility to cancer. Extreme expansion of the tract underlies Kennedy disease, and in vitro the AR Q tract length correlates inversely with transactivation capacity. However, whether normal variation influences physiology or the etiology of disease has been controversial. To assess directly the functional significance of Q tract variation, we converted the mouse AR to the human sequence by germline gene targeting, introducing alleles with 12, 21, or 48 glutamines. These three "humanized" AR (h/mAR) mouse lines were grossly normal in growth, behavior, fertility, and reproductive tract morphology. Phenotypic analysis revealed traits that varied subtly with Q tract length, including body fat amount and, more notably, seminal vesicle weight. Upon molecular analysis, tissue-specific differences in AR levels and target gene expression were detected between mouse lines. In the prostate, probasin, Nkx3.1, and clusterin mRNAs trended in directions predicted for inverse correlation of Q tract length with AR activation. Remarkably, when crossed with transgenic adenocarcinoma of mouse prostate (TRAMP) mice, striking genotype-dependent differences in prostate cancer initiation and progression were revealed. This link between Q tract length and prostate cancer, likely due to differential activation of AR targets, corroborates human epidemiological studies. This h/mAR allelic series in a homogeneous mouse genetic background allows examination of numerous physiological traits for Q tract influences and provides an animal model to test novel drugs targeted specifically to human AR.

Calcium-containing crystals such as basic calcium phosphate (BCP) 1 and calcium pyrophosphate dihydrate (CPPD) are two of the most common forms of pathologic articular materials that are associated with destructive arthropathies involving cartilage degeneration (1, 2). At concentrations found in pathologic human joint fluids, these crystals exert biological effects on cultured cells in a manner similar to growth factors like platelet-derived growth factor, epidermal growth factor, and serum. It has been demonstrated that BCP crystals stimulate fibroblast, synoviocyte, and chondrocyte mitogenesis in vitro (3); stimulate the production of prostaglandin via the phospholipase A 2 /cyclo-oxygenase pathway (4); activate phospholipase C and inositol phospholipid hydrolysis (5); induce the expression of the proto-oncogenes, c-fos and c-myc (6, 7); and induce the synthesis and secretion of metalloproteinases (MMPs) 1, 3, 8, and 13 (8 -12).In contrast to other mitogenic and growth factors, BCP crystal-elicited signal transduction pathways have not been completely studied. However, we have identified some of the component molecules involved in calcium-containing crystal signal transduction mechanisms. One pathway activated upon crystal stimulation of human fibroblasts (HF) is the p44 and p42 mitogen-activated protein kinase (p44/42 MAPK) pathway, also known as extracellular signal-related mitogen protein kinases 1 and 2 (ERK1 and ERK2), respectively. The MAPK cascade can be blocked by the selective inhibitors, PD98059 (13) and U0126 (14), which hinder the activation and phosphorylation of MEK (MAPK/ERK kinase). Co-treatment of HF with BCP crystals and PD98059 blocks crystal-induced p44/42 MAPK activation and mitogenesis (15) in addition to crystalinduced up-regulation of MMP-1 and MMP-3 mRNA and protein expressions (16). Moreover, phosphocitrate (PC), a specific inhibitor of the biological effects of BCP and CPPD crystals (17), also blocks crystal-induced activation of p44/42 MAPK, further supporting the role of this signal pathway in crystalinduced responses in HF (15).Another messenger with an apparent role in crystal-activated signal transduction is calcium. We have previously shown that treatment of HF with BCP crystals induces a rapid transient rise of intracellular calcium levels in seconds due to calcium influx from outside the cell, followed by a slow and sustained increase of intracellular calcium within 60 min after stimulation, due to crystal dissolution (18). Removal of calcium from the cell culture medium attenuates the BCP crystal in-

Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. These crystals are mitogenic and induce protooncogene expression and matrix metalloproteinase (MMP) synthesis and secretion in human fibroblasts, effects that are specifically blocked by phosphocitrate (PC). We have recently determined that crystals transduce signals to the nucleus via the activation of the p42 and p44 mitogen-activated protein (MAP) kinases (Nair et al., 1997, J Biol Chem 272:18920-18925). Treatment of human fibroblasts (HF) with BCP induces phosphorylation of p42/44 MAPK, which is inhibited by PC in a dose-dependent manner. Blocking of p42/44 MAPK signal transduction with an inhibitor (PD98059) of MEK1, an upstream activator of MAPKs, reduces crystal-induced p42/44 MAPK activation and significantly inhibits crystal-induced cell proliferation. Based on these findings, we sought to determine the role of the p42/44 MAPK signal transduction pathway in crystal-induced expression of matrix MMPs. We demonstrate suppression of crystal-induced MMPs via the utilization of two different MEK inhibitors: PD98059 and the recently described U0126, a novel inhibitor of MEK1 and MEK2. Treatment of HF with PD98059 blocks the induction of crystal-stimulated collagenase 1 (MMP-1) and stromelysin (MMP-3) expression. PD98059 and PC reduced the level of crystal-induced MMP-1 and MMP-3 mRNA expression to that observed in nonstimulated cells. Likewise, PD98059 treatment of HF blocked the epidermal growth factor (EGF)- and crystal-induced increases in MMP-1 and MMP-3 protein expression and secretion as demonstrated by Western blotting and zymography. Treatment of HF with U0126 inhibits EGF-induced phosphorylation of p42/44 MAPK as well as crystal- and EGF-induced upregulation of MMP-1 mRNA. Additionally, we demonstrate that treatment of HF with BCP, EGF, or PD98059 does not significantly alter levels of gelatinase A (MMP-2) mRNA and protein expression.

A well established functional polymorphism of the human androgen receptor (hAR) is the length of AR’s N-terminal glutamine tract (Q-tract). This tract is encoded by a CAG trinucleotide repeat and varies from 8 to 33 codons in the healthy population. Q-tract length is inversely correlated with AR transcriptional activity in vitro, but whether endogenous androgen action is affected is not consistently supported by results of clinical and epidemiological studies. To test whether Q-tract length influences androgen sensitivity in vivo, we examined effects of controlled androgen exposure in “humanized” mice with hAR knock-in alleles bearing 12, 21 or 48 CAGs. Mature male mice were analyzed before or 2 weeks after orchidectomy, with or without a subdermal dihydrotestosterone (DHT) implant to attain stable levels of this non-aromatizable androgen. The validity of this DHT clamp was demonstrated by similar serum levels of DHT and its two primary 3α/β-Diol metabolites, regardless of AR Q-tract length. Q-tract length was inversely related to DHT-induced suppression of castrate serum LH (p=0.005), as well as seminal vesicle (SV) weight (p=0.005) and prostate lobe weights (p<0.006). This confirms that the hAR Q-tract polymorphism mediates in vivo tissue androgen sensitivity by impacting negative hypothalamic feedback and trophic androgen effects on target organs. In this manner, AR Q-tract length variation may influence numerous aspects of male health, from virilization to fertility, as well as androgen-dependent diseases, such as prostate cancer.

The androgen receptor (AR) is involved in the initiation and progression of prostate cancer and its transition to androgen independence. Genetic variation in AR may contribute to disease risk and has been studied for a polymorphic N-terminal glutamine (Q) tract that shows population heterogeneity. While the length of this tract is known to affect AR in vitro, association with disease is complicated by genetic and environmental factors that have led to discordant epidemiological findings. To clarify the effect of Q tract polymorphism on prostate cancer, we created mice bearing humanized AR genes (h/mAr) varying in Q tract length. ARs with short Q tracts (12Q), which are transcriptionally more active, induce earlier disease in the transgene-induced TRAMP prostate cancer model than alleles with median (21Q) or long (48Q) tracts. Disease length varies within each genotype, with greater differentiation and AR expression in slower growing tumors. Remarkably, following androgen ablation, Q tract length has effects that are also allele-dependent and in directions opposite to those in hormone intact mice. Differences in AR activity conferred by Q tract length thus appear to direct distinct pathways of androgen-independent as well as androgen-dependent progression, and highlight substantial risk that may be associated with alterations in the androgen axis. This AR allelic series in humanized mice provides an experimental paradigm to dissect the role of AR in prostate cancer initiation and progression, to model response to treatment and to test therapies targeted specifically to the human AR.