Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

Estrogen is a known growth promoter for estrogen receptor (ER)-positive breast cancer cells. Paradoxically, in breast cancer cells that have been chronically deprived of estrogen stimulation, re-introduction of the hormone can induce apoptosis.Here, we sought to identify signaling networks that are triggered by estradiol (E2) in isogenic MCF-7 breast cancer cells that undergo apoptosis (MCF-7:5C) versus cells that proliferate upon exposure to E2 (MCF-7). The nuclear receptor co-activator AIB1 (Amplified in Breast Cancer-1) is known to be rate-limiting for E2-induced cell survival responses in MCF-7 cells and was found here to also be required for the induction of apoptosis by E2 in the MCF-7:5C cells. Proteins that interact with AIB1 as well as complexes that contain tyrosine phosphorylated proteins were isolated by immunoprecipitation and identified by mass spectrometry (MS) at baseline and after a brief exposure to E2 for two hours. Bioinformatic network analyses of the identified protein interactions were then used to analyze E2 signaling pathways that trigger apoptosis versus survival. Comparison of MS data with a computationally-predicted AIB1 interaction network showed that 26 proteins identified in this study are within this network, and are involved in signal transduction, transcription, cell cycle regulation and protein degradation.G-protein-coupled receptors, PI3 kinase, Wnt and Notch signaling pathways were most strongly associated with E2-induced proliferation or apoptosis and are integrated here into a global AIB1 signaling network that controls qualitatively distinct responses to estrogen

MiR-137 Targets Estrogen-Related Receptor Alpha and Impairs the Proliferative and Migratory Capacity of Breast Cancer Cells

ERRα is an orphan nuclear receptor emerging as a novel biomarker of breast cancer. Over-expression of ERRα in breast tumor is considered as a prognostic factor of poor clinical outcome. The mechanisms underlying the dysexpression of this nuclear receptor, however, are poorly understood. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play important roles in tumor initiation and progression. In the present study, we have identified that the expression of ERRα is regulated by miR-137, a potential tumor suppressor microRNA. The bioinformatics search revealed two putative and highly conserved target-sites for miR-137 located within the ERRα 3′UTR at nt 480–486 and nt 596–602 respectively. Luciferase-reporter assay demonstrated that the two predicted target sites were authentically functional. They mediated the repression of reporter gene expression induced by miR-137 in an additive manner. Moreover, ectopic expression of miR-137 down-regulated ERRα expression at both protein level and mRNA level, and the miR-137 induced ERRα-knockdown contributed to the impaired proliferative and migratory capacity of breast cancer cells. Furthermore, transfection with miR-137mimics suppressed at least two downstream target genes of ERRα–CCNE1 and WNT11, which are important effectors of ERRα implicated in tumor proliferation and migration. Taken together, our results establish a role of miR-137 in negatively regulating ERRα expression and breast cancer cell proliferation and migration. They suggest that manipulating the expression level of ERRα by microRNAs has the potential to influence breast cancer progression

Heat shock protein90 in lobular neoplasia of the breast

<p>Abstract</p> <p>Background</p> <p>Heat shock protein 90 (Hsp90) overexpression has been implicated in breast carcinogenesis, with putative prognostic and therapeutic implications. The purpose of this study is to evaluate the immunohistochemical expression of Hsp90 and to examine whether Hsp90 expression is associated with estrogen receptor alpha (ER-alpha) and beta (ER-beta) immunostaining in lobular neoplasia (LN) of the breast.</p> <p>Methods</p> <p>Tissue specimens were taken from 44 patients with LN. Immunohistochemical assessment of Hsp90, ER-alpha and ER-beta was performed both in the lesion and the adjacent normal breast ducts and lobules; the latter serving as control. As far as Hsp90 evaluation is concerned: i) the percentage of positive cells, and ii) the intensity was separately analyzed. Additionally, the Allred score was adopted and calculated. Accordingly, Allred score was separately evaluated for ER-alpha and ER-beta. The intensity was treated as an ordinal variable-score (0: negative, low: 1, moderate: 2, high: 3). Statistical analysis followed.</p> <p>Results</p> <p>Hsp90 immunoreactivity was mainly cytoplasmic in both the epithelial cells of normal breast (ducts and lobules) and LN. Some epithelial cells of LN also showed nuclear staining, but all the LN foci mainly disclosed a positive cytoplasmic immunoreaction for Hsp90. In addition, rare intralobular inflammatory cells showed a slight immunoreaction. The percentage of Hsp90 positive cells in the LN areas was equal to 67.1 ± 12.2%, whereas the respective percentage in the normal adjacent breast tissue was 69.1 ± 11.6%; the difference was not statistically significant. The intensity score of Hsp90 staining was 1.82 ± 0.72 in LN foci, while in the normal adjacent tissue the intensity score was 2.14 ± 0.64. This difference was statistically significant (p = 0.029, Wilcoxon matched-pairs signed-ranks test). The Hsp90 Allred score was 6.46 ± 1.14 in the LN foci, significantly lower than in the normal adjacent tissue (6.91 ± 0.92, p = 0.049, Wilcoxon matched-pairs signed-ranks test). Within the LN foci, the Hsp90 Allred score was neither associated with ER-alpha, nor with ER-beta percentage.</p> <p>Conclusion</p> <p>Hsp90 was lower in LN foci both at the level of intensity and Allred score, a finding contrary to what might have been expected, given that high Hsp90 expression is detected in invasive breast carcinomas. Hsp90 deregulation does not seem to be a major event in LN pathogenesis.</p

Genetic profiling of chromosome 1 in breast cancer: mapping of regions of gains and losses and identification of candidate genes on 1q

Chromosome 1 is involved in quantitative anomalies in 50–60% of breast tumours. However, the structure of these anomalies and the identity of the affected genes remain to be determined. To characterise these anomalies and define their consequences on gene expression, we undertook a study combining array-CGH analysis and expression profiling using specialised arrays. Array-CGH data showed that 1p was predominantly involved in losses and 1q almost exclusively in gains. Noticeably, high magnitude amplification was infrequent. In an attempt to fine map regions of copy number changes, we defined 19 shortest regions of overlap (SROs) for gains (one at 1p and 18 at 1q) and of 20 SROs for losses (all at 1p). These SROs, whose sizes ranged from 170 kb to 3.2 Mb, represented the smallest genomic intervals possible based on the resolution of our array. The elevated incidence of gains at 1q, added to the well-established concordance between DNA copy increase and augmented RNA expression, made us focus on gene expression changes at this chromosomal arm. To identify candidate oncogenes, we studied the RNA expression profiles of 307 genes located at 1q using a home-made built cDNA array. We identified 30 candidate genes showing significant overexpression correlated to copy number increase. In order to substantiate their involvement, RNA expression levels of these candidate genes were measured by quantitative (Q)-RT–PCR in a panel of 25 breast cancer cell lines previously typed by array-CGH. Q–PCR showed that 11 genes were significantly overexpressed in the presence of a genomic gain in these cell lines, and 20 overexpressed when compared to normal breast

Response of Estrogen Receptor-Positive Breast Cancer Tumorspheres to Antiestrogen Treatments

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment

Breast cancer chemoprevention: beyond tamoxifen

A large number of new potential chemoprevention agents are available that target molecular abnormalities found in estrogen receptor (ER)-negative and/or ER-positive precancerous breast tissue and have side effect profiles that differ from tamoxifen. Classes of agents currently undergoing evaluation in clinical prevention trials or those for which testing is planned in the near future include new selective ER modulators, aromatase inactivators/inhibitors, gonadotrophin-releasing hormone agonists, monoterpenes, isoflavones, retinoids, rexinoids, vitamin D derivatives, and inhibitors of tyrosine kinase, cyclooxygenase-2, and polyamine synthesis. New clinical testing models will use morphological and molecular biomarkers to select candidates at highest short-term risk, to predict the response to a particular class of agent, and to assess the response in phase II prevention trials. If validated, morphological and molecular markers could eventually replace cancer incidence as an indicator of efficacy in future phase III trials

ERRα promotes breast cancer cell dissemination to bone by increasing RANK expression in primary breast tumors

Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-of-function analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases

MicroRNA-mediated drug resistance in breast cancer

Chemoresistance is one of the major hurdles to overcome for the successful treatment of breast cancer. At present, there are several mechanisms proposed to explain drug resistance to chemotherapeutic agents, including decreased intracellular drug concentrations, mediated by drug transporters and metabolic enzymes; impaired cellular responses that affect cell cycle arrest, apoptosis, and DNA repair; the induction of signaling pathways that promote the progression of cancer cell populations; perturbations in DNA methylation and histone modifications; and alterations in the availability of drug targets. Both genetic and epigenetic theories have been put forward to explain the mechanisms of drug resistance. Recently, a small non-coding class of RNAs, known as microRNAs, has been identified as master regulators of key genes implicated in mechanisms of chemoresistance. This article reviews the role of microRNAs in regulating chemoresistance and highlights potential therapeutic targets for reversing miRNA-mediated drug resistance. In the future, microRNA-based treatments, in combination with traditional chemotherapy, may be a new strategy for the clinical management of drug-resistant breast cancers

The oral selective oestrogen receptor degrader (SERD) AZD9496 is comparable to fulvestrant in antagonising ER and circumventing endocrine resistance.

BACKGROUND: The oestrogen receptor (ER) is an important therapeutic target in ER-positive (ER+) breast cancer. The selective ER degrader (SERD), fulvestrant, is effective in patients with metastatic breast cancer, but its intramuscular route of administration and low bioavailability are major clinical limitations. METHODS: Here, we studied the pharmacology of a new oral SERD, AZD9496, in a panel of in vitro and in vivo endocrine-sensitive and -resistant breast cancer models. RESULTS: In endocrine-sensitive models, AZD9496 inhibited cell growth and blocked ER activity in the presence or absence of oestrogen. In vivo, in the presence of oestrogen, short-term AZD9496 treatment, like fulvestrant, resulted in tumour growth inhibition and reduced expression of ER-dependent genes. AZD9496 inhibited cell growth in oestrogen deprivation-resistant and tamoxifen-resistant cell lines and xenograft models that retain ER expression. AZD9496 effectively reduced ER levels and ER-induced transcription. Expression analysis of short-term treated tumours showed that AZD9496 potently inhibited classic oestrogen-induced gene transcription, while simultaneously increasing expression of genes negatively regulated by ER, including genes potentially involved in escape pathways of endocrine resistance. CONCLUSIONS: These data suggest that AZD9496 is a potent anti-oestrogen that antagonises and degrades ER with anti-tumour activity in both endocrine-sensitive and endocrine-resistant models