Combination of bazedoxifene with chemotherapy and SMAC-mimetics for the treatment of colorectal cancer.

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Mechanisms of cellular crosstalk in the gastric tumor microenvironment are mediated by YAP1 and STAT3.

Deregulation of the Hippo pathway is a driver for cancer progression and treatment resistance. In the context of gastric cancer, YAP1 is a biomarker for poor patient prognosis. Although genomic tumor profiling provides information of Hippo pathway activation, the present study demonstrates that inhibition of Yap1 activity has anti-tumor effects in gastric tumors driven by oncogenic mutations and inflammatory cytokines. We show that Yap1 is a key regulator of cell metabolism, proliferation, and immune responses in normal and neoplastic gastric epithelium. We propose that the Hippo pathway is targetable across gastric cancer subtypes and its therapeutic benefits are likely to be mediated by both cancer cell-intrinsic and -extrinsic mechanisms.

STAT3 Signaling in Breast Cancer: Multicellular Actions and Therapeutic Potential.

Interleukin (IL)-6 family cytokines, such as IL-6 and IL-11, are defined by the shared use of the gp130 receptor for the downstream activation of STAT3 signaling and the activation of genes which contribute to the "hallmarks of cancer", including proliferation, survival, invasion and metastasis. Increased expression of these cytokines, or the ligand-specific receptors IL-6R and IL-11RA, in breast tumors positively correlate to disease progression and poorer patient outcome. In this review, we examine evidence from pre-clinical studies that correlate enhanced IL-6 and IL-11 mediated gp130/STAT3 signaling to the progression of breast cancer. Key processes by which the IL-6 family cytokines contribute to the heterogeneous nature of breast cancer, immune evasion and metastatic potential, are discussed. We examine the latest research into the therapeutic targeting of IL-6 family cytokines that inhibit STAT3 transcriptional activity as a potential breast cancer treatment, including current clinical trials. The importance of the IL-6 family of cytokines in cellular processes that promote the development and progression of breast cancer warrants further understanding of the molecular basis for its actions to help guide the development of future therapeutic targets.

Whole blood GRHL2 expression as a prognostic biomarker in metastatic hormone-sensitive and castration-resistant prostate cancer.

BACKGROUND: As potent systemic therapies transition earlier in the prostate cancer disease course, molecular biomarkers are needed to guide optimal treatment selection for metastatic hormone-sensitive prostate cancer (mHSPC). The value of whole blood RNA to detect candidate biomarkers in mHSPC remains largely undefined. METHODS: In this cohort study, we used a previously optimised whole blood reverse transcription polymerase chain reaction assay to assess the prognostic utility [measured by seven-month undetectable prostate-specific antigen (PSA) and time to castration-resistance (TTCR)] of eight prostate cancer-associated gene transcripts in 43 mHSPC patients. Transcripts with statistically significant associations (P<0.05) were further investigated in a metastatic castration-resistant prostate cancer (mCRPC) cohort (n=119) receiving contemporary systemic therapy, exploring associations with PSA >50% response (PSA50), progression-free survival (PFS) and overall survival (OS). Clinical outcomes were prospectively collected in a protected digital database. Kaplan-Meier estimates and multivariable Cox proportional-hazards models assessed associations between gene transcripts and clinical outcomes (mHSPC covariates: disease volume, docetaxel use and haemoglobin level; mCRPC covariates: prior exposure to chemotherapy or ARPIs, haemoglobin, performance status and presence of visceral disease). Follow-up was performed monthly during ARPI treatment, three-weekly during taxane chemotherapy, and three-monthly during androgen deprivation therapy (ADT) monotherapy. Serial PSA measurements were performed before each follow-up visit and repeat imaging was at the discretion of the investigator. RESULTS: Detection of circulating Grainyhead-like 2 (GRHL2) transcript was associated with poor outcomes in mHSPC and mCRPC patients. Detectable GRHL2 expression in mHSPC was associated with a lower rate of seven-month undetectable PSA levels (25% vs. 65%, P=0.059), and independently associated with shorter TTCR (HR 7.3, 95% CI: 1.5-36, P=0.01). In the mCRPC cohort, GRHL2 expression predicted significantly lower PSA50 response rates (46% vs. 69%, P=0.01), and was independently associated with shorter PFS (HR 3.1, 95% CI: 1.8-5.2, P<0.001) and OS (HR 2.9, 95% CI: 1.6-5.1, P<0.001). Associations were most apparent in patients receiving ARPIs. CONCLUSIONS: Detectable circulating GRHL2 was a negative prognostic biomarker in our mHSPC and mCRPC cohorts. These data support further investigation of GRHL2 as a candidate prognostic biomarker in metastatic prostate cancer, in addition to expanding efforts to better understand a putative role in therapeutic resistance to AR targeted therapies.

Therapeutic Targeting of the Tumour Microenvironment in Metastatic Colorectal Cancer.

Liver metastasis is the primary contributor to the death of patients with colorectal cancer. Despite the overall success of current treatments including targeted therapy, chemotherapy, and immunotherapy combinations in colorectal cancer patients, the prognosis of patients with liver metastasis remains poor. Recent studies have highlighted the importance of the tumour microenvironment and the crosstalk within that determines the fate of circulating tumour cells in distant organs. Understanding the interactions between liver resident cells and tumour cells colonising the liver opens new therapeutic windows for the successful treatment of metastatic colorectal cancer. Here we discuss critical cellular interactions within the tumour microenvironment in primary tumours and in liver metastases that highlight potential therapeutic targets. We also discuss recent therapeutic advances for the treatment of metastatic colorectal cancer.

Prognostic Utility of a Whole-blood Androgen Receptor-based Gene Signature in Metastatic Castration-resistant Prostate Cancer.

BACKGROUND: The treatment paradigm for metastatic castration-resistant prostate cancer (mCRPC) has evolved significantly in recent years. Identifying predictive and/or prognostic biomarkers in the context of this rapidly expanding therapeutic armamentarium remains a pressing and unmet clinical need. OBJECTIVE: To develop a prognostic whole-blood gene signature for mCRPC patients. DESIGN, SETTING, AND PARTICIPANTS: As part of an ongoing prospective, multicentre biomarker research study (Australian Prostate Biomarker Alliance), we enrolled 115 mCRPC patients commencing chemotherapy (n = 34) or androgen receptor (AR) pathway inhibitors therapy (n = 81) and obtained pretreatment whole-blood samples in PAXgene RNA tubes. Gene expression was assessed using reverse transcription-polymerase chain reaction. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Gene transcripts correlating with overall survival (OS) at p < 0.10 in univariate Cox regression models were incorporated into a multigene signature. Kaplan-Meier survival estimates and multivariate analyses were used to assess association with clinical outcomes. Prognostic strength of the signature was estimated using a concordance probability estimate (CPE). RESULTS AND LIMITATIONS: Based on univariate analysis for OS, the following genes were incorporated into a multigene signature: AR splice variant 7 (AR-V7), and three androgen-regulated genes: GRHL2, HOXB13, and FOXA1. The number of positive transcripts clearly stratified survival outcomes (median OS: not reached vs 24.8 mo vs 16.2 mo for 0, 1, and ≥2 transcripts, respectively; p = 0.0052). Notably, this multigene signature retained prognostic significance on multivariable analysis (hazard ratio, 2.1; 95% confidence interval, 1.1-4.0; p = 0.019). Moreover, CPE for this model was 0.78, indicating strong discriminative capacity. Limitations include short follow-up time. CONCLUSIONS: Our data demonstrate the prognostic utility of a novel whole-blood AR-based signature in mCRPC patients commencing contemporary systemic therapies. Our pragmatic assay requires minimal processing, can be performed in most hospital laboratories, and could represent a key prognostic tool for risk stratification in mCRPC. PATIENT SUMMARY: We found that expression of certain genes associated with the androgen receptor could help determine how long men with advanced prostate cancer survive after starting modern drug therapies.

Expression of Androgen Receptor Splice Variant 7 or 9 in Whole Blood Does Not Predict Response to Androgen-Axis-targeting Agents in Metastatic Castration-resistant Prostate Cancer.

In 2014, a landmark study was published demonstrating that the expression of androgen receptor splice variant (AR-V) 7 was a negative predictive biomarker for response to abiraterone acetate and enzalutamide in metastatic castration-resistant prostate cancer (mCRPC) patients. However, these results were not supported by the recently reported ARMOR3-SV phase III clinical trial, which employed an identical circulating tumour cell assay to assess AR-V7 expression. Therefore, the predictive utility of AR-V7 expression in mCRPC remains uncertain, as does any potential association between other AR-Vs and treatment response. To further investigate, we designed a highly sensitive and specific whole blood assay for detecting AR-V7 and AR-V9. We then examined for a correlation between baseline AR-V7/V9 status and treatment outcome in 37 mCRPC patients commencing abiraterone or enzalutamide. Of the patients, 24% (9/37) were AR-V-positive. Notably, prostate-specific antigen (PSA) response rates did not significantly differ between AR-V-positive (6/9) and AR-V-negative (18/28) patients (66% vs 64%, p=0.9). Likewise, median PSA progression-free survival was not significantly different between AR-V-positive and AR-V-negative patients (9.2 mo vs not reached; p=0.9). These data, which support the findings of the pivotal ARMOR3-SV clinical trial, suggest that baseline AR-V expression does not predict outcomes in mCRPC patients receiving abiraterone or enzalutamide. PATIENT SUMMARY: Detection of androgen receptor splice variants (AR-Vs) in circulating tumour cells of advanced prostate cancer patients has been linked to resistance to abiraterone and enzalutamide. We designed a blood test to detect AR-Vs that can be performed more routinely than tests involving circulating tumour cells and found that patients with AR-Vs still benefit from these effective treatments.

Transcriptional control of local estrogen formation by aromatase in the breast.

Aromatase is the critical enzyme that converts androgens to estrogens. It is frequently highly expressed in the tumour bearing breast of women diagnosed with estrogen receptor positive tumours, resulting in dramatically increased local estrogen production to drive tumour progression. Expression of aromatase is regulated primarily at the transcriptional level of its encoding gene CYP19A1, located on chromosome 15 of the human genome. A characteristic feature of CYP19A1 expression is its use of alternative promoters to regulate transcription in a tissue-specific manner. In breast cancer, the increase in aromatase expression is mediated via higher expression of the distal adipose-specific promoter I.4 and a switch to the preferential use of proximal promoters I.3 and II. This results in a net increase of CYP19A1 transcripts in tumour-bearing breast up to 3-4-fold higher than normal breast. Current aromatase inhibitors - whilst efficacious - exhibit significant side effects that reduce patient compliance. Understanding the transcription factors and signalling pathways that control aromatase expression will lead to opportunities to develop breast-specific inhibitors with an improved side-effects profile. This article is part of a Special Issue entitled 'Essential role of DHEA'.

Estradiol regulates Tumor Necrosis Factor-α expression and secretion in Estrogen Receptor positive breast cancer cells.

The cytokine Tumor Necrosis Factor-α is critical to Estrogen Receptor positive breast cancer pathology, stimulating estrogen-biosynthesis pathways and preventing the differentiation of estrogen-producing fibroblasts. High concentrations of TNFα are detected in the tumor microenvironment, and infiltrating immune cells are thought to be a major source. This study identifies that TNFα is also a tumor-derived factor, expressed in ER+ tumour epithelial cells and regulated by 17-ß-estradiol (E2). Treatment of MCF-7, T47D and ZR-75 breast cancer cells with E2 increased TNFα mRNA and protein expression and secretion. This effect was mitigated with the use of ERα inhibitors 4-hydroy-tamoxifen and ICI-182780, indicating that E2-mediated TNFα induction was via the actions of ERα. Chromatin immunoprecipitation reveals ERα binding to the TNFα promoter upon stimulation with E2. This study demonstrates for the first time a positive feedback loop between estradiol and TNFα, critical in maintaining high levels of the hormone within the ER+ breast tumour microenvironment.

Endocrine disruption of the epigenome: a breast cancer link.

The heritable component of breast cancer accounts for only a small proportion of total incidences. Environmental and lifestyle factors are therefore considered to among the major influencing components increasing breast cancer risk. Endocrine-disrupting chemicals (EDCs) are ubiquitous in the environment. The estrogenic property of EDCs has thus shown many associations between ongoing exposures and the development of endocrine-related diseases, including breast cancer. The environment consists of a heterogenous population of EDCs and despite many identified modes of action, including that of altering the epigenome, drawing definitive correlations regarding breast cancer has been a point of much discussion. In this review, we describe in detail well-characterized EDCs and their actions in the environment, their ability to disrupt mammary gland formation in animal and human experimental models and their associations with exposure and breast cancer risk. We also highlight the susceptibility of early-life exposure to each EDC to mediate epigenetic alterations, and where possible describe how these epigenome changes influence breast cancer risk.

Hepatic glucose intolerance precedes hepatic steatosis in the male aromatase knockout (ArKO) mouse.

Estrogens are known to play a role in modulating metabolic processes within the body. The Aromatase knockout (ArKO) mice have been shown to harbor factors of Metabolic syndrome with central adiposity, hyperinsulinemia and male-specific hepatic steatosis. To determine the effects of estrogen ablation and subsequent replacement in males on whole body glucose metabolism, three- and six-month-old male ArKO mice were subjected to whole body glucose, insulin and pyruvate tolerance tests and analyzed for ensuing metabolic changes in liver, adipose tissue, and skeletal muscle. Estrogen-deficient male ArKO mice showed increased gonadal adiposity which was significantly reduced upon 17ß-estradiol (E2) treatment. Concurrently, elevated ArKO serum leptin levels were significantly reduced upon E2 treatment and lowered serum adiponectin levels were restored to wild type levels. Three-month-old male ArKO mice were hyperglycemic, and both glucose and pyruvate intolerant. These phenotypes continued through to 6 months of age, highlighting a loss of glycemic control. ArKO livers displayed changes in gluconeogenic enzyme expression, and in insulin signaling pathways upon E2 treatment. Liver triglycerides were increased in the ArKO males only after 6 months of age, which could be reversed by E2 treatment. No differences were observed in insulin-stimulated ex vivo muscle glucose uptake nor changes in ArKO adipose tissue and muscle insulin signaling pathways. Therefore, we conclude that male ArKO mice develop hepatic glucose intolerance by the age of 3 months which precedes the sex-specific development of hepatic steatosis. This can be reversed upon the administration of exogenous E2.

Oestradiol reduces liver receptor homolog-1 mRNA transcript stability in breast cancer cell lines.

The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E2), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER- cells. However, the presence of LRH-1 protein in ER- cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER- breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER- compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E2, showed increased mRNA stability in ER- versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E2 treatment, this effect mediated by ERα. Our data demonstrates that in ER- cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER- cells as well as ER- tumors suggests a possible role in the development of ER- tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER- and ER+ breast cancer.

Origins and actions of tumor necrosis factor α in postmenopausal breast cancer.

Tumor necrosis factor α (TNFα) has many roles in both physiological and pathological states. Initially thought to cause necrosis of tumors, research has shown that in many tumor types, including breast cancer, TNFα contributes to growth and proliferation. The presence of TNFα-derived from the tumor and infiltrating immune cells-within a breast tumor microenvironment has been correlated with a more aggressive phenotype, and the postmenopausal ER+ subtype of breast cancers appears to strongly respond to its many pro-growth signaling functions. We discuss how TNFα regulates estrogen biosynthesis within the breast, affecting the activity of the key estrogen-synthesizing enzymes aromatase, estrone sulfatase, and 17ß-HSD type 1. Additionally, we describe the anti-adipogenic actions of TNFα that are critical in preventing adjacent estrogen-producing adipose fibroblasts from differentiating, ensuring that the tumor maintains a constant source of estrogen-producing cells. We examine how the increased risk of developing breast cancer in older and obese individuals may be linked to the levels of TNFα in the body. Finally, we evaluate the feasibility of targeting TNFα and its associated pathways as a novel approach to breast cancer therapeutics.

NFκB and MAPK signalling pathways mediate TNFα-induced Early Growth Response gene transcription leading to aromatase expression.

The Early Growth Response genes EGR2 and EGR3 play an important role in mediating TNFα induced aromatase expression via the adipose specific promoter PI.4. The upstream signalling pathway stimulated by TNFα to initiate this is unknown. The aim of this present study was to determine the signalling pathways activated by TNFα which result in EGR2 and EGR3 transcription, and ultimately activation of PI.4. The NFκB inhibitor BAY-11-7082 dose-dependently inhibited transcription of EGR2 and EGR3 mRNA as well as total and PI.4-specific CYP19A1 mRNA. The MAPK pathway inhibitor U0126, inhibitor of MEK1/2 had the same effect, however inhibition of c-Jun and JNK1,2,3 with SP600125 did not lead to down-regulation. We provide evidence for the first time that EGR2 and EGR3 are regulated by NFκB and MAPK signalling pathways downstream of TNFα stimulation in breast adipose fibroblasts, and that this in turn is upstream of CYP19A1 transcription via PI.4.

Intracrine oestrogen production and action in breast cancer: an epigenetic focus.

Epigenome changes have been widely demonstrated to contribute to the initiation and progression of a vast array of cancers including breast cancer. The reversible process of many epigenetic modifications is thus an attractive feature for the development of novel therapeutic measures. In oestrogen receptor α (hereinafter referred to as ER) positive tumours, endocrine therapies have proven beneficial in patient care, particularly in postmenopausal women where two-thirds of tumours are oestrogen dependent. However, resistance to such therapies is a common feature amongst individuals. In the current review, we discuss the influence that epigenetics has on oestrogen dependent breast cancers, in particular (i) the production of intracrine oestrogen in postmenopausal women, (ii) the action of oestrogen on epigenetic processes, and (iii) the links between epigenetics and endocrine resistance and the current advancements in epigenetic therapy that target this process. This article is part of a Special Issue entitled 'CSR 2013'.

Involvement of early growth response factors in TNFα-induced aromatase expression in breast adipose.

Expression of the oestrogen producing enzyme, aromatase, is regulated in a tissue-specific manner by its encoding gene CYP19A1. In post-menopausal women, the major site for oestrogen production in the breast is the adipose, where CYP19A1 transcription is driven by the distal promoter I.4 (PI.4). Transcripts via this promoter are also elevated in breast adipose fibroblasts (BAFs) adjacent to a tumour. PI.4 expression is stimulated by a number of cytokines, and TNFα is one such factor. The transcriptional mechanisms induced by TNFα to stimulate PI.4 are poorly characterised. We show that the early growth response (Egr) transcription factors play an important role in the TNFα-induced signalling pathway resulting in elevated PI.4 transcription. TNFα treatment of BAFs increases mRNA levels of all four Egr family members, with EGR2 being the most highly expressed. Overexpression of EGR2 causes an increase in endogenous CYP19A1 expression in preadipocyte Simpson-Golabi-Behmel syndrome cells, driven by increases in PI.4-specific transcripts. PI.4 luciferase reporter activity is increased in a dose-dependent manner by EGR2, EGR3 and EGR4, with EGR2 showing the most potent activation of promoter activity. Deletion analysis indicates that this promoter activity is being indirectly mediated by a short region of the promoter not containing any previously characterised binding sites, and we further show that EGR2 does not bind directly or indirectly to this promoter region. However, siRNA knockdown of the Egrs reduces the total and PI.4-derived CYP19A1 transcription in BAFs. These studies unveil a novel component of the aromatase gene regulatory network and further enhance the complexity of oestrogen production in the breast.

Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer.

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.

Melatonin suppresses aromatase expression and activity in breast cancer associated fibroblasts.

The main biological active substance secreted by the pineal gland, melatonin (MLT), counteracts the effects of estrogens in breast cancer via exerting a number of its own oncostatic properties. Recent studies of postmenopausal women have identified that the major metabolite of MLT is statistically significantly associated with a lower risk of developing breast cancer. While MLT production decreases with age, breast cancer risk, however, increases with age and obesity. We hypothesize that MLT inhibits estrogen production in breast adipose fibroblasts (BAFs), the main local source of estrogen in breast tumors of postmenopausal women, by inhibiting transcription of the CYP19A1 gene that encodes the key enzyme aromatase. Normal BAFs were cultured from women undergoing breast reduction surgery, while breast cancer-associated fibroblasts (CAFs) were isolated from three women with estrogen receptor (ER) positive invasive ductal carcinomas. MTNR1A and MTNR1B receptor expression and CYP19A1 mRNA expression following MLT treatments were determined by qRT-PCR. BAFs express the G-protein coupled MLT receptors MTNR1A and MTNR1B with elevated levels of MTNR1A found in CAFs. Treatment of BAFs and CAFs with MLT resulted in significant suppression of CYP19A1 transcription and aromatase activity at pharmacological, physiological and sub-physiological concentrations. MLT suppression occurred through promoter-specific PI.4-, PI.3- and PII-derived CYP19A1 mRNA. Stimulation of CYP19A1 PII-mRNA and aromatase activity by prostaglandin E(2) (PGE(2)) were significantly attenuated by physiological doses of MLT. Lower levels of MLT in aging women may increase the risk of progressing ER-positive breast cancer through a decreased ability to suppress CYP19A1 expression and subsequent local estrogen production in BAFs/CAFs.

Epigenetic mechanisms regulating CYP19 transcription in human breast adipose fibroblasts.

Cytochrome aromatase p450, encoded by the gene CYP19, catalyzes the synthesis of estrogens from androgens. In post-menopausal women, adipose becomes the major site for estrogen production, where basal CYP19 transcription is driven by distal promoter I.4. In breast adipose fibroblasts (BAFs), CYP19 expression is elevated in the presence of tumour-derived factors through use of promoters I.3 and II. We show for the first time that DNA methylation contributes to CYP19 regulation in BAFs and breast cell lines. Promoter I.4 and I.3/II-derived mRNA were not dependent on the CpG methylation status within respective promoters. However, inhibition of DNA methylation with 5-aza-2'-deoxycytidine resulted in a significant approximately 40-fold induction in CYP19 mRNA expression in BAFs and breast cell lines. These studies uncover a new layer of complexity in the regulation of aromatase where CYP19 appears to be inhibited by DNA methylation and evokes the possibility that disruption to this epigenetic regulation may give rise to an increase in aromatase levels in the breast.