Mechanisms of resistance in estrogen receptor positive breast cancer: overcoming resistance to tamoxifen/aromatase inhibitors.

Several mechanisms of resistance have been identified, underscoring the complex nature of estrogen receptor (ER) signaling and the many connections between this pathway and other essential signaling pathways in breast cancer cells. Many therapeutic targets of cell signaling and cell cycle pathways have met success with endocrine therapy and remain an ongoing area of investigation. This review focuses on two major pathways that have recently emerged as important opportunities for therapeutic intervention in endocrine resistant breast tumors: PI3K/AKT/mTOR cell signaling and cyclinD1/cyclin-dependent kinase 4/6 cell cycle pathways. Additionally, we highlight individual and combination strategies in current clinical trials that target these pathways and others under investigation for the treatment of ER positive breast cancer.

Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3.

PURPOSE: NSD3 has been implicated as a candidate driver oncogene from the 8p11-p12 locus, and we have previously published evidence for its amplification and overexpression in human breast cancer. This aim of this study was to further characterize the transforming function of NSD3 in vivo. METHODS: We generated a transgenic mouse model in which NSD3 gene expression was driven by the MMTV promoter and expressed in mammary epithelium of FVB mice. Mammary glands were fixed and whole mounts were stained with carmine to visualize gland structure. Mammary tumors were formalin-fixed, and paraffin embedded (FFPE) tumors were stained with hematoxylin and eosin. RESULTS: Pups born to transgenic females were significantly underdeveloped compared to pups born to WT females due to a lactation defect in transgenic female mice. Whole mount analysis of the mammary glands of transgenic female mice revealed a profound defect in functional differentiation of mammary gland alveoli that resulted in the lactation defect. We followed parous and virgin NSD3 transgenic and control mice to 50 weeks of age and observed that several NSD3 parous females developed mammary tumors. Whole mount analysis of the mammary glands of tumor-bearing mice revealed numerous areas of mammary hyperplasia and ductal dysplasia. Histological analysis showed that mammary tumors were high-grade ductal carcinomas, and lesions present in other mammary glands exhibited features of alveolar hyperplasia, ductal dysplasia, and carcinoma in situ. CONCLUSIONS: Our results are consistent with our previous studies and demonstrate that NSD3 is a transforming breast cancer oncogene.

Amplification of WHSC1L1 regulates expression and estrogen-independent activation of ERα in SUM-44 breast cancer cells and is associated with ERα over-expression in breast cancer.

The 8p11-p12 amplicon occurs in approximately 15% of breast cancers in aggressive luminal B-type tumors. Previously, we identified WHSC1L1 as a driving oncogene from this region. Here, we demonstrate that over-expression of WHSC1L1 is linked to over-expression of ERα in SUM-44 breast cancer cells and in primary human breast cancers. Knock-down of WHSC1L1, particularly WHSC1L1-short, had a dramatic effect on ESR1 mRNA and ERα protein levels. SUM-44 cells do not require exogenous estrogen for growth in vitro; however, they are dependent on ERα expression, as ESR1 knock-down or exposure to the selective estrogen receptor degrader fulvestrant resulted in growth inhibition. ChIP-Seq experiments utilizing ERα antibodies demonstrated extensive ERα binding to chromatin in SUM-44 cells under estrogen-free conditions. ERα bound to ERE and FOXA1 motifs under estrogen-free conditions and regulated expression of estrogen-responsive genes. Short-term treatment with estradiol enhanced binding of ERα to chromatin and influenced expression of many of the same genes to which ERα was bound under estrogen-free conditions. Finally, knock-down of WHSC1L1 in SUM-44 cells resulted in loss of ERα binding to chromatin under estrogen-free conditions, which was restored upon exposure to estradiol. These results indicate the SUM-44 cells are a good model of a subset of luminal B breast cancers that have the 8p11-p12 amplicon, over-express WHSC1L1, and over-express ERα, but are independent of estrogen for binding to chromatin and regulation of gene expression. Breast cancers such as these, that are dependent on ERα activity but independent of estradiol, are a major cause of breast cancer mortality.

MicroRNA-510 promotes cell and tumor growth by targeting peroxiredoxin1 in breast cancer.

INTRODUCTION: MicroRNAs are small non-coding RNAs that are involved in the post-transcriptional negative regulation of mRNAs. MicroRNA 510 (miR-510) was initially shown to have a potential oncogenic role in breast cancer by the observation of its elevated levels in human breast tumor samples when compared to matched non-tumor samples. Few targets have been identified for miR-510. However, as microRNAs function through the negative regulation of their direct targets, the identification of those targets is critical for the understanding of their functional role in breast cancer. METHODS: Breast cancer cell lines were transfected with pre-miR-510 or antisense miR-510 and western blotting and quantitative real time PCR were performed. Functional assays performed included cell growth, migration, invasion, colony formation, cytotoxicity and in vivo tumor growth. We performed a PCR assay to identify novel direct targets of miR-510. The study focused on peroxiredoxin 1 (PRDX1) as it was identified through our screen and was bioinformatically predicted to contain a miR-510 seed site in its 3' untranslated region (3'UTR). Luciferase reporter assays and site-directed mutagenesis were performed to confirm PRDX1 as a direct target. The Student's two-sided, paired t-test was used and a P-value less than 0.05 was considered significant. RESULTS: We show that miR-510 overexpression in non-transformed and breast cancer cells can increase their cell growth, migration, invasion and colony formation in vitro. We also observed increased tumor growth when miR-510 was overexpressed in vivo. We identified PRDX1 through a novel PCR screen and confirmed it as a direct target using luciferase reporter assays. The reintroduction of PRDX1 into breast cancer cell lines without its regulatory 3'UTR confirmed that miR-510 was mediating its migratory phenotype at least in part through the negative regulation of PRDX1. Furthermore, the PI3K/Akt pathway was identified as a positive regulator of miR-510 both in vitro and in vivo. CONCLUSIONS: In this study, we provide evidence to support a role for miR-510 as a novel oncomir. We show that miR-510 directly binds to the 3'UTR of PRDX1 and blocks its protein expression, thereby suppressing migration of human breast cancer cells. Taken together, these data support a pivotal role for miR-510 in breast cancer progression and suggest it as a potential therapeutic target in breast cancer patients.