Vikram B. Wali

miR-34a Silences c-SRC to Attenuate Tumor Growth in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no clinically proven biologically targeted treatment options. The molecular heterogeneity of TNBC and lack of high frequency driver mutations other than TP53 have hindered the development of new and effective therapies that significantly improve patient outcomes. miRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are becoming promising therapeutic agents. We performed miRNA-profiling studies in different TNBC subtypes to identify miRNAs that significantly contribute to disease progression. We found that miR-34a was lost in TNBC, specifically within mesenchymal and mesenchymal stem cell-like subtypes, whereas expression of miR-34a targets was significantly enriched. Furthermore, restoration of miR-34a in cell lines representing these subtypes inhibited proliferation and invasion, activated senescence, and promoted sensitivity to dasatinib by targeting the proto-oncogene c-SRC. Notably, SRC depletion in TNBC cell lines phenocopied the effects of miR-34a reintroduction, whereas SRC overexpression rescued the antitumorigenic properties mediated by miR-34a. miR-34a levels also increased when cells were treated with c-SRC inhibitors, suggesting a negative feedback exists between miR-34a and c-SRC. Moreover, miR-34a administration significantly delayed tumor growth of subcutaneously and orthotopically implanted tumors in nude mice, and was accompanied by c-SRC downregulation. Finally, we found that miR-34a and SRC levels were inversely correlated in human tumor specimens. Together, our results demonstrate that miR-34a exerts potent antitumorigenic effects in vitro and in vivo and suggests that miR-34a replacement therapy, which is currently being tested in human clinical trials, represents a promising therapeutic strategy for TNBC.

Predictors of Chemosensitivity in Triple Negative Breast Cancer: An Integrated Genomic Analysis.

Background Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease, and although no effective targeted therapies are available to date, about one-third of patients with TNBC achieve pathologic complete response (pCR) from standard-of-care anthracycline/taxane (ACT) chemotherapy. The heterogeneity of these tumors, however, has hindered the discovery of effective biomarkers to identify such patients. Methods and Findings We performed whole exome sequencing on 29 TNBC cases from the MD Anderson Cancer Center (MDACC) selected because they had either pCR (n = 18) or extensive residual disease (n = 11) after neoadjuvant chemotherapy, with cases from The Cancer Genome Atlas (TCGA; n = 144) and METABRIC (n = 278) cohorts serving as validation cohorts. Our analysis revealed that mutations in the AR- and FOXA1-regulated networks, in which BRCA1 plays a key role, are associated with significantly higher sensitivity to ACT chemotherapy in the MDACC cohort (pCR rate of 94.1% compared to 16.6% in tumors without mutations in AR/FOXA1 pathway, adjusted p = 0.02) and significantly better survival outcome in the TCGA TNBC cohort (log-rank test, p = 0.05). Combined analysis of DNA sequencing, DNA methylation, and RNA sequencing identified tumors of a distinct BRCA-deficient (BRCA-D) TNBC subtype characterized by low levels of wild-type BRCA1/2 expression. Patients with functionally BRCA-D tumors had significantly better survival with standard-of-care chemotherapy than patients whose tumors were not BRCA-D (log-rank test, p = 0.021), and they had significantly higher mutation burden (p < 0.001) and presented clonal neoantigens that were associated with increased immune cell activity. A transcriptional signature of BRCA-D TNBC tumors was independently validated to be significantly associated with improved survival in the METABRIC dataset (log-rank test, p = 0.009). As a retrospective study, limitations include the small size and potential selection bias in the discovery cohort. Conclusions The comprehensive molecular analysis presented in this study directly links BRCA deficiency with increased clonal mutation burden and significantly enhanced chemosensitivity in TNBC and suggests that functional RNA-based BRCA deficiency needs to be further examined in TNBC.

Global Gene Expression Changes Induced By Prolonged Cold Ischemic Stress and Preservation Method of Breast Cancer Tissue

Tissue handling can alter global gene expression potentially affecting the analytical performance of genomic signatures, but such effects have not been systematically evaluated.

Statistical measures of transcriptional diversity capture genomic heterogeneity of cancer

BackgroundMolecular heterogeneity of tumors suggests the presence of multiple different subclones that may limit response to targeted therapies and contribute to acquisition of drug resistance, but its quantification has remained challenging.ResultsWe performed simulations to evaluate statistical measures that best capture the molecular diversity within a group of tumors for either continuous (gene expression) or discrete (mutations, copy number alterations) molecular data. Dispersion based metrics in the principal component space best captured the underlying heterogeneity. To demonstrate utility of these measures, we characterized the diversity in transcriptional and genomic profiles of different breast tumor subtypes, and showed that basal-like or triple-negative breast cancers (TNBC) are significantly more heterogeneous molecularly than other subtypes. Our analysis also suggests that transcriptional diversity is a global characteristic of the tumors observed across the majority of molecular pathways. Among basal-like tumors, those that were resistant to multi-agent chemotherapy showed greater transcriptional diversity compared to chemotherapy-sensitive tumors, suggesting that potentially multiple mechanisms may be contributing to chemotherapy resistance.ConclusionsWe proposed and validated measures of transcriptional and genomic diversity that can quantify the molecular diversity of tumors. We applied the new measures to genomic data from breast tumors and demonstrated that basal-like breast cancers are significantly more diverse than other breast cancers. The observation that chemo-resistant tumors are significantly more diverse molecularly than chemosensitive tumors implies that multiple resistance mechanisms may be active, thus limiting the sensitivity and accuracy of predictive markers of chemotherapy response.

Systematic drug screening identifies tractable targeted combination therapies in triple-negative breast cancer

Triple-negative breast cancer (TNBC) remains an aggressive disease without effective targeted therapies. In this study, we addressed this challenge by testing 128 FDA-approved or investigational drugs as either single agents or in 768 pairwise drug combinations in TNBC cell lines to identify synergistic combinations tractable to clinical translation. Medium-throughput results were scrutinized and extensively analyzed for sensitivity patterns, synergy, anticancer activity, and were validated in low-throughput experiments. Principal component analysis revealed that a fraction of all upregulated or downregulated genes of a particular targeted pathway could partly explain cell sensitivity toward agents targeting that pathway. Combination therapies deemed immediately tractable to translation included ABT-263/crizotinib, ABT-263/paclitaxel, paclitaxel/JQ1, ABT-263/XL-184, and paclitaxel/nutlin-3, all of which exhibited synergistic antiproliferative and apoptotic activity in multiple TNBC backgrounds. Mechanistic investigations of the ABT-263/crizotinib combination offering a potentially rapid path to clinic demonstrated RTK blockade, inhibition of mitogenic signaling, and proapoptotic signal induction in basal and mesenchymal stem-like TNBC. Our findings provide preclinical proof of concept for several combination treatments of TNBC, which offer near-term prospects for clinical translation. Cancer Res; 77(2); 566-78. ©2016 AACR.

Convergent and Divergent Cellular Responses by ErbB4 Isoforms in Mammary Epithelial Cells

Associations of ErbB4 (ERBB4/HER4), the fourth member of the EGFR family, with cancer are variable, possibly as a result of structural diversity of this receptor. There are multiple structural isoforms of ERBB4 arising by alternative mRNA splicing, and a subset undergo proteolysis that releases membrane-anchored and soluble isoforms that associate with transcription factors and coregulators to modulate transcription. To compare the differential and common signaling activities of full-length (FL) and soluble intracellular isoforms of ERBB4, four JM-a isoforms (FL and soluble intracellular domain (ICD) CYT-1 and CYT-2) were expressed in isogenic MCF10A cells and their biologic activities were analyzed. Both FL and ICD CYT-2 promoted cell proliferation and invasion, and CYT-1 suppressed cell growth. Transcriptional profiling revealed several new and underexplored ERBB4-regulated transcripts, including: proteases/protease inhibitors (MMP3 and SERPINE2), the YAP/Hippo pathway (CTGF, CYR61, and SPARC), the mevalonate/cholesterol pathway (HMGCR, HMGCS1, LDLR, and DHCR7), and cytokines (IL8, CCL20, and CXCL1). Many of these transcripts were subsequently validated in a luminal breast cancer cell line that normally expresses ERBB4. Furthermore, ChIP-seq experiments identified ADAP1, APOE, SPARC, STMN1, and MXD1 as novel molecular targets of ERBB4. These findings clarify the diverse biologic activities of ERBB4 isoforms, and reveal new and divergent functions. Implications: ErbB4 as a regulator of Hippo and mevalonate pathways provides new insight into milk production and anabolic processes in normal mammary epithelia and cancer. Mol Cancer Res; 12(8); 1140–55. ©2014 AACR.

Overexpression of ERBB4 JM-a CYT-1 and CYT-2 isoforms in transgenic mice reveals isoform-specific roles in mammary gland development and carcinogenesis

IntroductionHuman Epidermal Growth Factor Receptor (ERBB4/HER4) belongs to the Epidermal Growth Factor receptor/ERBB family of receptor tyrosine kinases. While ERBB1, ERBB2 and ERBB3 are often overexpressed or activated in breast cancer, and are oncogenic, the role of ERBB4 in breast cancer is uncertain. Some studies suggest a tumor suppressor role of ERBB4, while other reports suggest an oncogenic potential. Alternative splicing of ERBB4 yields four major protein products, these spliced isoforms differ in the extracellular juxtamembrane domain (JM-a versus JM-b) and cytoplasmic domain (CYT-1 versus CYT-2). Two of these isoforms, JM-a CYT-1 and JM-a CYT-2, are expressed in the mammary gland. Failure to account for isoform-specific functions in previous studies may account for conflicting reports on the role of ERBB4 in breast cancer.MethodsWe have produced mouse mammary tumour virus (MMTV) -ERBB4 transgenic mice to evaluate potential developmental and carcinogenic changes associated with full length (FL) JM-a ERBB4 CYT-1 versus ERBB4 CYT-2. Mammary tissue was isolated from transgenic mice and sibling controls at various developmental stages for whole mount analysis, RNA extraction, and immunohistochemistry. To maintain maximal ERBB4 expression, transgenic mice were bred continuously for a year after which mammary glands were isolated and analyzed.ResultsOverexpressing FL CYT-1 isoform resulted in suppression of mammary ductal morphogenesis which was accompanied by decreased number of mammary terminal end buds (TEBs) and Ki-67 positive cells within TEBs, while FL CYT-2 isoform had no effect on ductal growth in pubescent mice. The suppressive ductal phenotype in CYT-1 mice disappeared after mid-pregnancy, and subsequent developmental stages showed no abnormality in mammary gland morphology or function in CYT-1 or CYT-2 transgenic mice. However, sustained expression of FL CYT-1 isoform resulted in formation of neoplastic mammary lesions, suggesting a potential oncogenic function for this isoform.ConclusionsTogether, we present isoform-specific roles of ERBB4 during puberty and early pregnancy, and reveal a novel oncogenic property of CYT-1 ERBB4. The results may be exploited to develop better therapeutic strategies in breast cancer.

Mitotic read-out genes confer poor outcome in luminal A breast cancer tumors

Luminal breast tumors have been classified into A and B subgroups, with the luminal A being associated with a more favorable clinical outcome. Unfortunately, luminal A tumors do not have a universally good prognosis. We used transcriptomic analyses using public datasets to evaluate the differential expression between normal breast tissue and breast cancer, focusing on upregulated genes included in cell cycle function. Association of selected genes with relapse free survival (RFS) and overall survival (OS) was performed using the KM Plotter Online Tool (http://www.kmplot.com). Seventy-seven genes were differentially expressed between normal and malignant breast tissue. Only five genes were associated with poor RFS and OS. The mitosis-related genes GTSE1, CDCA3, FAM83D and SMC4 were associated with poor outcome specifically in Luminal A tumors. The combination of FAM83D and CDCA3 for RFS and GTSE1 alone for OS showed the better prediction for clinical outcome. CDCA3 was amplified in 3.4% of the tumors, and FAM83D and SMC4 in 2.3% and 2.2%, respectively. In conclusion, we describe a set of genes that predict detrimental outcome in Luminal A tumors. These genes may have utility for stratification in trials of antimitotic agents or cytotoxic chemotherapy, or as candidates for direct target inhibition.

Metabolic isoenzyme shifts in cancer as potential novel therapeutic targets

The functional redundancy of metabolic enzyme expression may present a new strategy for developing targeted therapies in cancer. To satisfy the increased metabolic demand required during neoplastic transformations and proliferation, cancer cells may rely on additional isoforms of a metabolic enzyme to satisfy the increased demand for metabolic precursors, which could subsequently render cancer cells more vulnerable to isoform-specific inhibitors. In this review, we provide a survey of common isoenzyme shifts that have been reported to be important in cancer metabolism and link those to metabolic pathways that currently have drugs in various stages of development. This phenomenon suggests a potentially new therapeutic strategy for the treatment of cancer by identifying shifts in the expression of metabolic isoenzymes between cancer and normal cells. We also delineate other putative metabolic isoenzymes that could be targets for novel targeted therapies for cancer. Changes in isoenzyme expression that occur during neoplastic transformations or in response to environmental pressure in cancer cells may result in isoenzyme diversity that may subsequently render cancer cells more vulnerable to isoform-specific inhibitors due to reliance on a single isoform to perform a vital enzymatic function.

Characterization of DNA variants in the human kinome in breast cancer

Kinases play a key role in cancer biology, and serve as potential clinically useful targets for designing cancer therapies. We examined nucleic acid variations in the human kinome and several known cancer-related genes in breast cancer. DNA was extracted from fine needle biopsies of 73 primary breast cancers and 19 metastatic lesions. Targeted sequencing of 518 kinases and 68 additional cancer related genes was performed using the SOLiD sequencing platform. We detected 1561 unique, non-synonymous variants in kinase genes in the 92 cases, and 74 unique variants in 43 kinases that were predicted to have major functional impact on the protein. Three kinase groups—CMGC, STE and TKL—showed greater mutational load in metastatic compared to primary cancer samples, however, after correction for multiple testing the difference was significant only for the TKL group (P = 0.04). We also observed that a higher proportion of histologic grade 1 and 2 cases had high functional impact variants in the SCYL2 gene compared with grade 3 cases. Our findings indicate that individual breast cancers harbor a substantial number of potentially functionally important nucleotide variations in kinase genes, most of which are present in unique combinations and include both somatic and germline functional variants.