GATA2 facilitates steroid receptor coactivator recruitment to the androgen receptor complex.

The androgen receptor (AR) is a key driver of prostate cancer (PC), even in the state of castration-resistant PC (CRPC) and frequently even after treatment with second-line hormonal therapies such as abiraterone and enzalutamide. The persistence of AR activity via both ligand-dependent and ligand-independent mechanisms (including constitutively active AR splice variants) highlights the unmet need for alternative approaches to block AR signaling in CRPC. We investigated the transcription factor GATA-binding protein 2 (GATA2) as a regulator of AR signaling and an actionable therapeutic target in PC. We demonstrate that GATA2 directly promotes expression of both full-length and splice-variant AR, resulting in a strong positive correlation between GATA2 and AR expression in both PC cell lines and patient specimens. Conversely, GATA2 expression is repressed by androgen and AR, suggesting a negative feedback regulatory loop that, upon androgen deprivation, derepresses GATA2 to contribute to AR overexpression in CRPC. Simultaneously, GATA2 is necessary for optimal transcriptional activity of both full-length and splice-variant AR. GATA2 colocalizes with AR and Forkhead box protein A1 on chromatin to enhance recruitment of steroid receptor coactivators and formation of the transcriptional holocomplex. In agreement with these important functions, high GATA2 expression and transcriptional activity predicted worse clinical outcome in PC patients. A GATA2 small molecule inhibitor suppressed the expression and transcriptional function of both full-length and splice-variant AR and exerted potent anticancer activity against PC cell lines. We propose pharmacological inhibition of GATA2 as a first-in-field approach to target AR expression and function and improve outcomes in CRPC.

Metabolomic profiling identifies biochemical pathways associated with castration-resistant prostate cancer.

Despite recent developments in treatment strategies, castration-resistant prostate cancer (CRPC) is still the second leading cause of cancer-associated mortality among American men, the biological underpinnings of which are not well understood. To this end, we measured levels of 150 metabolites and examined the rate of utilization of 184 metabolites in metastatic androgen-dependent prostate cancer (AD) and CRPC cell lines using a combination of targeted mass spectrometry and metabolic phenotyping. Metabolic data were used to derive biochemical pathways that were enriched in CRPC, using Oncomine concept maps (OCM). The enriched pathways were then examined in-silico for their association with treatment failure (i.e., prostate specific antigen (PSA) recurrence or biochemical recurrence) using published clinically annotated gene expression data sets. Our results indicate that a total of 19 metabolites were altered in CRPC compared to AD cell lines. These altered metabolites mapped to a highly interconnected network of biochemical pathways that describe UDP glucuronosyltransferase (UGT) activity. We observed an association with time to treatment failure in an analysis employing genes restricted to this pathway in three independent gene expression data sets. In summary, our studies highlight the value of employing metabolomic strategies in cell lines to derive potentially clinically useful predictive tools.

AR collaborates with ERα in aromatase inhibitor-resistant breast cancer.

Androgen receptor (AR) is an attractive target in breast cancer because of its frequent expression in all the molecular subtypes, especially in estrogen receptor (ER)-positive luminal breast cancers. We have previously shown a role for AR overexpression in tamoxifen resistance. We engineered ER-positive MCF-7 cells to overexpress aromatase and AR (MCF-7 AR Arom cells) to explore the role of AR in aromatase inhibitor (AI) resistance. Androstendione (AD) was used as a substrate for aromatization to estrogen. The nonsteroidal AI anastrazole (Ana) inhibited AD-stimulated growth and ER transcriptional activity in MCF-7 Arom cells, but not in MCF-7 AR Arom cells. Enhanced activation of pIGF-1R and pAKT was found in AR-overexpressing cells, and their inhibitors restored sensitivity to Ana, suggesting that these pathways represent escape survival mechanisms. Sensitivity to Ana was restored with AR antagonists, or the antiestrogen fulvestrant. These results suggest that both AR and ERα must be blocked to restore sensitivity to hormonal therapies in AR-overexpressing ERα-positive breast cancers. AR contributed to ERα transcriptional activity in MCF-7 AR Arom cells, and AR and ERα co-localized in AD + Ana-treated cells, suggesting cooperation between the two receptors. AR-mediated resistance was associated with a failure to block ER transcriptional activity and enhanced up-regulation of AR and ER-responsive gene expression. Clinically, it may be necessary to block both AR and ERα in patients whose tumors express elevated levels of AR. In addition, inhibitors to the AKT/IGF-1R signaling pathways may provide alternative approaches to block escape pathways and restore hormone sensitivity in resistant breast tumors.

Cistrome and transcriptome analysis identifies unique androgen receptor (AR) and AR-V7 splice variant chromatin binding and transcriptional activities.

The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.

Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients.

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.

1α,25-dihydroxyvitamin D3 inhibits C4-2 prostate cancer cell growth via a retinoblastoma protein (Rb)-independent G1 arrest.

BACKGROUND: The active metabolite of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25D) reduces the growth of several prostate cancer cell lines, most commonly by inducing a cell-cycle arrest in G(1). This is mediated, in part, through down-regulation of c-Myc, a positive regulator of the transcription factor, E2F. There is evidence that prostate cancer cells lacking functional retinoblastoma protein (Rb), a negative regulator of E2F activity, are poorly responsive to 1,25D treatment. Since up to 60% of prostate cancers demonstrate a loss of heterozygosity for Rb, we sought to determine whether Rb is required for the growth inhibitory effects of 1,25D. METHODS: Using siRNA, Rb was reduced in C4-2 prostate cancer cells, and the response of cells to 1,25D treatment or depletion of c-myc measured by [(3)H]-thymidine incorporation and flow cytometry. The effects of 1,25D treatment on E2F levels and activity, and E2F target gene expression were also measured. RESULTS: 1,25D treatment and c-Myc depletion both cause a G(1) arrest inhibiting C4-2 cell proliferation independently of Rb. 1,25D reduces c-Myc expression and causes a decrease in E2F and E2F target genes. Bcl-2, an E2F target and positive regulator of C4-2 cell growth, also is down-regulated by 1,25D independently of Rb. CONCLUSIONS: Redundant growth inhibitory pathways compensate for the loss of Rb, and tumors lacking functional Rb may be responsive to 1,25D.

Androgen receptor and its splice variant, AR-V7, differentially induce mRNA splicing in prostate cancer cells.

Prostate cancer (PCa) is dependent on the androgen receptor (AR). Advanced PCa is treated with an androgen deprivation therapy-based regimen; tumors develop resistance, although they typically remain AR-dependent. Expression of constitutively active AR variants lacking the ligand-binding domain including the variant AR-V7 contributes to this resistance. AR and AR-V7, as transcription factors, regulate many of the same genes, but also have unique activities. In this study, the capacity of the two AR isoforms to regulate splicing was examined. RNA-seq data from models that endogenously express AR and express AR-V7 in response to doxycycline were used. Both AR isoforms induced multiple changes in splicing and many changes were isoform-specific. Analyses of two endogenous genes, PGAP2 and TPD52, were performed to examine differential splicing. A novel exon that appears to be a novel transcription start site was preferentially induced by AR-V7 in PGAP2 although it is induced to a lesser extent by AR. The previously described AR induced promoter 2 usage that results in a novel protein derived from TPD52 (PrLZ) was not induced by AR-V7. AR, but not AR-V7, bound to a site proximal to promoter 2, and induction was found to depend on FOXA1.

Isoform-specific Activities of Androgen Receptor and its Splice Variants in Prostate Cancer Cells.

Androgen receptor (AR) signaling continues to drive castration-resistant prostate cancer (CRPC) in spite of androgen deprivation therapy (ADT). Constitutively active shorter variants of AR, lacking the ligand binding domain, are frequently expressed in CRPC and have emerged as a potential mechanism for prostate cancer to escape ADT. ARv7 and ARv567es are 2 of the most commonly detected variants of AR in clinical samples of advanced, metastatic prostate cancer. It is not clear if variants of AR merely act as weaker substitutes for AR or can mediate unique isoform-specific activities different from AR. In this study, we employed LNCaP prostate cancer cell lines with inducible expression of ARv7 or ARv567es to delineate similarities and differences in transcriptomics, metabolomics, and lipidomics resulting from the activation of AR, ARv7, or ARv567es. While the majority of target genes were similarly regulated by the action of all 3 isoforms, we found a clear difference in transcriptomic activities of AR versus the variants, and a few differences between ARv7 and ARv567es. Some of the target gene regulation by AR isoforms was similar in the VCaP background as well. Differences in downstream activities of AR isoforms were also evident from comparison of the metabolome and lipidome in an LNCaP model. Overall our study implies that shorter variants of AR are capable of mediating unique downstream activities different from AR and some of these are isoform specific.

Androgen receptor overexpression induces tamoxifen resistance in human breast cancer cells.

Although the androgen receptor (AR) is a known clinical target in prostate cancer, little is known about its possible role in breast cancer. We have investigated the role of AR expression in human breast cancer in response to treatment with the antiestrogen tamoxifen. Resistance to tamoxifen is a major problem in treating women with breast cancer. By gene expression profiling, we found elevated AR and reduced estrogen receptor (ER) alpha mRNA in tamoxifen-resistant tumors. Exogenous overexpression of AR rendered ERalpha-positive MCF-7 breast cancer cells resistant to the growth-inhibitory effects of tamoxifen in anchorage-independent growth assays and in xenograft studies in athymic nude mice. AR-overexpressing cells remained sensitive to growth stimulation with dihydrotestosterone. Treatment with the AR antagonist Casodex (bicalutamide) reversed this resistance, demonstrating the involvement of AR signaling in tamoxifen resistance. In AR-overexpressing cells, tamoxifen induced transcriptional activation by ERalpha that could be blocked by Casodex, suggesting that AR overexpression enhances tamoxifen's agonistic properties. Our data suggest a role for AR overexpression as a novel mechanism of hormone resistance, so that AR may offer a new clinical therapeutic target in human breast cancers.

Target gene-specific regulation of androgen receptor activity by p42/p44 mitogen-activated protein kinase.

Evidence that the androgen receptor (AR) is not only important in androgen-dependent prostate cancer, but also continues to play a role in tumors that become resistant to androgen deprivation therapies, highlights the need to find alternate means to block AR activity. AR, a hormone-activated transcription factor, and its coactivators are phosphoproteins. Thus, we sought to determine whether inhibition of specific cell signaling pathways would reduce AR function. We found that short-term inhibition of p42/p44 MAPK activity either by a MAPK kinase inhibitor, U0126, or by depletion of kinase with small interfering RNA caused target gene-specific reductions in AR activity. AR enhances histone H3 acetylation of target genes that are sensitive to U0126 including prostate-specific antigen and TMPRSS2, but does not increase histone H3 acetylation of the U0126-resistant PMEPA1 gene. Thus, although AR induces transcription of many target genes, the molecular changes induced by AR at the chromatin level are target gene specific. Long-term treatment (24-48 h) with U0126 causes a G1 cell cycle arrest and reduces AR expression both through a decrease in AR mRNA and a reduction in AR protein stability. Thus, treatments that reduce p42/p44 MAPK activity in prostate cancer have the potential to reduce AR activity through a reduction in expression levels as well as by target gene-selective inhibition of AR function.

Inositol polyphosphate 4-phosphatase type II regulation of androgen receptor activity.

Activation and transcriptional reprogramming of AR in advanced prostate cancer frequently coincides with the loss of two tumor suppressors, INPP4B and PTEN, which are highly expressed in human and mouse prostate epithelium. While regulation of AR signaling by PTEN has been described by multiple groups, it is not known whether the loss of INPP4B affects AR activity. Using prostate cancer cell lines, we showed that INPP4B regulates AR transcriptional activity and the oncogenic signaling pathways Akt and PKC. Analysis of gene expression in prostate cancer patient cohorts showed a positive correlation between INPP4B expression and both AR mRNA levels and AR transcriptional output. Using an Inpp4b-/- mouse model, we demonstrated that INPP4B suppresses Akt and PKC signaling pathways and modulates AR transcriptional activity in normal mouse prostate. Remarkably, PTEN protein levels and phosphorylation of S380 were the same in Inpp4b-/- and WT males, suggesting that the observed changes were due exclusively to the loss of INPP4B. Our data show that INPP4B modulates AR activity in normal prostate and its loss contributes to the AR-dependent transcriptional profile in prostate cancer.

Human progesterone receptor displays cell cycle-dependent changes in transcriptional activity.

The human progesterone receptor (PR) contains multiple Ser-Pro phosphorylation sites that are potential substrates for cyclin-dependent kinases, suggesting that PR activity might be regulated during the cell cycle. Using T47D breast cancer cells stably transfected with an mouse mammary tumor virus (MMTV) chloramphenicol acetyltransferase reporter (Cat0) synchronized in different phases of the cell cycle, we found that PR function and phosphorylation is remarkably cell cycle dependent, with the highest activity in S phase. Although PR expression was reduced in the G2/M phase, the activity per molecule of receptor was markedly reduced in both G1 and G2/M phases compared to the results seen with the S phase of the cell cycle. Although PR is recruited to the MMTV promoter equivalently in the G1 and S phases, recruitment of SRC-1, SRC-3, and, consequently, CBP is reduced in G1 phase despite comparable expression levels of SRC-1 and SRC-3. In G2/M phase, site-specific phosphorylation of PR at Ser162 and at Ser294, a site previously reported to be critical for transcriptional activity and receptor turnover, was abolished. Treatment with the histone deacetylase inhibitor trichostatin A elevated G1 and G2/M activity to that of the S phase, indicating that the failure to recruit sufficient levels of active histone acetyltransferase is the primary defect in PR-mediated transactivation.

Cyclin-dependent kinase activity is required for progesterone receptor function: novel role for cyclin A/Cdk2 as a progesterone receptor coactivator.

Our studies examining the role of the cell cycle-regulated kinase cyclin A/Cdk2 in progesterone receptor (PR) action have demonstrated that cyclin-dependent kinase activity is required for PR function and that cyclin A/Cdk2 functions as a PR coactivator. Although Cdk2 can phosphorylate PR, elimination of these phosphorylation sites has little effect on the ability of cyclin A/Cdk2 to stimulate PR activity. PR interacts with cyclin A and recruits cyclin A/Cdk2 to progestin-responsive promoters, stimulating transcription. Inhibition of Cdk2 activity abolishes progesterone-dependent activation of PR target genes in part through inhibition of PR-dependent recruitment of steroid receptor coactivator 1 (SRC-1) and subsequent histone H4 acetylation at the target promoter. In vitro studies revealed that the interaction between SRC-1 and PR is dependent upon phosphorylation of SRC-1. This heretofore-unknown mechanism provides a potential means for integrating the regulation of PR activity with cell cycle progression. Moreover, the ability of PR to recruit cyclin A/Cdk2 to target promoters provides locally elevated levels of kinase, which can preferentially facilitate phosphorylation-dependent interactions and enzymatic activities of coactivators at the target promoter.

c-Jun N-terminal kinase contributes to aberrant retinoid signaling in lung cancer cells by phosphorylating and inducing proteasomal degradation of retinoic acid receptor alpha.

Retinoic acid (RA) is the ligand for nuclear RA receptors (RARs and RXRs) and is crucial for normal epithelial cell growth and differentiation. During malignant transformation, human bronchial epithelial cells acquire a block in retinoid signaling caused in part by a transcriptional defect in RARs. Here, we show that activation of c-Jun N-terminal kinase (JNK) contributes to RAR dysfunction by phosphorylating RARalpha and inducing degradation through the ubiquitin-proteasomal pathway. Analysis of RARalpha mutants and phosphopeptide mapping revealed that RARalpha residues Thr181, Ser445, and Ser461 are phosphorylated by JNK. Mutation of these residues to alanines prevented efficient ubiquitination of RARalpha and increased the stability of the protein. We investigated the importance of RARalpha phosphorylation by JNK as a mediator of retinoid resistance in lung cancer. Mice that develop lung cancer from activation of a latent K-ras oncogene had high intratumoral JNK activity and low RARalpha levels and were resistant to treatment with an RAR ligand. JNK inhibition in a human lung cancer cell line enhanced RARalpha levels, ligand-induced activity of RXR-RAR dimers, and growth inhibition by RA. These findings point to JNK as a key mediator of aberrant retinoid signaling in lung cancer cells.

Steroid receptor phosphorylation: a key modulator of multiple receptor functions.

Steroid receptors are hormone-activated transcription factors, the expression and activities of which are also highly dependent upon posttranslational modifications including phosphorylation. The remarkable number of phosphorylation sites in these receptors and the wide variety of kinases participating in their phosphorylation facilitate integration between cell-signaling pathways and steroid receptor action. Sites have been identified in all of the functional domains although the sites are predominantly in the amino-terminal portions of the receptors. Regulation of function is receptor specific, site specific, and often dependent upon activation of a specific cell-signaling pathway. This complexity explains, in part, the early difficulties in identifying roles for phosphorylation in receptor function. With increased availability of phosphorylation site-specific antibodies and better means to measure receptor activities, numerous roles for site-specific phosphorylation have been identified including sensitivity of response to hormone, DNA binding, expression, stability, subcellular localization, and protein-protein interactions that determine the level of regulation of specific target genes. This review summarizes current knowledge regarding receptor phosphorylation and regulation of function. As functional assays become more sophisticated, it is likely that additional roles for phosphorylation in receptor function will be identified.

Androgens modulate expression of transcription intermediary factor 2, an androgen receptor coactivator whose expression level correlates with early biochemical recurrence in prostate cancer.

Prostate cancer is an androgen-dependent disease; metastatic prostate cancer is typically treated by androgen receptor (AR) blockade. Recurrence after androgen ablation and evidence that AR continues to play a role in many prostate cancers has led to an examination of other factors that potentiate AR activity. AR is a ligand-activated transcription factor whose activity is regulated not only by hormone but also by the levels of coactivators recruited by AR to facilitate transcription. We sought to assess the consequences of reducing expression of the transcription intermediary factor 2 (TIF2) coactivator on prostate cancer cell growth and AR action in cell lines to examine TIF2 expression in prostate cancer and to correlate expression with clinical outcome. Depletion of TIF2 reduced expression of AR-induced target genes and slowed proliferation of AR-dependent and AR-independent prostate cancer cells. Remarkably, we found that TIF2 expression is directly repressed by high levels of androgens in multiple AR-expressing cell lines. Expression of a reporter containing 5'-flanking region of the TIF2 was repressed both by androgens and by the antagonist, Casodex. Expression of TIF2 correlates with biochemical (prostate-specific antigen) recurrence (P = 0.0136). In agreement with our in vitro findings, the highest expression of TIF2 was found in patients whose cancer relapsed after androgen ablation therapy, supporting the idea that AR blockade might activate pathways that lead to stimulation of AR-dependent and AR-independent proliferation of prostate epithelium. The elevated expression of TIF2 at low hormone levels likely aids in inducing AR activity under these conditions; treatment with Casodex has the potential to counteract this induction.

Targeting the Hsp40/Hsp70 Chaperone Axis as a Novel Strategy to Treat Castration-Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is characterized by reactivation of androgen receptor (AR) signaling, in part by elevated expression of AR splice variants (ARv) including ARv7, a constitutively active, ligand binding domain (LBD)-deficient variant whose expression has been correlated with therapeutic resistance and poor prognosis. In a screen to identify small-molecule dual inhibitors of both androgen-dependent and androgen-independent AR gene signatures, we identified the chalcone C86. Binding studies using purified proteins and CRPC cell lysates revealed C86 to interact with Hsp40. Pull-down studies using biotinylated-C86 found Hsp40 present in a multiprotein complex with full-length (FL-) AR, ARv7, and Hsp70 in CRPC cells. Treatment of CRPC cells with C86 or the allosteric Hsp70 inhibitor JG98 resulted in rapid protein destabilization of both FL-AR and ARv, including ARv7, concomitant with reduced FL-AR- and ARv7-mediated transcriptional activity. The glucocorticoid receptor, whose elevated expression in a subset of CRPC also leads to androgen-independent AR target gene transcription, was also destabilized by inhibition of Hsp40 or Hsp70. In vivo, Hsp40 or Hsp70 inhibition demonstrated single-agent and combinatorial activity in a 22Rv1 CRPC xenograft model. These data reveal that, in addition to recognized roles of Hsp40 and Hsp70 in FL-AR LBD remodeling, ARv lacking the LBD remain dependent on molecular chaperones for stability and function. Our findings highlight the feasibility and potential benefit of targeting the Hsp40/Hsp70 chaperone axis to treat prostate cancer that has become resistant to standard antiandrogen therapy.Significance: These findings highlight the feasibility of targeting the Hsp40/Hsp70 chaperone axis to treat CRPC that has become resistant to standard antiandrogen therapy. Cancer Res; 78(14); 4022-35. ©2018 AACR.

Role of Androgen Receptor Variants in Prostate Cancer: Report from the 2017 Mission Androgen Receptor Variants Meeting.

CONTEXT: Although a number of studies have demonstrated the importance of constitutively active androgen receptor variants (AR-Vs) in prostate cancer, questions still remain about the precise role of AR-Vs in the progression of castration-resistant prostate cancer (CRPC). OBJECTIVE: Key stakeholders and opinion leaders in prostate cancer convened on May 11, 2017 in Boston to establish the current state of the field of AR-Vs. EVIDENCE ACQUISITION: The meeting "Mission Androgen Receptor Variants" was the second of its kind sponsored by the Prostate Cancer Foundation (PCF). This invitation-only event was attended by international leaders in the field and representatives from sponsoring organizations (PCF and industry sponsors). Eighteen faculty members gave short presentations, which were followed by in-depth discussions. Discussions focused on three thematic topics: (1) potential of AR-Vs as biomarkers of therapeutic resistance; (2) role of AR-Vs as functionally active CRPC progression drivers; and (3) utility of AR-Vs as therapeutic targets in CRPC. EVIDENCE SYNTHESIS: The three meeting organizers synthesized this meeting report, which is intended to summarize major data discussed at the meeting and identify key questions as well as strategies for addressing these questions. There was a critical consensus that further study of the AR-Vs is an important research focus in CRPC. Contrasting views and emphasis, each supported by data, were presented at the meeting, discussed among the participants, and synthesized in this report. CONCLUSIONS: This article highlights the state of knowledge and outlines the most pressing questions that need to be addressed to advance the AR-V field. PATIENT SUMMARY: Although further investigation is needed to delineate the role of androgen receptor (AR) variants in metastatic castration-resistant prostate cancer, advances in measurement science have enabled development of blood-based tests for treatment selection. Detection of AR variants (eg, AR-V7) identified a patient population with poor outcomes to existing AR-targeting therapies, highlighting the need for novel therapeutic agents currently under development.

Cyclin dependent kinase 2 and the regulation of human progesterone receptor activity.

The function of the S phase kinase cyclin A/Cdk2 in maintaining and regulating cell cycle kinetics is well established. However an alternative role in the regulation of progesterone receptor (PR) signaling is emerging. PR and its coactivators are phosphoproteins. Cyclin A/Cdk2 phosphorylates several of the PR phosphorylation sites in vitro and there is evidence that it participates in PR phosphorylation in vivo. Cyclin A/Cdk2 also functions as a PR coactivator. Overexpression increases PR transcriptional activity independent of PR phosphorylation. In the presence of hormone, cyclin A/Cdk2 is recruited to PR bound to DNA of target genes. Inhibition of Cdk activity prevents recruitment of the p160 coactivator steroid receptor coactivator-1 (SRC-1), suggesting that Cdk2 phosphorylates SRC-1. Consistent with this finding, phosphatase treatment of SRC-1 reduces its ability to interact with PR in vitro. Moreover, PR transcriptional activity is highest in S phase where cyclin A is expressed. In G1, PR activity is reduced and the capacity to recruit SRC-1 to a progestin responsive promoter is diminished. Future studies will focus on the importance of cyclin A/Cdk2 phosphorylation of other components of the PR transcription complex, such as the p160 coactivator SRC-1, and the specific role of Cdk2 target sites in the regulation of PR activity.

PTHrP contributes to the anti-proliferative and integrin alpha6beta4-regulating effects of 1,25-dihydroxyvitamin D(3).

Parathyroid hormone-related protein (PTHrP) increases the growth and metastatic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] suppresses PTHrP expression and exerts an anti-proliferative effect in prostate carcinoma cells. We used the human prostate cancer cell line C4-2 as a model system to ask whether down-regulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the anti-proliferative effects of 1,25(OH)(2)D(3). Since PTHrP increases the expression of the pro-invasive integrin alpha6beta4, we also asked whether 1,25(OH)(2)D(3) decreases integrin alpha6beta4 expression in C4-2 cells, and whether modulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the effects of 1,25(OH)(2)D(3) on integrin alpha6beta4 expression. Two strategies were utilized to modulate PTHrP levels: overexpression of PTHrP (-36 to +139) and suppression of endogenous PTHrP expression using siRNAs. We report a direct correlation between PTHrP expression, C4-2 cell proliferation and integrin alpha6beta4 expression at the mRNA and cell surface protein level. Treatment of parental C4-2 cells with 1,25(OH)(2)D(3) decreased cell proliferation and integrin alpha6 and beta4 expression. These 1,25(OH)(2)D(3) effects were significantly attenuated in cells with suppressed PTHrP expression. 1,25(OH)(2)D(3) regulates PTHrP expression via a negative vitamin D response element (nVDRE) within the noncoding region of the PTHrP gene. The effects of 1,25(OH)(2)D(3) on cell proliferation and integrin alpha6beta4 expression were significantly attenuated in cells overexpressing PTHrP (-36 to +139), which lacks the nVDRE. These findings suggest that one of the pathways via which 1,25(OH)(2)D(3) exerts its anti-proliferative effects is through down-regulation of PTHrP expression.