Rachel Ruoff

Androgen receptor levels are upregulated by Akt in prostate cancer.

Multiple lines of evidence suggest a functional link between the androgen receptor (AR) and the serine/threonine kinase Akt in the development and progression of prostate cancer. To investigate the impact of Akt activity on AR homeostasis, we treated androgen-dependent LNCaP and LAPC-4 prostate cancer cells with Akt inhibitor. Akt inhibition decreased AR expression, suggesting that Akt activity was required for regulation of AR protein levels. However, while androgen-independent LNCaP-abl cells also showed diminished AR protein levels in response to Akt inhibition, treatment of androgen-independent LNCaP-AI cells failed to alter AR protein levels upon similar treatment, suggesting that AR protein levels in these androgen-independent prostate cells were regulated by mechanisms independent of Akt activation. Regulation of AR, downstream of activated Akt, also was observed in vivo when examining transgenic mice that overexpress constitutively active mutant myristoylated (myr)-Akt1 in the prostate. Transgenic mice expressing activated myr-Akt1 exhibited higher levels of AR mRNA and protein. Expression of activated myr-Akt1 did not alter prostate cell growth and no significant size differences between prostate tissues derived from transgenic animals were observed when comparing transgenic mice with wild-type mice. Still, transgenic mice overexpressing Akt exhibited higher levels of γH2AX and phosphorylated Chk2 in prostate tissue. These changes in markers associated with oncogene-induced senescence confirmed significant altered signaling in the transgenic mouse model. Overall, results presented here suggest that AR levels are regulated by the Akt pathway.

Phosphorylation of the androgen receptor by PIM1 in hormone refractory prostate cancer

Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the AR at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild-type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine-to-alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand-independent manner and cell type-specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression, AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR-mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild-type AR, but not S213A mutant AR. Androgen-mediated transcription of endogenous PSA, Nkx3.1 and IGFBP5 was also decreased in the presence of PIM1, whereas IL6, cyclin A1 and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.

Sin3B Expression Is Required for Cellular Senescence and Is Up-regulated upon Oncogenic Stress

Serial passage of primary mammalian cells or strong mitogenic signals induce a permanent exit from the cell cycle called senescence. A characteristic of senescent cells is the heterochromatinization of loci encoding pro-proliferative genes, leading to their transcriptional silencing. Senescence is thought to represent a defense mechanism against uncontrolled proliferation and cancer. Consequently, genetic alterations that allow senescence bypass are associated with susceptibility to oncogenic transformation. We show that fibroblasts genetically inactivated for the chromatin-associated Sin3B protein are refractory to replicative and oncogene-induced senescence. Conversely, overexpression of Sin3B triggers senescence and the formation of senescence-associated heterochromatic foci. Although Sin3B is strongly up-regulated upon oncogenic stress, decrease in expression of Sin3B is associated with tumor progression in vivo, suggesting that expression of Sin3B may represent a barrier against transformation. Together, these results underscore the contribution of senescence in tumor suppression and suggest that expression of chromatin modifiers is modulated at specific stages of cellular transformation. Consequently, these findings suggest that modulation of Sin3B-associated activities may represent new therapeutic opportunities for treatment of cancers.

Regulation of Androgen Receptor-Mediated Transcription by RPB5 Binding Protein URI/RMP

ABSTRACT Androgen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes.

Genome-wide impact of Androgen Receptor Trapped Clone-27 Loss on Androgen-regulated Transcription in Prostate Cancer Cells

The androgen receptor (AR) directs diverse biological processes through interaction with coregulators such as AR trapped clone-27 (ART-27). Our results show that ART-27 is recruited to AR-binding sites by chromatin immunoprecipitation analysis. In addition, the effect of ART-27 on genome-wide transcription was examined. The studies indicate that loss of ART-27 enhances expression of many androgen-regulated genes, suggesting that ART-27 inhibits gene expression. Surprisingly, classes of genes that are up-regulated upon ART-27 depletion include regulators of DNA damage checkpoint and cell cycle progression, suggesting that ART-27 functions to keep expression levels of these genes low. Consistent with this idea, stable reduction of ART-27 by short-hairpin RNA enhances LNCaP cell proliferation compared with control cells. The effect of ART-27 loss was also examined in response to the antiandrogen bicalutamide. Unexpectedly, cells treated with ART-27 siRNA no longer exhibited gene repression in response to bicalutamide. To examine ART-27 loss in prostate cancer progression, immunohistochemistry was conducted on a tissue array containing samples from primary tumors of individuals who were clinically followed and later shown to have either recurrent or nonrecurrent disease. Comparison of ART-27 and AR staining indicated that nuclear ART-27 expression was lost in the majority of AR-positive recurrent prostate cancers. Our studies show that reduction of ART-27 protein levels in prostate cancer may facilitate antiandrogen-resistant disease.

Expression of androgen receptor and its phosphorylated forms in breast cancer progression

Androgen receptor (AR) expression in breast cancers may serve as a prognostic and predictive marker. We examined the expression pattern of AR and its phosphorylated forms, Ser‐213 (AR‐Ser[P]‐213) and Ser‐650 (AR‐Ser[P]‐650), in breast cancer and evaluated their association with clinicopathological parameters.

The Heterochromatin Protein 1 Family is Regulated in Prostate Development and Cancer

PURPOSE The HP1 family of evolutionarily conserved proteins regulates heterochromatin packaging, in addition to a less defined role in the regulation of euchromatic genes. To examine the possible role of HP1 proteins in fetal prostate development and prostate cancer the protein expression of HP1alpha, beta and gamma was evaluated in human archival tissue. MATERIALS AND METHODS Tissue sections from human prostate cancer and fetal prostate were examined using antibodies against HP1 isoforms to evaluate HP1 modulation in cancer and development. Western blot analysis of HP1 proteins was also performed in extracts of cultured prostate cancer cells. RESULTS HP1alpha, beta and gamma are differentially regulated in various cellular compartments in prostate development. HP1alpha is not expressed at 14 or 24 weeks of prostate development but it is expressed in adult prostate tissue. HP1beta is highly expressed at 14 and 24 weeks, and it appears predominantly in epithelial cells compared to HP1gamma, which is expressed at equal levels in epithelial and stromal cells. All 3 HP1 isoforms show altered expression in prostate cancer compared to that in normal adult prostate tissue. CONCLUSIONS HP1 proteins are tightly regulated during prostate development. In the adult prostate HP1alpha, beta and gamma antibodies detect high levels of HP1 antigen in a contiguous layer of epithelial cells. However, the detection of HP1 in prostate cancer ranges from undetectable to inconsistent staining of noncontiguous epithelial cells.

Phosphoproteomics of Fibroblast Growth Factor 1 (FGF1) Signaling in Chondrocytes: Identifying the Signature of Inhibitory Response

Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes. Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however, its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We could localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3β (GSK3β) as a novel key mediator of FGF inhibitory response in chondrocytes.

Increased activity of chondrocyte translational apparatus accompanies osteoarthritis

Degeneration of articular cartilage is central to the pathology of osteoarthritis (OA). However, the molecular mechanisms leading to these irreversible changes are still poorly understood. This study was undertaken to investigate how changes in the chondrocyte translational apparatus may contribute to the development and progression of knee OA.