Differential modulation of the androgen receptor for prostate cancer therapy depends on the DNA response element.

Androgen receptor (AR) action is a hallmark of prostate cancer (PCa) with androgen deprivation being standard therapy. Yet, resistance arises and aberrant AR signaling promotes disease. We sought compounds that inhibited genes driving cancer but not normal growth and hypothesized that genes with consensus androgen response elements (cAREs) drive proliferation but genes with selective elements (sAREs) promote differentiation. In a high-throughput promoter-dependent drug screen, doxorubicin (dox) exhibited this ability, acting on DNA rather than AR. This dox effect was observed at low doses for multiple AR target genes in multiple PCa cell lines and also occurred in vivo. Transcriptomic analyses revealed that low dox downregulated cell cycle genes while high dox upregulated DNA damage response genes. In chromatin immunoprecipitation (ChIP) assays with low dox, AR binding to sARE-containing enhancers increased, whereas AR was lost from cAREs. Further, ChIP-seq analysis revealed a subset of genes for which AR binding in low dox increased at pre-existing sites that included sites for prostate-specific factors such as FOXA1. AR dependence on cofactors at sAREs may be the basis for differential modulation by dox that preserves expression of genes for survival but not cancer progression. Repurposing of dox may provide unique opportunities for PCa treatment.

Genomic Structure, Evolutionary Origins, and Reproductive Function of a Large Amplified Intrinsically Disordered Protein-Coding Gene on the X Chromosome (Laidx) in Mice.

Mouse sex chromosomes are enriched for co-amplified gene families, present in tens to hundreds of copies. Co-amplification of Slx/Slxl1 on the X chromosome and Sly on the Y chromosome are involved in dose-dependent meiotic drive, however the role of other co-amplified genes remains poorly understood. Here we demonstrate that the co-amplified gene family on the X chromosome, Srsx, along with two additional partial gene annotations, is actually part of a larger transcription unit, which we name LaidxLaidx is harbored in a 229 kb amplicon that represents the ancestral state as compared to a 525 kb Y-amplicon containing the rearranged LaidyLaidx contains a 25,011 nucleotide open reading frame, predominantly expressed in round spermatids, predicted to encode an 871 kD protein. Laidx has orthologous copies with the rat and also the 825-MY diverged parasitic Chinese liver fluke, Clonorchis sinensis, the likely result of a horizontal gene transfer of rodent Laidx to an ancestor of the liver fluke. To assess the male reproductive functions of Laidx, we generated mice carrying a multi-megabase deletion of the Laidx-ampliconic region. Laidx-deficient male mice do not show detectable reproductive defects in fertility, fecundity, testis histology, and offspring sex ratio. We speculate that Laidx and Laidy represent a now inactive X vs. Y chromosome conflict that occurred in an ancestor of present day mice.

Profiling human androgen receptor mutations reveals treatment effects in a mouse model of prostate cancer.

Gain-of-function mutations in the androgen receptor (AR) are found in prostate cancer and are implicated in the failure of hormone therapy. Most studies have emphasized the ligand-binding domain (LBD) where mutations can create promiscuous receptors, but mutations in the NH(2)-terminal transactivation domain have also been found. To assess AR alteration as a mechanism of treatment resistance, a mouse model (h/mAR-TRAMP) was used in which the murine AR coding region is replaced by human sequence and prostate cancer initiated by a transgenic oncogene. Mice received either no treatment, androgen depletion by castration, or treatment with antiandrogens, and 20 AR transcripts were sequenced per end-stage tumor. All tumors expressed several mutant alleles, although most mutations were low frequency. Some mutations that occurred multiple times within the population were differentially located dependent on treatment. Mutations in castrated or antiandrogen-treated mice were widely dispersed but with a prominent cluster in the LBD (amino acids 736-771), whereas changes in intact mice centered near the NH(2)-terminal polymorphic glutamine tract. Functional characterization of selected LBD mutant alleles showed diverse effects on AR activity, with about half of the mutations reducing transactivation in vitro. One receptor, AR-R753Q, behaved in a cell- and promoter-dependent manner, although as a germ-line mutation it causes androgen insensitivity syndrome. This suggests that alleles that are loss of function during development may still activate a subset of AR targets to become gain of function in tumorigenesis. Mutant ARs may thus use multiple mechanisms to evade cancer treatment.

A Neofunctionalized X-Linked Ampliconic Gene Family Is Essential for Male Fertility and Equal Sex Ratio in Mice.

The mammalian sex chromosomes harbor an abundance of newly acquired ampliconic genes, although their functions require elucidation [1-9]. Here, we demonstrate that the X-linked Slx and Slxl1 ampliconic gene families represent mouse-specific neofunctionalized copies of a meiotic synaptonemal complex protein, Sycp3. In contrast to the meiotic role of Sycp3, CRISPR-loxP-mediated multi-megabase deletions of the Slx (5 Mb) and Slxl1 (2.3Mb) ampliconic regions result in post-meiotic defects, abnormal sperm, and male infertility. Males carrying Slxl1 deletions sire more male offspring, whereas males carrying Slx and Slxl1 duplications sire more female offspring, which directly correlates with Slxl1 gene dosage and gene expression levels. SLX and SLXL1 proteins interact with spindlin protein family members (SPIN1 and SSTY1/2) and males carrying Slxl1 deletions downregulate a sex chromatin modifier, Scml2, leading us to speculate that Slx and Slxl1 function in chromatin regulation. Our study demonstrates how newly acquired X-linked genes can rapidly evolve new and essential functions and how gene amplification can increase sex chromosome transmission.

Male mice with large inversions or deletions of X-chromosome palindrome arms are fertile and express their associated genes during post-meiosis.

Large (>10 kb) palindromic sequences are enriched on mammalian sex chromosomes. In mice, these palindromes harbor gene families (≥2 gene copies) expressed exclusively in post-meiotic testicular germ cells, a time when most single-copy sex-linked genes are transcriptionally repressed. This observation led to the hypothesis that palindromic structures or having ≥2 gene copies enable post-meiotic gene expression. We tested these hypotheses by using CRISPR to precisely engineer large (10's of kb) inversions and deletions of X-chromosome palindrome arms for two regions that carry the mouse 4930567H17Rik and Mageb5 palindrome gene families. We found that 4930567H17Rik and Mageb5 gene expression is unaffected in mice carrying palindrome arm inversions and halved in mice carrying palindrome arm deletions. We assessed whether palindrome-associated genes were sensitive to reduced expression in mice carrying palindrome arm deletions. Male mice carrying palindrome arm deletions are fertile and show no defects in post-meiotic spermatogenesis. Together, these findings suggest palindromic structures on the sex chromosomes are not necessary for their associated genes to evade post-meiotic transcriptional repression and that these genes are not sensitive to reduced expression levels. Large sex chromosome palindromes may be important for other reasons, such as promoting gene conversion between palindrome arms.

Replacing the mouse androgen receptor with human alleles demonstrates glutamine tract length-dependent effects on physiology and tumorigenesis in mice.

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.

Interaction of the Androgen Receptor, ETV1, and PTEN Pathways in Mouse Prostate Varies with Pathological Stage and Predicts Cancer Progression.

To examine the impact of common somatic mutations in prostate cancer (PCa) on androgen receptor (AR) signaling, mouse models were designed to perturb sequentially the AR, ETV1, and PTEN pathways. Mice with "humanized" AR (hAR) alleles that modified AR transcriptional strength by varying polyglutamine tract (Q-tract) length were crossed with mice expressing a prostate-specific, AR-responsive ETV1 transgene (ETV1(Tg)). While hAR allele did not grossly affect ETV1-induced neoplasia, ETV1 strongly antagonized global AR regulation and repressed critical androgen-induced differentiation and tumor suppressor genes, such as Nkx3-1 and Hoxb13. When Pten was varied to determine its impact on disease progression, mice lacking one Pten allele (Pten(+/-) ) developed more frequent prostatic intraepithelial neoplasia (PIN). Yet, only those with the ETV1 transgene progressed to invasive adenocarcinoma. Furthermore, progression was more frequent with the short Q-tract (stronger) AR, suggesting that the AR, ETV1, and PTEN pathways cooperate in aggressive disease. On the Pten(+/-) background, ETV1 had markedly less effect on AR target genes. However, a strong inflammatory gene expression signature, notably upregulation of Cxcl16, was induced by ETV1. Comparison of mouse and human patient data stratified by the presence of E26 transformation-specific ETS fusion genes highlighted additional factors, some not previously associated with prostate cancer but for which targeted therapies are in development for other diseases. In sum, concerted use of these mouse models illuminates the complex interplay of AR, ETV1, and PTEN pathways in pre-cancerous neoplasia and early tumorigenesis, disease stages difficult to analyze in man.

Length of the human androgen receptor glutamine tract determines androgen sensitivity in vivo.

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 2weeks 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 3αDiol and 3ßDiol 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.

Treatment-dependent androgen receptor mutations in prostate cancer exploit multiple mechanisms to evade therapy.

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.

Glutamine tract length of human androgen receptors affects hormone-dependent and -independent prostate cancer in mice.

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.

Molecular mechanism of the induction of metalloproteinases 1 and 3 in human fibroblasts by basic calcium phosphate crystals. Role of calcium-dependent protein kinase C alpha.

Synovial fluid basic calcium phosphate (BCP) crystals are common in osteoarthritis and are often associated with destructive arthropathies involving cartilage degeneration. These crystals are mitogenic and induce oncogene expression and matrix metalloproteinase (MMP) synthesis and secretion in human fibroblasts. To date, BCP crystal-elicited signal transduction pathways have not been completely studied. Because protein kinase C (PKC) is known to play an important role in signal transduction, we investigated the participation of this pathway in the BCP crystal induction of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts. Using reverse transcription/polymerase chain reaction (RT-PCR) and Northern and Western blotting techniques, we show here that BCP crystal stimulation of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts is dependent upon the calcium-dependent PKC signal transduction pathway and that the PKC alpha isozyme is specifically involved in the pathway. We have previously shown that BCP crystal induction of MMP-1 and MMP-3 is also dependent on the p44/42 mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We now show that these two pathways operate independently and seem to complement each other. This leads to our hypothesis that the two pathways initially function independently, ultimately leading to an increase in mitogenesis and MMP synthesis, and may converge downstream of PKC and p44/42 MAPK to mediate BCP crystal-induced cellular responses.