Purpose Anti-endocrine therapy remains the most effective treatment for ER+ breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer-associated fibroblasts (CAFs), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and anti-endocrine resistance. Experimental Design Tissues from ER+ breast cancer patients were analyzed for the presence of CD146 positive (CD146pos) and CD146 negative (CD146neg) fibroblasts. ER dependent proliferation and tamoxifen sensitivity were evaluated in ER+ tumor cells co-cultured with CD146pos or CD146neg fibroblasts. RNAseq was used to develop a high confidence gene signature that predicts for disease recurrence in tamoxifen treated patients with ER+ breast cancer. Results We demonstrate that ER+ breast cancers contain two CAF subtypes defined by CD146 expression. CD146neg CAFs suppress ER expression in ER+ breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146pos CAFs maintains ER expression in ER+ breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146pos CAFs with tamoxifen-resistant breast cancer cells is sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146neg CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes. Conclusions Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER+ breast cancer, and should be considered a target for drug development.

Recurrent mutations in core splicing factors have been reported in several clonal disorders, including cancers. Mutations in SF3B1, a component of the U2 splicing complex, are the most common. SF3B1 mutations are associated with aberrant pre-mRNA splicing using cryptic 3’ splice sites (3’SS) but the mechanism of their selection is not clear. To understand how cryptic 3’SS are selected, we performed comprehensive analysis of transcriptome-wide changes to splicing and gene expression associated with SF3B1 mutations in patient samples as well as an experimental model of inducible expression. Hundreds of cryptic 3’SS were detectable across the genome in cells expressing mutant SF3B1. These 3’SS are typically sequestered within RNA secondary structures and poorly accessible compared to their corresponding canonical 3’SS. We hypothesized that these cryptic 3’SS are inaccessible during normal splicing catalysis and that this constraint is overcome in spliceosomes containing mutant SF3B1. This model of secondary structure-dependent selection of cryptic 3’SS was found across multiple clonal processes associated with SF3B1 mutations (myelodysplastic syndrome and chronic lymphocytic leukemia). We validated our model predictions in mini-gene splicing assays. Additionally, we found deregulated expression of proteins with relevant functions in splicing factor-related diseases both in association with aberrant splicing and without corresponding splicing changes. Our results show that SF3B1 mutations are associated with a distinct splicing program shared across multiple clonal processes and define a biochemical mechanism for altered 3’SS choice.

Purpose miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA binding sites. This results in only circumstantial evidence of miRNA-target interaction and typically leads to large numbers of false positive predictions. Methods Here, we used a genome-wide approach (High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, or HITS-CLIP) to define direct miRNA-mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified and triple negative). Results Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. Conclusions We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.