Austin E. Gillen

HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer.

AbstractmiRNAs 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. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA–mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). 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. 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.

Fibroblast Subtypes Regulate Responsiveness of Luminal Breast Cancer to Estrogen

Purpose: Antiendocrine therapy remains the most effective treatment for estrogen receptor–positive (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 (CAF), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and antiendocrine resistance. Experimental Design: Tissues from patients with ER+ breast cancer 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 cocultured with CD146pos or CD146neg fibroblasts. RNA sequencing 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 are 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. Clin Cancer Res; 23(7); 1710–21. ©2016 AACR.

valr: Reproducible genome interval analysis in R

New tools for reproducible exploratory data analysis of large datasets are important to address the rising size and complexity of genomic data. We developed the valr R package to enable flexible and efficient genomic interval analysis. valr leverages new tools available in the ”tidyverse”, including dplyr. Benchmarks of valr show it performs similar to BEDtools and can be used for interactive analyses and incorporated into existing analysis pipelines.

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1,313 to 2,002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.

Dynamic temperature-sensitive A-to-I RNA editing in the brain of a heterothermic mammal during hibernation

RNA editing diversifies genomically encoded information to expand the complexity of the transcriptome. In ectothermic organisms, including Drosophila and Cephalopoda, where body temperature mirrors ambient temperature, decreases in environmental temperature lead to increases in A-to-I RNA editing and cause amino acid recoding events that are thought to be adaptive responses to temperature fluctuations. In contrast, endothermic mammals, including humans and mice, typically maintain a constant body temperature despite environmental changes. Here, A-to-I editing primarily targets repeat elements, rarely results in the recoding of amino acids, and plays a critical role in innate immune tolerance. Hibernating ground squirrels provide a unique opportunity to examine RNA editing in a heterothermic mammal whose body temperature varies over 30°C and can be maintained at 5°C for many days during torpor. We profiled the transcriptome in three brain regions at six physiological states to quantify RNA editing and determine whether cold-induced RNA editing modifies the transcriptome as a potential mechanism for neuroprotection at low temperature during hibernation. We identified 5165 A-to-I editing sites in 1205 genes with dynamically increased editing after prolonged cold exposure. The majority (99.6%) of the cold-increased editing sites are outside of previously annotated coding regions, 82.7% lie in SINE-derived repeats, and 12 sites are predicted to recode amino acids. Additionally, A-to-I editing frequencies increase with increasing cold-exposure, demonstrating that ADAR remains active during torpor. Our findings suggest that dynamic A-to-I editing at low body temperature may provide a neuroprotective mechanism to limit aberrant dsRNA accumulation during torpor in the mammalian hibernator.

FGFR1 underlies obesity-associated progression of estrogen receptor–positive breast cancer after estrogen deprivation

Obesity increases breast cancer mortality by promoting resistance to therapy. Here, we identified regulatory pathways in estrogen receptor-positive (ER-positive) tumors that were shared between patients with obesity and those with resistance to neoadjuvant aromatase inhibition. Among these was fibroblast growth factor receptor 1 (FGFR1), a known mediator of endocrine therapy resistance. In a preclinical model with patient-derived ER-positive tumors, diet-induced obesity promoted a similar gene expression signature and sustained the growth of FGFR1-overexpressing tumors after estrogen deprivation. Tumor FGFR1 phosphorylation was elevated with obesity and predicted a shorter disease-free and disease-specific survival for patients treated with tamoxifen. In both human and mouse mammary adipose tissue, FGF1 ligand expression was associated with metabolic dysfunction, weight gain, and adipocyte hypertrophy, implicating the impaired response to a positive energy balance in growth factor production within the tumor niche. In conjunction with these studies, we describe a potentially novel graft-competent model that can be used with patient-derived tissue to elucidate factors specific to extrinsic (host) and intrinsic (tumor) tissue that are critical for obesity-associated tumor promotion. Taken together, we demonstrate that obesity and excess energy establish a tumor environment with features of endocrine therapy resistance and identify a role for ligand-dependent FGFR1 signaling in obesity-associated breast cancer progression.

Alternative Polyadenylation of PRELID1 Regulates Mitochondrial ROS Signaling and Cancer Outcomes

Disruption of posttranscriptional gene regulation is a critical step in oncogenesis that can be difficult to observe using traditional molecular techniques. To overcome this limitation, a modified polyadenylation site sequencing (PAS-seq) protocol was used to generate a genome-wide map of alternative polyadenylation (APA) events in human primary breast tumor specimens and matched normal tissue. This approach identified an APA event in the PRELID1 mRNA that enhances its steady-state level and translational efficiency, and is a strong breast cancer subtype-dependent predictor of patient clinical outcomes. Furthermore, it has been demonstrated that PRELID1 regulates stress response and mitochondrial reactive oxygen species (ROS) production in a cell type–specific manner. Modulation of PRELID1 expression, including its posttranscriptional control, appears to be a common stress response across different cancer types. These data reveal that PRELID1 mRNA processing is an important regulator of cell type–specific responses to stress used by multiple cancers and is associated with patient outcomes. Implications: This study suggests that the regulation of PRELID1 expression, by APA and other mechanisms, plays a role in mitochondrial ROS signaling and represents a novel prognostic factor and therapeutic target in cancer. Mol Cancer Res; 15(12); 1741–51. ©2017 AACR.

Abstract P4-04-06: CD146 positive and negative stroma direct breast tumor estrogen receptor levels, therapeutic response and metastatic potential

Background: Cellular heterogeneity within all breast cancer subtypes remains a major cause of treatment failure and development of metastatic disease. Currently, both preclinical studies and drug development efforts focus almost exclusively on the epithelial component of breast cancers. Despite strong preclinical data novel therapies often fail in clinical testing. We propose that this failure is, in part, due to taking tumor cells out of their context and using pre-clinical models that fail to capture the complexity of human disease. We hypothesize that tumors hijack normal components of the tissue microenvironment and use it to their advantage. Here we demonstrate that similar to the normal hematopoetic niche, two major subtypes of breast cancer stroma can be defined by CD146 expression. We further show that the ratio of the stromal subtypes alters the response to therapy and increases the metastatic potential of breast cancer cells (BCC). Results: Tumor associated stroma, from all breast cancer subtypes, contains a mixture of CD146+ and CD146- fibroblasts. We isolated and derived pure human CD146+ and CD146- clonal lines. Both subtypes expressed markers of activated fibroblasts and clustered by gene expression profiling with normal stromal cell lines HS27A (CD146+) or HS5 (CD146-) according to CD146 expression. Although both stromal subtypes were derived from tumor associated tissue, CD146+ breast cancer stroma clustered with normal breast associated stroma and correlated with good clinical outcome (Finak et. al. Nature Med 2008). CD146- stroma clustered with breast cancer associated stroma and predicted worse outcome. Using cell line and patient-derived xenograft models of estrogen receptor (ER) positive breast cancer, we demonstrated that CD146+ compared to CD146- stroma supported significantly higher ER expression in BCCs. BCC co-cultured with CD146+ stroma responded more robustly to estrogen treatment and anti-endocrine therapy with tamoxifen. Next we used expression profiling data to predict stromal influence on treatment response. CD146+ stroma expressed 3-fold more TGFβ than CD146- stroma. Inhibiting TGFβ decreased proliferation 2-fold in BCCs grown in media conditioned by CD146+ stroma, but not by CD146- stroma. Conversely, HBEGF expression was 3-fold higher in CD146- stroma compared to CD146+ stroma. Inhibiting EGFR decreased proliferation 1.5-fold in BCCs grown in conditioned by CD146- stroma, but not by CD146+ stroma. In addition, intracardiac injection of stroma resulted in distant metastases in our primary orthotopic PDX model of breast cancer. Lastly we confirmed our preclinical observation using a small set of clinical samples. Patients with high CD146+ to CD146- stromal ratio had better clinical outcomes than patients with high CD146- to CD146+ stromal ratio. Conclusion: We conclude that stromal subtypes defined by CD146 expression direct the heterogeneity of ER expression, response to therapy and the metastatic potential of breast cancer cells. Citation Format: Heather M Brechbuhl, Jessica J Finlay-Schultz, Tomomi M Yamamoto, Austin E Gillen, Anthony Elias, Carol A Sartorius, Peter Kabos. CD146 positive and negative stroma direct breast tumor estrogen receptor levels, therapeutic response and metastatic potential [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-06.