Activity and safety of enobosarm, a novel, oral, selective androgen receptor modulator, in androgen receptor-positive, oestrogen receptor-positive, and HER2-negative advanced breast cancer (Study G200802): a randomised, open-label, multicentre, multinational, parallel design, phase 2 trial.

BACKGROUND: The androgen receptor is a tumour suppressor in oestrogen receptor-positive breast cancer. The activity and safety of enobosarm, an oral selective androgen receptor modulator, was evaluated in women with oestrogen receptor (ER)-positive, HER2-negative, and androgen receptor (AR)-positive disease. METHODS: Women who were postmenopausal (aged ≥18 years) with previously treated ER-positive, HER2-negative, locally advanced or metastatic breast cancer with an Eastern Cooperative Oncology Group performance status of 0-2 were enrolled in a randomised, open-label, multicentre, multinational, parallel design, phase 2 trial done at 35 cancer treatment centres in nine countries. Participants were stratified on the setting of immediately preceding endocrine therapy and the presence of bone-only metastasis and randomly assigned (1:1) to 9 mg or 18 mg oral enobosarm daily using an interactive web response system. The primary endpoint was clinical benefit rate at 24 weeks in those with centrally confirmed AR-positive disease (ie, the evaluable population). This trial is registered with ClinicalTrials.gov (NCT02463032). FINDINGS: Between Sept 10, 2015, and Nov 28, 2017, 136 (79%) of 172 patients deemed eligible were randomly assigned to 9 mg (n=72) or 18 mg (n=64) oral enobosarm daily. Of these 136 patients, 102 (75%) patients formed the evaluable population (9 mg, n=50; 18 mg, n=52). The median age was 60·5 years (IQR 52·3-69·3) in the 9 mg group and 62·5 years (54·0-69·3) in the 18 mg group. The median follow-up was 7·5 months (IQR 2·9-14·1). At 24 weeks, 16 (32%, 95% CI 20-47) of 50 in the 9 mg group and 15 (29%, 17-43) of 52 in the 18 mg group had clinical benefit. Six (8%) of 75 patients who received 9 mg and ten (16%) of 61 patients who received 18 mg had grade 3 or grade 4 drug-related adverse events, most frequently increased hepatic transaminases (three [4%] of 75 in the 9 mg group and two [3%] of 61 in the 18 mg group), hypercalcaemia (two [3%] and two [3%]), and fatigue (one [1%] and two [3%]). Four deaths (one in the 9 mg group and three in the 18 mg group) were deemed unrelated to the study drug. INTERPRETATION: Enobosarm has anti-tumour activity in patients with ER-positive, HER2-negative advanced breast cancer, showing that AR activation can result in clinical benefit, supporting further clinical investigation of selective AR activation strategies for the treatment of AR-positive, ER-positive, HER2-negative advanced breast cancer. FUNDING: GTx.

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing.

Arming androgen receptors to oppose oncogenic estrogen receptor activity in breast cancer.

Most breast cancers are driven by oncogenic activity of the estrogen receptor alpha (ER). Resistance to ER target therapies is the major cause of breast cancer death. Recently, there has been renewed interest in targeting the androgen receptor (AR) to treat ER-driven breast cancers. Herein, we discuss evidence for an AR agonist, not antagonist, treatment strategy.

Progesterone receptor modulates ERα action in breast cancer.

Progesterone receptor (PR) expression is used as a biomarker of oestrogen receptor-α (ERα) function and breast cancer prognosis. Here we show that PR is not merely an ERα-induced gene target, but is also an ERα-associated protein that modulates its behaviour. In the presence of agonist ligands, PR associates with ERα to direct ERα chromatin binding events within breast cancer cells, resulting in a unique gene expression programme that is associated with good clinical outcome. Progesterone inhibited oestrogen-mediated growth of ERα(+) cell line xenografts and primary ERα(+) breast tumour explants, and had increased anti-proliferative effects when coupled with an ERα antagonist. Copy number loss of PGR, the gene coding for PR, is a common feature in ERα(+) breast cancers, explaining lower PR levels in a subset of cases. Our findings indicate that PR functions as a molecular rheostat to control ERα chromatin binding and transcriptional activity, which has important implications for prognosis and therapeutic interventions.

MDM2 inhibition in combination with endocrine therapy and CDK4/6 inhibition for the treatment of ER-positive breast cancer.

BACKGROUND: Resistance to endocrine therapy is a major clinical challenge in the management of oestrogen receptor (ER)-positive breast cancer. In this setting, p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment-resistant ER-positive breast cancer. METHODS: We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate anti-tumour effects in p53 wildtype and p53 mutant ER-positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and endocrine-resistant ER-positive breast cancer. RESULTS: We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant-resistant patient-derived xenograft model. CONCLUSIONS: We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine-resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programmes.

Elevated levels of tumour apolipoprotein D independently predict poor outcome in breast cancer patients.

AIMS: Apolipoprotein D (ApoD) is a protein that is regulated by androgen and oestrogen, and is a major constituent of breast cysts. Although ApoD has been reported to be a marker of breast cancer, its prognostic importance in invasive breast cancer is unclear. The aim of this study was to investigate the relationship between ApoD protein expression, oestrogen receptor-α (ERα) expression and androgen receptor (AR) expression in predicting breast cancer outcome. METHODS AND RESULTS: ApoD levels were measured by the use of immunohistochemistry and video image analysis on tissue sections from a breast cancer cohort (n = 214). We assessed the associations of ApoD expression with disease-free survival (DFS), metastasis-free survival (MFS), and overall survival (OS). We also assessed the relationship between ApoD expression, AR expression and ERα expression in predicting OS. ApoD expression (>1% ApoD positivity) was found in 72% (154/214) of tissues. High ApoD positivity (≥20.7%, fourth quartile) was an independent predictor of MFS and OS, and conferred a 2.2-fold increased risk of developing metastatic disease and a 2.1-fold increased risk of breast cancer-related death. ApoD positivity was not associated with AR or ERα nuclear positivity. However, patients with (≥1%) ERα-positive cancers with low (<20.7%) ApoD positivity, or those showing high (≥78%) AR positivity and low (<20.7%) ApoD positivity had better OS than other patient groups. CONCLUSIONS: ApoD expression could be used to predict breast cancer prognosis independently of ERα and AR expression.

Androgen Receptor Signalling Promotes a Luminal Phenotype in Mammary Epithelial Cells.

Androgens influence mammary gland development but the specific role of the androgen receptor (AR) in mammary function is largely unknown. We identified cell subsets that express AR in vivo and determined the effect of AR activation and transgenic AR inhibition on sub-populations of the normal mouse mammary epithelium by flow cytometry and immunohistochemistry. Immunolocalisation of AR with markers of lineage identity was also performed in human breast tissues. AR activation in vivo significantly decreased the proportion of basal cells, and caused an accumulation of cells that expressed a basal cell marker but exhibited morphological features of luminal identity. Conversely, in AR null mice the proportion of basal mammary epithelial cells was significantly increased. Inhibition of AR increased basal but not luminal progenitor cell activity in vitro. A small population of AR-positive cells in a basal-to-luminal phenotype transition was also evident in human breast lobules. Collectively, these data support a role for AR in promoting a luminal phenotype in mammary epithelial cells.

Improved relapse-free survival on aromatase inhibitors in breast cancer is associated with interaction between oestrogen receptor-α and progesterone receptor-b.

BACKGROUND: Recent pre-clinical studies indicate that activated progesterone receptor (PR) (particularly the PR-B isoform) binds to oestrogen receptor-α (ER) and reprogrammes transcription toward better breast cancer outcomes. We investigated whether ER and PR-B interactions were present in breast tumours and associated with clinical parameters including response to aromatase inhibitors. METHODS: We developed a proximity ligation assay to detect ER and PR-B (ER:PR-B) interactions in formalin-fixed paraffin-embedded tissues. The assay was validated in a cell line and patient-derived breast cancer explants and applied to a cohort of 229 patients with ER-positive and HER2-negative breast cancer with axillary nodal disease. RESULTS: Higher frequency of ER:PR-B interaction correlated with increasing patient age, lower tumour grade and mitotic index. A low frequency of ER:PR-B interaction was associated with higher risk of relapse. In multivariate analysis, ER:PR-B interaction frequency was an independent predictive factor for relapse, whereas PR expression was not. In subset analysis, low frequency of ER:PR-B interaction was predictive of relapse on adjuvant aromatase inhibitor (HR 4.831, p = 0.001), but not on tamoxifen (HR 1.043, p = 0.939). CONCLUSIONS: This study demonstrates that ER:PR-B interactions have utility in predicting patient response to adjuvant AI therapy.

Targeting chromatin binding regulation of constitutively active AR variants to overcome prostate cancer resistance to endocrine-based therapies.

Androgen receptor (AR) variants (AR-Vs) expressed in prostate cancer (PCa) lack the AR ligand binding domain (LBD) and function as constitutively active transcription factors. AR-V expression in patient tissues or circulating tumor cells is associated with resistance to AR-targeting endocrine therapies and poor outcomes. Here, we investigated the mechanisms governing chromatin binding of AR-Vs with the goal of identifying therapeutic vulnerabilities. By chromatin immunoprecipitation and sequencing (ChIP-seq) and complementary biochemical experiments, we show that AR-Vs display a binding preference for the same canonical high-affinity androgen response elements (AREs) that are preferentially engaged by AR, albeit with lower affinity. Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation. Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth in vitro and in vivo. Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression. Overall, this study demonstrates that AR-Vs broadly restore AR chromatin binding events that are otherwise suppressed during endocrine therapy, and provides pre-clinical rationale for BET inhibition as a strategy for inhibiting expression and chromatin binding of AR-Vs in PCa.

IκBα mediates prostate cancer cell death induced by combinatorial targeting of the androgen receptor.

BACKGROUND: Combining different clinical agents to target multiple pathways in prostate cancer cells, including androgen receptor (AR) signaling, is potentially an effective strategy to improve outcomes for men with metastatic disease. We have previously demonstrated that sub-effective concentrations of an AR antagonist, bicalutamide, and the histone deacetylase inhibitor, vorinostat, act synergistically when combined to cause death of AR-dependent prostate cancer cells. METHODS: In this study, expression profiling of human prostate cancer cells treated with bicalutamide or vorinostat, alone or in combination, was employed to determine the molecular mechanisms underlying this synergistic action. Cell viability assays and quantitative real time PCR were used to validate identified candidate genes. RESULTS: A substantial proportion of the genes modulated by the combination of bicalutamide and vorinostat were androgen regulated. Independent pathway analysis identified further pathways and genes, most notably NFKBIA (encoding IκBα, an inhibitor of NF-κB and p53 signaling), as targets of this combinatorial treatment. Depletion of IκBα by siRNA knockdown enhanced apoptosis of prostate cancer cells, while ectopic overexpression of IκBα markedly suppressed cell death induced by the combination of bicalutamide and vorinostat. CONCLUSION: These findings implicate IκBα as a key mediator of the apoptotic action of this combinatorial AR targeting strategy and a promising new therapeutic target for prostate cancer.

Hormone-sensing mammary epithelial progenitors: emerging identity and hormonal regulation.

The hormone-sensing mammary epithelial cell (HS-MEC-expressing oestrogen receptor-alpha (ERα) and progesterone receptor (PGR)) is often represented as being terminally differentiated and lacking significant progenitor activity after puberty. Therefore while able to profoundly influence the proliferation and function of other MEC populations, HS-MECs are purported not to respond to sex hormone signals by engaging in significant cell proliferation during adulthood. This is a convenient and practical simplification that overshadows the sublime, and potentially critical, phenotypic plasticity found within the adult HS-MEC population. This concept is exemplified by the large proportion (~80 %) of human breast cancers expressing PGR and/or ERα, demonstrating that HS-MECs clearly proliferate in the context of breast cancer. Understanding how HS-MEC proliferation and differentiation is driven could be key to unraveling the mechanisms behind uncontrolled HS-MEC proliferation associated with ERα- and/or PGR-positive breast cancers. Herein we review evidence for the existence of a HS-MEC progenitor and the emerging plasticity of the HS-MEC population in general. This is followed by an analysis of hormones other than oestrogen and progesterone that are able to influence HS-MEC proliferation and differentiation: androgens, prolactin and transforming growth factor-beta1.

Androgen receptor driven transcription in molecular apocrine breast cancer is mediated by FoxA1.

Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumours were recently identified as an additional subgroup, characterised as oestrogen receptor negative and androgen receptor positive (ER- AR+), but with an expression profile resembling ER+ luminal breast cancer. One possible explanation for the apparent incongruity is that ER gene expression programmes could be recapitulated by AR. Using a cell line model of ER- AR+ molecular apocrine tumours (termed MDA-MB-453 cells), we map global AR binding events and find a binding profile that is similar to ER binding in breast cancer cells. We find that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a nuclear receptor. AR functionality is dependent on FoxA1, since silencing of FoxA1 inhibits AR binding, expression of the majority of the molecular apocrine gene signature and growth cell growth. These findings show that AR binds and regulates ER cis-regulatory elements in molecular apocrine tumours, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers.

Hsp90: still a viable target in prostate cancer.

Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the maturation, activation and stability of critical signaling proteins that drive the development and progression of prostate cancer, including the androgen receptor. Despite robust preclinical data demonstrating anti-tumor activity of first-generation Hsp90 inhibitors in prostate cancer, poor clinical responses initially cast doubt over the clinical utility of this class of agent. Recent advances in compound design and development, use of novel preclinical models and further biological insights into Hsp90 structure and function have now stimulated a resurgence in enthusiasm for these drugs as a therapeutic option. This review highlights how the development of new-generation Hsp90 inhibitors with improved physical and pharmacological properties is unfolding, and discusses the potential contexts for their use either as single agents or in combination, for men with metastatic prostate cancer.

Selective inhibition of CDK9 in triple negative breast cancer.

Targeted therapy for triple-negative breast cancers (TNBC) remains a clinical challenge due to tumour heterogeneity. Since TNBC have key features of transcriptionally addicted cancers, targeting transcription via regulators such as cyclin-dependent kinase 9 (CDK9) has potential as a therapeutic strategy. Herein, we preclinically tested a new selective CDK9 inhibitor (CDDD11-8) in TNBC using cell line, patient-derived organoid, and patient-derived explant models. In vitro, CDDD11-8 dose-dependently inhibited proliferation (IC50 range: 281-734 nM), induced cell cycle arrest, and increased apoptosis of cell lines, which encompassed the three major molecular subtypes of TNBC. On target inhibition of CDK9 activity was demonstrated by reduced RNAPII phosphorylation at a CDK9 target peptide and down-regulation of the MYC and MCL1 oncogenes at the mRNA and protein levels in all cell line models. Drug induced RNAPII pausing was evident at gene promoters, with strongest pausing at MYC target genes. Growth of five distinct patient-derived organoid models was dose-dependently inhibited by CDDD11-8 (IC50 range: 272-771 nM), including three derived from MYC amplified, chemo-resistant TNBC metastatic lesions. Orally administered CDDD11-8 also inhibited growth of mammary intraductal TNBC xenograft tumours with no overt toxicity in vivo (mice) or ex vivo (human breast tissues). In conclusion, our studies indicate that CDK9 is a viable therapeutic target in TNBC and that CDDD11-8, a novel selective CDK9 inhibitor, has efficacy in TNBC without apparent toxicity to normal tissues.

The androgen receptor interacts with GATA3 to transcriptionally regulate a luminal epithelial cell phenotype in breast cancer.

BACKGROUND: The androgen receptor (AR) is a tumor suppressor in estrogen receptor (ER) positive breast cancer, a role sustained in some ER negative breast cancers. Key factors dictating AR genomic activity in a breast context are largely unknown. Herein, we employ an unbiased chromatin immunoprecipitation-based proteomic technique to identify endogenous AR interacting co-regulatory proteins in ER positive and negative models of breast cancer to gain new insight into mechanisms of AR signaling in this disease. RESULTS: The DNA-binding factor GATA3 is identified and validated as a novel AR interacting protein in breast cancer cells irrespective of ER status. AR activation by the natural ligand 5α-dihydrotestosterone (DHT) increases nuclear AR-GATA3 interactions, resulting in AR-dependent enrichment of GATA3 chromatin binding at a sub-set of genomic loci. Silencing GATA3 reduces but does not prevent AR DNA binding and transactivation of genes associated with AR/GATA3 co-occupied loci, indicating a co-regulatory role for GATA3 in AR signaling. DHT-induced AR/GATA3 binding coincides with upregulation of luminal differentiation genes, including EHF and KDM4B, established master regulators of a breast epithelial cell lineage. These findings are validated in a patient-derived xenograft model of breast cancer. Interaction between AR and GATA3 is also associated with AR-mediated growth inhibition in ER positive and ER negative breast cancer. CONCLUSIONS: AR and GATA3 interact to transcriptionally regulate luminal epithelial cell differentiation in breast cancer regardless of ER status. This interaction facilitates the tumor suppressor function of AR and mechanistically explains why AR expression is associated with less proliferative, more differentiated breast tumors and better overall survival in breast cancer.

Library size confounds biology in spatial transcriptomics data.

Spatial molecular data has transformed the study of disease microenvironments, though, larger datasets pose an analytics challenge prompting the direct adoption of single-cell RNA-sequencing tools including normalization methods. Here, we demonstrate that library size is associated with tissue structure and that normalizing these effects out using commonly applied scRNA-seq normalization methods will negatively affect spatial domain identification. Spatial data should not be specifically corrected for library size prior to analysis, and algorithms designed for scRNA-seq data should be adopted with caution.

ACSM1 and ACSM3 regulate fatty acid metabolism to support prostate cancer growth and constrain ferroptosis.

Solid tumors are highly reliant on lipids for energy, growth, and survival. In prostate cancer, the activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes. Here, we identified acyl-CoA synthetase medium chain family members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 were upregulated in prostate tumors compared to non-malignant tissues and other cancer types. Both enzymes enhanced proliferation and protected prostate cancer cells from death in vitro, while silencing ACSM3 led to reduced tumor growth in an orthotopic xenograft model. ACSM1 and ACSM3 were major regulators of the prostate cancer lipidome and enhanced energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation and cell death by ferroptosis. Conversely, elevated ACSM1/3 activity enabled prostate cancer cells to survive toxic levels of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, this study reveals a tumor-promoting function for medium chain acyl-CoA synthetases and positions ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance.

Knockdown of the cochaperone SGTA results in the suppression of androgen and PI3K/Akt signaling and inhibition of prostate cancer cell proliferation.

Solid tumors have an increased reliance on Hsp70/Hsp90 molecular chaperones for proliferation, survival and maintenance of intracellular signaling systems. An underinvestigated component of the chaperone system is the tetratricopeptide repeat (TPR)-containing cochaperone, which coordinates Hsp70/Hsp90 involvement on client proteins as well as having diverse individual actions. A potentially important cochaperone in prostate cancer (PCa) is small glutamine-rich TPR-containing protein alpha (SGTA), which interacts with the androgen receptor (AR) and other critical cancer-related client proteins. In this study, the authors used small interfering RNA coupled with genome-wide expression profiling to investigate the biological significance of SGTA in PCa and its influence on AR signaling. Knockdown of SGTA for 72 hr in PCa C4-2B cells significantly altered expression of >1,900 genes (58% decreased) and reduced cell proliferation (p < 0.05). The regulation of 35% of 5α-dihydrotestosterone (DHT) target genes was affected by SGTA knockdown, with gene-specific effects on basal or DHT-induced expression or both. Pathway analysis revealed a role for SGTA in p53, generic PCa and phosphoinositol kinase (PI3K) signaling pathways; the latter evident by a reduction in PI3K subunit p100ß levels and decreased phosphorylated Akt. Immunohistochemical analysis of 64 primary advanced PCa samples showed a significant increase in the AR:SGTA ratio in cancerous lesions compared to patient-matched benign prostatic hyperplasia tissue (p < 0.02). This study not only provides insight into the biological actions of SGTA and its effect on genome-wide AR transcriptional activity and other therapeutically targeted intracellular signaling pathways but also provides evidence for PCa-specific alterations in SGTA expression.

Characterization of the prostate cancer susceptibility gene KLF6 in human and mouse prostate cancers.

BACKGROUND: Krüppel-like factor (KLF) 6 is a candidate tumor suppressor gene in prostate cancer, but the mechanisms contributing to its loss of expression are poorly understood. We characterized KLF6 expression and DNA methylation status during prostate tumorigenesis in humans and mice. METHODS: KLF6 expression was assessed in matched human non-malignant (NM) and tumor prostate tissues (n = 22) by quantitative real-time PCR (qPCR) and in three independent human prostate cancer cohorts bioinformatically. QPCR for KLF6 expression and methylation-sensitive PCR (MSP) were performed in human prostate LNCaP cancer cells after 5-aza-2'-deoxycytidine treatment. Klf6 protein levels and DNA promoter methylation were assessed in TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors by immunohistochemistry and MSP, respectively. RESULTS: KLF6 splice variants expression was increased (P = 0.0015) in human prostate tumors compared to NM tissues. Overall, KLF6 was decreased in metastatic compared to primary prostate cancers and reduced expression in primary tumors was associated with a shorter time to relapse (P = 0.0028). Treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in up-regulation of KLF6 expression (two-fold; P = 0.002) and a decrease in DNA methylation of the KLF6 promoter in LNCaP cells. Klf6 protein levels significantly decreased with progression in the TRAMP model of prostate cancer (P < 0.05), but there was no difference in Klf6 promoter methylation. CONCLUSION: KLF6 expression was decreased in both clinical prostate cancer and the TRAMP model with disease progression, but this could not be explained by DNA methylation of the KLF6 promoter.