Hanan Elsarraj

Expression profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion

IntroductionThere are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized.MethodsMicroarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers.ResultsAnalysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification.ConclusionBCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.

A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance

Summary In this report, we have shown that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. MiR146b expression was significantly higher in the mammary glands of pregnant and lactating mice than in virgin mice. Furthermore, miR146b levels were significantly higher in mouse mammary glands exposed to the sex hormones, estrogen and progesterone, compared with those of untreated control animals. Pregnancy-derived primary mouse mammary epithelial cells in which miR146b was knocked down showed a significant reduction in the number of hollow acinar organoid structures formed on three-dimensional Matrigel and in &bgr;-casein expression. This demonstrates that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. It has been shown that mouse mammary luminal progenitors give rise to hollow organoid structures, whereas solid organoid structures are derived from stem cells. Among several miR146b targets, miR146b knockdown resulted in preferential STAT3&bgr; overexpression. In the primary mouse mammary epithelial cells, overexpression of STAT3&bgr; isoform caused mammary epithelial cell death and a significant reduction in &bgr;-casein mRNA expression. Therefore, we conclude that during pregnancy miR146b is involved in luminal alveolar progenitor cell maintenance, at least partially, by regulating STAT3&bgr;.

Emerging Functions of microRNA-146a/b in Development and Breast Cancer

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression through translational repression or mRNA degradation. These molecules play critical roles in regulating normal developmental processes, but when deregulated, are causally linked to the pathogenesis of numerous diseases, including cancer. MicroRNA-146a and -146b are encoded by two different genes, but differ by only two bases and appear to function redundantly in many systems. Initial studies branded miR-146a/b as important mediators of inflammatory signaling, documenting the ability of these miRNAs to influence differentiation, proliferation, apoptosis and effector immune mechanisms within the hematopoietic system. Numerous contemporary studies now implicate miR-146a/b as pleiotropic regulators of tumorigenesis, as a polymorphism in miR-146a and altered expression of both miR-146a/b have been linked with cancer risk, tumor histogenesis and invasive and metastatic capacity in diverse cancers. Despite the numerous reports concerning miR-146a/b in human cancers, the mechanistic contributions of these miRNAs in both normal and neoplastic mammary gland development and biology remains poorly characterized.

NEMO, a transcriptional target of estrogen and progesterone, is linked to tumor suppressor PML in breast cancer

The beneficial versus detrimental roles of estrogen plus progesterone (E+P) in breast cancer remains controversial. Here we report a beneficial mechanism of E+P treatment in breast cancer cells driven by transcriptional upregulation of the NFκB modulator NEMO, which in turn promotes expression of the tumor suppressor protein promyelocytic leukemia (PML). E+P treatment of patient-derived epithelial cells derived from ductal carcinoma in situ (DCIS) increased secretion of the proinflammatory cytokine IL6. Mechanistic investigations indicated that IL6 upregulation occurred as a result of transcriptional upregulation of NEMO, the gene that harbored estrogen receptor (ER) binding sites within its promoter. Accordingly, E+P treatment of breast cancer cells increased ER binding to the NEMO promoter, thereby increasing NEMO expression, NFκB activation, and IL6 secretion. In two mouse xenograft models of DCIS, we found that RNAi-mediated silencing of NEMO increased tumor invasion and progression. This seemingly paradoxical result was linked to NEMO-mediated regulation of NFκB and IL6 secretion, increased phosphorylation of STAT3 on Ser727, and increased expression of PML, a STAT3 transcriptional target. In identifying NEMO as a pivotal transcriptional target of E+P signaling in breast cancer cells, our work offers a mechanistic explanation for the paradoxical antitumorigenic roles of E+P in breast cancer by showing how it upregulates the tumor suppressor protein PML. Cancer Res; 77(14); 3802-13. ©2017 AACR.

Abstract B01: B cell lymphoma 9 mediates a cross talk between the canonical Wnt and EGFR signaling in breast cancer

Human ductal carcinoma in situ (DCIS) are the most common type of non-invasive breast cancers. The five-year survival rate for women diagnosed with non-invasive DCIS is 98% while the five-year survival plummets to 83-27% for breast cancers that have become invasive and have spread to distant sites [also referred to as invasive ductal carcinoma (IDC)]. To study DCIS pathobiology and factors that promote their transition to IDC, we have developed a novel in vivo DCIS model, MIND (mouse-intraductal), that involves intraductal injection of epithelial cells derived from primary human DCIS biopsy and surgical samples thus mimicking the entire process of DCIS to IDC transition. As a complementary approach, we have utilized human DCIS/IDC tandem lesions, which are patient DCIS that show a transition to IDC within the same breast. Analysis of RNA and protein at distinct stages of in situ to IDC using both models showed B cell lymphoma-9 (BCL9) up-regulation to be associated with DCIS transition to IDC. BCL9 is a recently identified co-activator of Wnt-stimulated beta-catenin-mediated transcription. Our studies showed that in vivo silencing of BCL9 led to inhibition of DCIS invasion and reversal of EMT. We have also demonstrated a direct binding interaction between BCL9 and beta-catenin and showed suppression of beta-catenin-mediated transcription by BCL9 knockdown. Analysis of patient DCIS samples revealed a significant correlation between high nuclear BCL9 expression and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) negative and Ki67. Furthermore, analysis of the TCGA data showed BCL9 gene to be upregulated in 26% of breast cancers. This is a significant gene alteration when compared to HER2 (ERBB2) gene (19%) and estrogen receptor (ESR1) gene (8%) alterations in breast cancers. Interestingly, a significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification suggesting that BCL9 may predispose to the development of basal breast cancers. We have performed an RPPA analysis on our DCIS cell lines KD BCL9 vs. control. This analysis indicated that BCL9 KD showed down-regulation in a number of genes in the EGFR signaling pathway including p-EGFR, p-HER2, p-STAT3, and p-Src. Conclusion: BCL9 is a molecular driver of DCIS invasive progression. The molecular mechanism for BCL99s role in breast cancer progression is through the enhancement in the canonical Wnt and EGFR signaling. Citation Format: Hanan Elsarraj, Hong Yan, Jennifer Knapp, Anna Tsimelzon, Shixia Huang, Andrew Godwin, Sue Hilsenbeck, Dean Edwards, Fariba Behbod. B cell lymphoma 9 mediates a cross talk between the canonical Wnt and EGFR signaling in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B01.

Abstract P2-05-05: Expression profiling of in vivo DCIS progression models identified BCL9 as a molecular driver of invasive progression

Introduction: There are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. At present, it is not clear why some DCIS remain non-invasive for decades while others become invasive. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of DCIS patients. To identify factors that promote DCIS invasion, we have profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using two in vivo models, MIND (mouse-intraductal) and DCIS/IDC tandem lesions. These studies led to the identification of B cell lymphoma-9 as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However its role in breast cancer progression had not been recognized. Methods: Microarray and RNA sequencing were utilized to characterize the sequential and temporal changes in mRNA expression during DCIS invasive transition. BCL9 shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, EMT and canonical Wnt signaling. Immunofluorescence of 28 patient DCIS samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. TCGA data was analyzed to assess the status of BCL9 gene alterations in 959 human breast cancers. Results: Analysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: ER and PR negative, high nuclear grade, and high HER2. In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of epithelial-mesenchymal transition (EMT). Analysis of the TCGA data showed BCL9 gene to be altered in 26% of breast cancers. This is a significant alteration when compared to ERBB2 (19%) and ESR1 (8%). A significantly higher proportion of basal like invasive breast cancers showed BCL9 amplification. Conclusion: BCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC. Citation Format: Behbod F, Elsarraj H, Hong Y, Valdez K, Chien J, Godwin A, Fields T. Expression profiling of in vivo DCIS progression models identified BCL9 as a molecular driver of invasive progression. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-05.

Breast Cancer Invasion and Metastasis

Breast cancer is the most common malignancy among western women, and 10–15 % of all breast cancer patients develop and ultimately succumb to metastatic disease. In breast cancer, malignant cells disseminate through lymphatic or hematogenous routes to distant organs. Over the last decades, the 5-year survival of breast cancer has increased due to early screening and advanced local and systemic treatments. Understanding the fundamental biology underlying the progression of breast cancer has fostered the identification and development of therapeutics. In this chapter, we discuss the morphologic and molecular heterogeneity of breast cancer and the relationship between breast cancer subtype and metastatic potential. Moreover, we detail different in vitro assays which provide simple and robust systems to study basic cellular processes that are critical to orchestrating metastatic progression of breast cancer. Lastly, we address the strengths and shortcomings of different in vivo models that allow integrated analysis of heterotypic signaling and tissue architecture in breast cancer progression.