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BackgroundHigher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines.ResultsOur studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16–22 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16–22 reveals pathways related to repression of WNT signaling. There are also differences in intra-chromosomal interactions between the cell lines; telomeric and sub-telomeric regions in the MCF-10A cells display more frequent interactions than are observed in the MCF-7 cells.ConclusionsWe show evidence of an intricate relationship between chromosomal organization and gene expression between epithelial and breast cancer cells. Importantly, this work provides a genome-wide view of higher-order chromatin dynamics and a resource for studying higher-order chromatin interactions in two cell lines commonly used to study the progression of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0768-0) contains supplementary material, which is available to authorized users.

Progression of breast cancer to metastatic bone disease is linked to deregulated expression of the transcription factor Runx2. Therefore, our goal was to evaluate the potential for clinical use of Runx2-targeting microRNAs (miRNAs) to reduce tumor growth and bone metastatic burden. Expression analysis of a panel of miRNAs regulating Runx2 revealed a reciprocal relationship between the abundance of Runx2 protein and two miRNAs, miR-135 and miR-203. These miRNAs are highly expressed in normal breast epithelial cells where Runx2 is not detected, and absent in metastatic breast cancer cells and tissue biopsies that express Runx2. Reconstituting metastatic MDA-MB-231-Luc cells with miR-135 and miR-203 reduced the abundance of Runx2 and expression of the metastasis-promoting Runx2 target genes IL-11, MMP-13, and PTHrP. Additionally, tumor cell viability was decreased and migration suppressed in vitro. Orthotopic implantation of MDA-MB-231-luc cells delivered with miR-135 or miR-203, followed by an intratumoral administration of the synthetic miRNAs reduced the tumor growth and spontaneous metastasis to bone. Furthermore, intratibial injection of these miRNA-delivered cells impaired tumor growth in the bone environment and inhibited bone resorption. Importantly, reconstitution of Runx2 in MDA-MB-231-luc cells delivered with miR-135 and miR-203 reversed the inhibitory effect of the miRNAs on tumor growth and metastasis. Thus, we have identified that aberrant expression of Runx2 in aggressive tumor cells is related to the loss of specific Runx2-targeting miRNAs and that a clinically relevant replacement strategy by delivery of synthetic miRNAs is a candidate therapeutic approach to prevent metastatic bone disease by this route.

Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFβ signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFβ and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression.

The Brahma (BRM) and Brahma-related Gene 1 (BRG1) ATPases are highly conserved homologues that catalyze the chromatin remodeling functions of the multi-subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in man cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor’s receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells.

Runx1 is a transcription factor essential for definitive hematopoiesis, and genetic abnormalities in Runx1 cause leukemia. Runx1 is functionally promiscuous and acts as either an oncogene or tumor suppressor gene in certain epithelial cancers. Recent evidence suggests that Runx1 is an important factor in breast cancer, however its role remains ambiguous. Here, we addressed whether Runx1 has a specific pathological role during breast cancer progression and show that Runx1 has an oncogenic function. We observed elevated Runx1 expression in a subset of human breast cancers. Furthermore, throughout the course of disease progression in a classical mouse model of breast cancer (i.e., the MMTV-PyMT transgenic model), Runx1 expression increases in the primary site (mammary gland) and is further upregulated in tumors and distal lung metastatic lesions. Ex vivo studies using tumor epithelial cells derived from these mice express significantly higher levels of Runx1 than normal mammary epithelial cells. The tumor cells exhibit increased rates of migration and invasion, indicative of an aggressive cancer phenotype. Inhibition of Runx1 expression using RNA interference significantly abrogates these cancer-relevant phenotypic characteristics. Importantly, our data establish that Runx1 contributes to murine mammary tumor development and malignancy and potentially represents a key disease-promoting and prognostic factor in human breast cancer progression and metastasis.

The onset and progression of breast cancer are linked to genetic and epigenetic changes that alter the normal programming of cells. Epigenetic modifications of DNA and histones contribute to chromatin structure that result in the activation or repression of gene expression. Several epigenetic pathways have been shown to be highly deregulated in cancer cells. Targeting specific histone modifications represents a viable strategy to prevent oncogenic transformation, tumor growth or metastasis. Methylation of histone H3 lysine 4 has been extensively studied and shown to mark genes for expression; however this residue can also be acetylated and the specific function of this alteration is less well known. To define the relative roles of histone H3 methylation (H3K4me3) and acetylation (H3K4ac) in breast cancer, we determined genomic regions enriched for both marks in normal-like (MCF10A), transformed (MCF7) and metastatic (MDA-MB-231) cells using a genome-wide ChIP-Seq approach. Our data revealed a genome-wide gain of H3K4ac associated with both early and late breast cancer cell phenotypes, while gain of H3K4me3 was predominantly associated with late stage cancer cells. Enrichment of H3K4ac was over-represented at promoters of genes associated with cancer-related phenotypic traits, such as estrogen response and epithelial-to-mesenchymal transition pathways. Our findings highlight an important role for H3K4ac in predicting epigenetic changes associated with early stages of transformation. In addition, our data provide a valuable resource for understanding epigenetic signatures that correlate with known breast cancer-associated oncogenic pathways.

Bone metastasis is a common and devastating complication of late stage breast and prostate cancer. Complex interactions between tumor cells, bone cells and a milieu of components in their microenvironment contribute to the osteolytic, osteoblastic or mixed lesions present in patients with metastasis to bone. In the last decade, miRNAs have emerged as key players in cancer progression yet the importance of miRNAs in regulating cancer metastasis to bone is now being appreciated. Here, we emphasize important concepts of bone biology and miRNAs in the context of breast and prostate cancer and focus on recent advances that have improved our understanding of the role of specific miRNAs with direct involvement in metastatic bone disease.