Damien A. Leach

Hic-5 influences genomic and non-genomic actions of the androgen receptor in prostate myofibroblasts.

There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regarding androgen action in stromal cells of the tumor microenvironment. We report here the genome-wide effects of a stromal cell specific molecular adapter and AR coregulator, hydrogen peroxide-inducible gene 5 (Hic-5/TGFB1I1), on AR function in prostate myofibroblasts. Following androgen stimulation, Hic-5 rapidly translocates to the nucleus, coincident with increased phosphorylation of focal adhesion kinase. As a coregulator, Hic-5 acted to amplify or inhibit regulation of approximately 50% of AR target genes, affected androgen regulation of growth, cell adhesion, motility and invasion. These data suggest Hic-5 as a transferable adaptor between focal adhesions and the nucleus of prostate myofibroblasts, where it acts a key mediator of the specificity and sensitivity of AR signaling. We propose a model in which Hic-5 coordinates AR signaling with adhesion and extracellular matrix contacts to regulate cell behavior in the tumor microenvironment.

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 p100β 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.

Stromal Androgen Receptor in Prostate Cancer Development and Progression

Prostate cancer development and progression is the result of complex interactions between epithelia cells and fibroblasts/myofibroblasts, in a series of dynamic process amenable to regulation by hormones. Whilst androgen action through the androgen receptor (AR) is a well-established component of prostate cancer biology, it has been becoming increasingly apparent that changes in AR signalling in the surrounding stroma can dramatically influence tumour cell behavior. This is reflected in the consistent finding of a strong association between stromal AR expression and patient outcomes. In this review, we explore the relationship between AR signalling in fibroblasts/myofibroblasts and prostate cancer cells in the primary site, and detail the known functions, actions, and mechanisms of fibroblast AR signaling. We conclude with an evidence-based summary of how androgen action in stroma dramatically influences disease progression.

Uncovering a new role for peroxidase enzymes as drivers of angiogenesis.

Peroxidases are heme-containing enzymes released by activated immune cells at sites of inflammation. To-date their functional role in human health has mainly been limited to providing a mechanism for oxidative defence against invading bacteria and other pathogenic microorganisms. Our laboratory has recently identified a new functional role for peroxidase enzymes in stimulating fibroblast migration and collagen biosynthesis, offering a new insight into the causative association between inflammation and the pro-fibrogenic events that mediate tissue repair and regeneration. Peroxidases are found at elevated levels within and near blood vessels however, their direct involvement in angiogenesis has never been reported. Here we report for the first time that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are readily internalised by human umbilical vein endothelial cells (HUVEC) where they promote cellular proliferation, migration, invasion, and stimulate angiogenesis both in vitro and in vivo. These pro-angiogenic effects were attenuated using the specific peroxidase inhibitor 4-ABAH, indicating the enzymes catalytic activity is essential in mediating this response. Mechanistically, we provide evidence that MPO and EPO regulate endothelial FAK, Akt, p38 MAPK, ERK1/2 phosphorylation and stabilisation of HIF-2α, culminating in transcriptional regulation of key angiogenesis pathways. These findings uncover for the first time an important and previously unsuspected role for peroxidases as drivers of angiogenesis, and suggest that peroxidase inhibitors may have therapeutic potential for the treatment of angiogenesis related diseases driven by inflammation.

VDR Activity Is Differentially Affected by Hic-5 in Prostate Cancer and Stromal Cells

Patients with prostate cancer treated with androgen deprivation therapy (ADT) eventually develop castrate-resistant prostate cancer (CRPC). 1,25-Dihydroxyvitamin D3 (1,25D3/calcitriol) is a potential adjuvant therapy that confers antiproliferative and pro-differentiation effects in vitro, but has had mixed results in clinical trials. The impact of the tumor microenvironment on 1,25D3 therapy in patients with CRPC has not been assessed. Transforming growth factor β (TGFβ), which is associated with the development of tumorigenic “reactive stroma” in prostate cancer, induced vitamin D3 receptor (VDR) expression in the human WPMY-1 prostate stromal cell line. Similarly, TGFβ enhanced 1,25D3-induced upregulation of CYP24A1, which metabolizes 1,25D3 and thereby limits VDR activity. Ablation of Hic-5, a TGFβ-inducible nuclear receptor coregulator, inhibited basal VDR expression, 1,25D3-induced CYP24A1 expression and metabolism of 1,25D3 and TGFβ-enhanced CYP24A1 expression. A Hic-5–responsive sequence was identified upstream (392–451 bp) of the CYP24A1 transcription start site that is occupied by VDR only in the presence of Hic-5. Ectopic expression of Hic-5 sensitized LNCaP prostate tumor cells to growth-inhibitory effects of 1,25D3 independent of CYP24A1. The sensitivity of Hic-5–expressing LNCaP cells to 1,25D3-induced growth inhibition was accentuated in coculture with Hic-5–ablated WPMY-1 cells. Therefore, these findings indicate that the search for mechanisms to sensitize prostate cancer cells to the antiproliferative effects of VDR ligands needs to account for the impact of VDR activity in the tumor microenvironment. Implications: Hic-5 acts as a coregulator with distinct effects on VDR transactivation, in prostate cancer and stromal cells, and may exert diverse effects on adjuvant therapy designed to exploit VDR activity in prostate cancer. Mol Cancer Res; 12(8); 1166–80. ©2014 AACR.

The unique transcriptional response produced by concurrent estrogen and progesterone treatment in breast cancer cells results in upregulation of growth factor pathways and switching from a Luminal A to a Basal-like subtype

BackgroundIn breast cancer, progesterone receptor (PR) positivity or abundance is positively associated with survival and treatment response. It was initially believed that PR was a useful diagnostic marker of estrogen receptor activity, but increasingly PR has been recognised to play an important biological role in breast homeostasis, carcinogenesis and metastasis. Although PR expression is almost exclusively observed in estrogen receptor positive tumors, few studies have investigated the cellular mechanisms of PR action in the context of ongoing estrogen signalling.MethodsIn this study, we contrast PR function in estrogen pretreated ZR-75-1 breast cancer cells with vehicle treated ZR-75-1 and T-47D breast cancer cells using expression microarrays and chromatin immunoprecipitation-sequencing.ResultsEstrogen cotreatment caused a dramatic increase in the number of genes regulated by progesterone in ZR-75-1 cells. In T-47D cells that have naturally high levels of PR, estrogen and progesterone cotreatment resulted in a reduction in the number of regulated genes in comparison to treatment with either hormone alone. At a genome level, estrogen pretreatment of ZR-75-1 cells led to a 10-fold increase in the number of PR DNA binding sites detected using ChIP-sequencing. Time course assessment of progesterone regulated genes in the context of estrogen pretreatment highlighted a series of important regulatory pathways, including those driven by epithelial growth factor receptor (EGFR). Importantly, progesterone applied to cells pretreated with estradiol resulted in switching of the PAM50-determined intrinsic breast cancer subtype from Luminal A to Basal-like, and increased the Oncotype DX® Unscaled Recurrence Score.ConclusionEstrogen pretreatment of breast cancer cells increases PR steady state levels, resulting in an unequivocal progesterone response that upregulates key members of growth factor pathways. The transformative changes progesterone exerts on the breast cancer subtype suggest that these subtyping tools should be used with caution in premenopausal women.

p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells

Abstract Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis.

The prognostic value of stromal FK506-binding protein 1 and androgen receptor in prostate cancer outcome

Improving our ability to predict cancer progression and response to conservative or radical intent therapy is critical if we are to prevent under or over treatment of individual patients. Whereas the majority of solid tumors now have a range of molecular and/or immunological markers to help define prognosis and treatment options, prostate cancer still relies mainly on histological grading and clinical parameters. We have recently reported that androgen receptor (AR) expression in stroma inversely associates with prostate cancer‐specific survival, and that stromal AR reduces metastasis. For this paper, we tested the hypothesis that the AR‐regulated gene FKBP51 could be used as a marker of AR activity to better predict outcome.

Myofibroblast androgen receptor expression determines cell survival in co-cultures of myofibroblasts and prostate cancer cells in vitro

Fibroblasts express androgen receptor (AR) in the normal prostate and during prostate cancer development. We have reported that loss of AR expression in prostate cancer-associated fibroblasts is a poor prognostic indicator. Here we report outcomes of direct and indirect co-cultures of immortalised AR-positive (PShTert-AR) or AR-negative (PShTert) myofibroblasts with prostate cancer cells. In the initial co-cultures the AR-negative PC3 cell line was used so AR expression and signalling were restricted to the myofibroblasts. In both direct and indirect co-culture with PShTert-AR myofibroblasts, paracrine signalling to the PC3 cells slowed proliferation and induced apoptosis. In contrast, PC3 cells proliferated with PShTert myofibroblasts irrespective of the co-culture method. In direct co-culture PC3 cells induced apoptosis in and destroyed PShTerts by direct signalling. Similar results were seen in direct co-cultures with AR-negative DU145 and AR-positive LNCaP and C4-2B prostate cancer cell lines. The AR ligand 5α-dihydrotestosterone (DHT) inhibited the proliferation of the PShTert-AR myofibroblasts, thereby reducing the extent of their inhibitory effect on cancer cell growth. These results suggest loss of stromal AR would favour prostate cancer cell growth in vivo, providing an explanation for the clinical observation that reduced stromal AR is associated with a poorer outcome.

Protocols for Studies on Stromal Cells in Prostate Cancer

Interactions between tumor cells and fibroblasts play a pivotal role in cancer development and progression. Indeed, the paracrine communication between these two cell types is known to have physiological effects that alter carcinogenic and metastatic potential. An often overlooked player in these interactions is the involvement of the extracellular matrix (ECM). The network of ECM proteins secreted from fibroblasts is reportedly altered with cancer initiation and progression, and in several cases has been associated with patient outcome. The androgen receptor (AR) is one such example and has been shown to be a dynamic and inducible regulator of ECM production. Contemporary assessment of dynamic multicellular interactions leading to cancer initiation and progression necessitates 3D in vitro modeling to better mimic the in vivo environment. In the current chapter, we describe some simple approaches to generate 3D models of fibroblast-produced ECM, how hormone manipulation of fibroblasts can lead to production of different ECMs, and how these ECM models can be used to test processes implicated in cancer progression and metastasis.