BRCA1 - A good predictive marker of drug sensitivity in breast cancer treatment?

There are currently only two predictive markers of response to chemotherapy for breast cancer in routine clinical use, namely the Estrogen receptor-alpha and the HER2 receptor. The breast and ovarian cancer susceptibility gene BRCA1 is an important genetic factor in hereditary breast and ovarian cancer and there is increasing evidence of an important role for BRCA1 in the sporadic forms of both cancer types. Our group and numerous others have shown in both preclinical and clinical studies that BRCA1 is an important determinant of chemotherapy responses in breast cancer. In this review we will outline the current understanding of the role of BRCA1 as a determinant of response to DNA damaging and microtubule damaging chemotherapy. We will then discuss how the known functions of this multifaceted protein may provide mechanistic explanations for its role in chemotherapy responses. (c) 2006 Elsevier B.V. All rights reserved.

Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials

Background: As trials of 5 years of tamoxifen in early breast cancer mature, the relevance of hormone receptor measurements (and other patient characteristics) to long-term outcome can be assessed increasingly reliably. We report updated meta-analyses of the trials of 5 years of adjuvant tamoxifen.
Methods: We undertook a collaborative meta-analysis of individual patient data from 20 trials (n=21457) in early breast cancer of about 5 years of tamoxifen versus no adjuvant tamoxifen, with about 80% compliance. Recurrence and death rate ratios (RRs) were from log-rank analyses by allocated treatment.
Findings: In oestrogen receptor (ER)-positive disease (n=10 645), allocation to about 5 years of tamoxifen substantially reduced recurrence rates throughout the first 10 years (RR 0.53 [SE 0.03] during years 0-4 and RR 0.68 [0.06] during years 5-9 [both 2p<0.00001]; but RR 0.97 [0.10] during years 10-14, suggesting no further gain or loss after year 10). Even in marginally ER-positive disease (10-19 fmol/mg cytosol protein) the recurrence reduction was substantial (RR 0.67 [0.08]). In ER-positive disease, the RR was approximately independent of progesterone receptor status (or level), age, nodal status, or use of chemotherapy. Breast cancer mortality was reduced by about a third throughout the first 15 years (RR 0.71 [0.05] during years 0-4, 0.66 [0.05] during years 5-9, and 0.68 [0.08] during years 10-14; p<0.0001 for extra mortality reduction during each separate time period). Overall non-breast-cancer mortality was little affected, despite small absolute increases in thromboembolic and uterine cancer mortality (both only in women older than 55 years), so all-cause mortality was substantially reduced. In ER-negative disease, tamoxifen had little or no effect on breast cancer recurrence or mortality.
Interpretation: 5 years of adjuvant tamoxifen safely reduces 15-year risks of breast cancer recurrence and death. ER status was the only recorded factor importantly predictive of the proportional reductions. Hence, the absolute risk reductions produced by tamoxifen depend on the absolute breast cancer risks (after any chemotherapy) without tamoxifen.
Funding: Cancer Research UK, British Heart Foundation, and Medical Research Council.

Breast Cancer Risk in Relation to Occupations with Exposure to Carcinogens and Endocrine Disruptors:a Canadian Case-Control Study

Background

Endocrine disrupting chemicals and carcinogens, some of which may not yet have been classified as such, are present in many occupational environments and could increase breast cancer risk. Prior research has identified associations with breast cancer and work in agricultural and industrial settings. The purpose of this study was to further characterize possible links between breast cancer risk and occupation, particularly in farming and manufacturing, as well as to examine the impacts of early agricultural exposures, and exposure effects that are specific to the endocrine receptor status of tumours.

Methods

1005 breast cancer cases referred by a regional cancer center and 1147 randomly-selected community controls provided detailed data including occupational and reproductive histories. All reported jobs were industry- and occupation-coded for the construction of cumulative exposure metrics representing likely exposure to carcinogens and endocrine disruptors. In a frequency-matched case?control design, exposure effects were estimated using conditional logistic regression.

Results

Across all sectors, women in jobs with potentially high exposures to carcinogens and endocrine disruptors had elevated breast cancer risk (OR = 1.42; 95% CI, 1.18-1.73, for 10 years exposure duration). Specific sectors with elevated risk included: agriculture (OR = 1.36; 95% CI, 1.01-1.82); bars-gambling (OR = 2.28; 95% CI, 0.94-5.53); automotive plastics manufacturing (OR = 2.68; 95% CI, 1.47-4.88), food canning (OR = 2.35; 95% CI, 1.00-5.53), and metalworking (OR = 1.73; 95% CI, 1.02-2.92). Estrogen receptor status of tumors with elevated risk differed by occupational grouping. Premenopausal breast cancer risk was highest for automotive plastics (OR = 4.76; 95% CI, 1.58-14.4) and food canning (OR = 5.70; 95% CI, 1.03-31.5).

Conclusions

These observations support hypotheses linking breast cancer risk and exposures likely to include carcinogens and endocrine disruptors, and demonstrate the value of detailed work histories in environmental and occupational epidemiology.

Identification of RBCK1 as a novel regulator of FKBPL: implications for tumor growth and response to tamoxifen

FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identifed a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-b-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-a (ERa). Furthermore, FKBPL and RBCK1 associate with ERa at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.

Endocrine disruptor activity of multiple environmental food chain contaminants

Industrial chemicals, antimicrobials, drugs and personal care products have been reported as global pollutants which enter the food chain. Some of them have also been classified as endocrine disruptors based on results of various studies employing a number of in vitro/. vivo tests. The present study employed a mammalian reporter gene assay to assess the effects of known and emerging contaminants on estrogen nuclear receptor transactivation.Out of fifty-nine compounds assessed, estrogen receptor agonistic activity was observed for parabens (. n= 3), UV filters (. n= 6), phthalates (. n= 4) and a metabolite, pyrethroids (. n= 9) and their metabolites (. n= 3). Two compounds were estrogen receptor antagonists while some of the agonists enhanced 17β-estradiol mediated response.This study reports five new compounds (pyrethroids and their metabolites) possessing estrogen agonist activity and highlights for the first time that pyrethroid metabolites are of particular concern showing much greater estrogenic activity than their parent compounds.

Biotransformation of zearalenone and zearalenols to their major glucuronide metabolites reduces estrogenic activity

Zearalenone (ZEN) is a mycotoxin produced by Fusarium fungi. Once ingested, ZEN may be absorbed andmetabolised to a- and b-zearalenol (a-ZOL, b-ZOL), and to a lesser extent a- and b-zearalanol (a-ZAL,b-ZAL). Further biotransformation to glucuronide conjugates also occurs to facilitate the elimination ofthese toxins from the body. Unlike ZEN and its metabolites, information regarding the estrogenic activityof these glucuronide conjugates in various tissues is lacking. ZEN-14-O-glucuronide, a-ZOL-14-O-glucuronide,a-ZOL-7-O-glucuronide, b-ZOL-14-O-glucuronide and b-ZOL-16-O-glucuronide, previouslyobtained as the major products from preparative enzymatic synthesis, were investigated for their potentialto cause endocrine disruption through interference with estrogen receptor transcriptional activity.All five glucuronide conjugates showed a very weak agonist response in an estrogen responsive reportergene assay (RGA), with activity ranging from 0.0001% to 0.01% of that of 17b-estradiol, and also lessthan that of ZEN, a-ZOL and b-ZOL which have previously shown estrogenic potencies of the order 17bestradiol> a-ZOL > ZEN > b-ZOL. Confirmatory mass spectrometry revealed that any activity observedwas likely a result of minor deconjugation of the glucuronide moiety. This study confirms that formationof ZEN and ZOL glucuronides is a detoxification reaction with regard to estrogenicity, serving as a potentialhost defence mechanism against ZEN-induced estrogenic activity.

The prognostic significance of protein tyrosine phosphatase 4A2 in breast cancer

Although PTP4A3 has been shown to be a very important factor in promoting cancer progression, the role of its close family member PTP4A2 is still largely unknown. Recent reports have shown contradicting results on the role of PTP4A2 in breast cancer progression. Considering this, we aimed to investigate the prognostic value of PTP4A2 in five independent breast cancer data sets (minimum 198 patients per cohort, totaling 1,124 patients) in the Gene Expression Omnibus Database. We found that high expression of PTP4A2 was a favorable prognostic marker in all five independent breast cancer data sets, as well as in the combined cohort, with a hazard ratio of 0.68 (95% confidence interval =0.56-0.83; P<0.001). Low PTP4A2 expression was associated with estrogen receptor-negative tumors and tumors with higher histological grading; furthermore, low expression was inversely correlated with the expression of genes involved in proliferation, including MKI67 and the MCM gene family encoding the minichromosome maintenance proteins. These findings suggest that PTP4A2 may play a role in breast cancer progression by dysregulating cell proliferation. PTP4A2 expression was positively correlated with ESR1, the gene encoding estrogen receptor-alpha, and inversely correlated with EGFR expression, suggesting that PTP4A2 may be involved in these two important oncogenic pathways. Together, our results suggest that expression of PTP4A2 is a favorable prognostic marker in breast cancer.

Gender-specific genomic profiling in metastatic colorectal cancer patients treated with 5-fluorouracil and oxaliplatin

AIMS: Survival and response rates in metastatic colorectal cancer remain poor, despite advances in drug development. There is increasing evidence to suggest that gender-specific differences may contribute to poor clinical outcome. We tested the hypothesis that genomic profiling of metastatic colorectal cancer is dependent on gender.

MATERIALS & METHODS: A total of 152 patients with metastatic colorectal cancer who were treated with oxaliplatin and continuous infusion 5-fluorouracil were genotyped for 21 polymorphisms in 13 cancer-related genes by PCR. Classification and regression tree analysis tested for gender-related association of polymorphisms with overall survival, progression-free survival and tumor response.

RESULTS: Classification and regression tree analysis of all polymorphisms, age and race resulted in gender-specific predictors of overall survival, progression-free survival and tumor response. Polymorphisms in the following genes were associated with gender-specific clinical outcome: estrogen receptor β, EGF receptor, xeroderma pigmentosum group D, voltage-gated sodium channel and phospholipase A2.

CONCLUSION: Genetic profiling to predict the clinical outcome of patients with metastatic colorectal cancer may depend on gender.

Chemoprevention in BRCA1 Mutation Carriers—A Proof-of-Concept Study

Background: Women with germline BRCA1 mutations have a high lifetime risk of breast cancer, with the only available risk-reduction strategies being risk-reducing surgery or chemoprevention. These women predominantly develop triple-negative breast cancers; hence, it is unlikely that selective estrogen receptor modulators (serms) will reduce the risk of developing cancer, as these have not been shown to reduce the incidence of estrogen receptor–negative breast cancers. Preclinical data from our laboratory suggest that exposure to estrogen and its metabolites is capable of causing dna double-strand breaks (dsbs) and thus driving genomic instability, an early hallmark of BRCA1-related breast cancer. Therefore, an approach that lowers circulating estrogen levels and reduces estrogen metabolite exposure may prove a successful chemopreventive strategy.

Aims: To provide proof of concept of the hypothesis that the combination of luteinizing-hormone releasing-hormone agonists (lhrha) and aromatase inhibitors (ais) can suppress circulating levels of estrogen and its metabolites in BRCA1 mutation carriers, thus reducing estrogen metabolite levels in breast cells, reducing dna dsbs, and potentially reducing the incidence of breast cancer.

Methods: 12 Premenopausal BRCA1 mutation carriers will undergo baseline ultrasound-guided breast core biopsy and plasma and urine sampling. Half the women will be treated for 3 months with combination goserelin (lhrha) plus anastrazole (ai), and the remainder with tamoxifen (serm) before repeat tissue, plasma, and urine sampling. After a 1-month washout period, groups will cross over for a further 3 months treatment before final biologic sample collection. Tissue, plasma, and urine samples will be examined using a combination of immunohistochemistry, comet assays, and ultrahigh performance liquid chromatography tandem mass spectrometry to assess the impact of lhrha plus ai compared with serm on levels of dna damage, estrogens, and genotoxic estrogen metabolites. Quality of life will also be assessed during the study.

Results: This trial is currently ongoing.

Adjuvant chemotherapy in oestrogen-receptor-poor breast cancer: patient-level meta-analysis of randomised trials

Background: The long-term effects of adjuvant polychemotherapy regimens in oestrogen-receptor-poor (ER-poor) breast cancer, and the extent to which these effects are modified by age or tamoxifen use, can be assessed by an updated meta-analysis of individual patient data from randomised trials. Methods: Collaborative meta-analyses of individual patient data for about 6000 women with ER-poor breast cancer in 46 trials of polychemotherapy versus not (non-taxane-based polychemotherapy, typically about six cycles; trial start dates 1975-96, median 1984) and about 14 000 women with ER-poor breast cancer in 50 trials of tamoxifen versus not (some trials in the presence and some in the absence of polychemotherapy; trial start dates 1972-93, median 1982). Findings: In women with ER-poor breast cancer, polychemotherapy significantly reduced recurrence, breast cancer mortality, and death from any cause, in those younger than 50 years and those aged 50-69 years at entry into trials of polychemotherapy versus not. In those aged younger than 50 years (1907 women, 15% node-positive), the 10-year risks were: recurrence 33% versus 45% (ratio of 10-year risks 0·73, 2p<0·00001), breast cancer mortality 24% versus 32% (ratio 0·73, 2p=0·0002), and death from any cause 25% versus 33% (ratio 0·75, 2p=0·0003). In women aged 50-69 years (3965 women, 58% node-positive), the 10-year risks were: recurrence 42% versus 52% (ratio 0·82, 2p<0·00001), breast cancer mortality 36% versus 42% (ratio 0·86, 2p=0·0004), and death from any cause 39% versus 45% (ratio 0·87, 2p=0·0009). Few were aged 70 years or older. Tamoxifen had little effect on recurrence or death in women who were classified in these trials as having ER-poor disease, and did not significantly modify the effects of polychemotherapy. Interpretation: In women who had ER-poor breast cancer, and were either younger than 50 years or between 50 and 69 years, these older adjuvant polychemotherapy regimens were safe (ie, had little effect on mortality from causes other than breast cancer) and produced substantial and definite reductions in the 10-year risks of recurrence and death. Current and future chemotherapy regimens could well yield larger proportional reductions in breast cancer mortality.

Androgens and estrogens stimulate ribosome biogenesis in prostate and breast cancer cells in receptor dependent manner

Ribosome biogenesis is a fundamental cellular process intimately linked to cell growth and proliferation, which is upregulated in most of cancers especially in aggressive cancers. In breast and prostate cancers steroid hormone receptor signalling is the principal stimulus for cancer growth and progression. Here we investigated the link between estrogen and androgen receptor signalling and the initial stage of ribosome biogenesis - transcription of rRNA genes. We have discovered that estrogen or androgen treatment can positively regulate rRNA synthesis in breast and prostate cancer cells respectively and that this effect is receptor dependent. This novel and interesting finding suggests a previously unidentified link between steroid hormone receptor signalling pathways and the regulation of ribosome biogenesis.

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.

Molecular pathways: Estrogen pathway in colorectal cancer.

Worldwide, colorectal cancer has a higher incidence rate in men than in women, suggesting a protective role for sex hormones in the development of the disease. Preclinical data support a role for estrogen and its receptors in the initiation and progression of colorectal cancer and establishes that protective effects of estrogen are exerted through ERβ. Hormone replacement therapy (HRT) in postmenopausal women as well as consumption of soy reduces the incidence of colorectal cancer. In the Women's Health Initiative trial, use of HRT in postmenopausal women reduced the risk of colon cancer by 56% [95% confidence interval (CI), 0.38-0.81; P = 0.003]. A recent meta-analysis showed that in women, consumption of soy reduced the risk of colon cancer by 21% (95% CI, 0.03-0.35; P = 0.026). In this review, using the preclinical data, we translate the findings in the clinical trials and observational studies to define the role of estrogen in the prevention of colorectal cancer. We hypothesize that sometime during the tumorigenesis process ERβ expression in colonocytes is lost and the estrogen ligand, HRT, or soy products, exerts its effects through preventing this loss. Thus, in the adenoma-to-carcinoma continuum, timing of HRT is a significant determinant of the observed benefit from this intervention. We further argue that the protective effects of estrogen are limited to certain molecular subtypes. Successful development of estrogen modulators for prevention of colorectal cancer depends on identification of susceptible colorectal cancer population(s). Thus, research to better understand the estrogen pathway is fundamental for clinical delivery of these agents.

ARB: Phase II window of opportunity study of short-term preoperative treatment with enzalutamide in ER-positive and triple-negative breast cancer

The androgen receptor (AR) is expressed in 60-80% of breast cancers (BC) across all molecular phenotypes, with a higher incidence in oestrogen receptor positive (ER+) BC compared to ER negative tumours. In ER+ disease, AR-expression has been linked to endocrine resistance which might be reversed with combined treatment targeting ER and AR. In triple negative BCs (TNBC), preclinical and clinical investigations have described a subset of patients that express the AR and are sensitive to androgen blockade, providing a novel therapeutic target. Enzalutamide, a potent 2nd generation anti-androgen, has demonstrated substantial preclinical and clinical anti-tumour activity in AR+ breast cancer. Short-term preoperative window of opportunity studies are a validated strategy for novel treatments to provide proof-of-concept and define the most appropriate patient population by directly assessing treatment effects in tumour tissue before and after treatment. The ARB study aims to assess the anti-tumour effects of enzalutamide in early ER+ breast cancer and TNBC, to identify the optimal target population for further studies and to directly explore the biologic effects of enzalutamide on BC and stromal cells. Methods: ARB is an international, investigator sponsored WOO phase II study in women with newly diagnosed primary ER+ BC or AR+ TNBC of ≥ 1cm. The study has two cohorts. In the ER+ cohort, postmenopausal patients will be randomised 2:1 to receive either enzalutamide (160mg OD) plus exemestane (50mg OD) or exemestane (25mg OD). In the TNBC cohort, AR+ will receive single agent treatment with enzalutamide (160mg OD). Study treatment is planned for 15–29 days, followed by surgery or neo-adjuvant therapy. Tissue and blood samples are collected before treatment and on the last day of study treatment. The primary endpoint is inhibition of tumour-cell proliferation, as measured by change in Ki67 expression, determined centrally by 2 investigators. Secondary endpoints include induction of apoptosis (Caspase3), circulating hormone levels and safety. ARB aims to recruit ≈235 patients from ≈40 sites in the UK, Germany, Spain and USA. The study is open to recruitment.