Identifying founder mutations in BRCA1 and BRCA2 in specific populations constitute a valuable opportunity for genetic screening. Several studies from different populations have reported recurrent and/or founder mutations representing a relevant proportion of BRCA mutation carriers. In Latin America, only few founder mutations have been described. We screened 453 Chilean patients with hereditary breast cancer for mutations in BRCA1 and BRCA2. For recurrent mutations, we genotyped 11 microsatellite markers in BRCA1 and BRCA2 in order to determine a founder effect through haplotype analysis. We found a total of 25 mutations (6 novel) in 71 index patients among which, nine are present exclusively in Chilean patients. Our analysis revealed the presence of nine founder mutations, 4 in BRCA1 and 5 in BRCA2, shared by 2 to 10 unrelated families and spread in different regions of Chile. Our panel contains the highest amount of founder mutations until today and represents the highest percentage (78%) of BRCA1 and BRCA2 mutation carriers. We suggest that the dramatic reduction of Amerindian population due to smallpox and wars with Spanish conquerors, a scarce population increase during 300 years, and the geographic position of Chile constituted a favorable scenario to establish founder genetic markers in our population.

Both miRNAs are potential biomarkers for improved overall survival in patients with BRCA1-deficient TNBC tumors.

Metastasis, the leading cause of cancer-associated deaths, is promoted by transcription factors SNAIL, SLUG, ZEB1 and TWIST through the activation of epithelial- mesenchymal transition (EMT). MicroRNAs can suppress EMT, emerging as candidate molecular biomarkers and novel therapeutic targets. Herein, we evaluated microRNAs downregulated in breast cancer tissues expressing EMT transcription factors, to find new potential regulators of EMT. MiR-30a, miR-1271, miR-196a, miR-202, miR-210, miR-22, miR-331 and miR-34b were validated. Seven microRNAs downregulated luciferase activity through EMT transcription factors 3′UTR, and all microRNAs decreased cell migration, invasion and/or proliferation. In MDA-MB-231 cells, miR-196a and miR-22 decreased endogenous ZEB1 levels, and miR-30a endogenous CCR7 levels. These results suggest that microRNAs studied are novel regulators of EMT through the control of SNAIL, SLUG, ZEB1 and TWIST. They also regulate the metastatic behavior of cancer cells, and may control the development of lymph node metastasis through the regulation of CCR7.

Metastases derived from primary tumors are responsible for the high cancer-associated death rates. Lymph nodes near to the primary breast tumor have a high chance to develop a secondary tumor, representing one of the first signs of metastasis. For this reason, inhibition of metastasis should be a major goal for breast cancer treatment. Metastasis is a non-random process that initiates with a phenotypic change of the tumor cells, named Epithelial-Mesenchymal Transition (EMT), and is leaded by the transcription factors SNAIL, SLUG, ZEB and TWIST. Because these changes in tumor cells must be plastic and reversible, epigenetic alterations are necessary for EMT. In this sense, microRNAs have emerged as candidate molecular biomarkers and novel therapeutic targets associated to metastasis. The aim of this study is to identify microRNAs that regulate the expression of EMT-transcription factors in breast cancer tumors, and that are involved in lymph node metastasis. For this purpose, we used microRNA microarray data from 50 fresh frozen breast tumors, 28 from patients with lymph node metastasis. Transcription factor expression was assayed by immunohistochemistry. Microarray data analysis using RankProd (R package) revealed approximately 40 microRNAs down-regulated in breast tumors expressing EMT-transcription factors (p<0.05). Using in silico analysis, we predicted 28 microRNAs to be regulators of SNAIL, SLUG and/or TWIST. Four not previously validated microRNAs were selected: miR-202, miR-210, miR-331 and miR-34b. We used luciferase reporter assays to assess the regulation of the 3´UTR of selected transcription factors by each microRNA, at two different concentrations: 10nM and 50nM. We found that miR-210 caused a 50% decrease in luciferase activity through SNAIL and SLUG 3'UTR when tested at 50nM. miR-331, predicted as regulator of SLUG 3'UTR, decreased luciferase activity at both evaluated concentrations in a dose-dependent manner, in a 20% and a 37%. In the case of TWIST, miR-34b at 50nM, decreased luciferase activity in a 65%. No inhibition of luciferase activity was found with mir202. In addition to transcription factors, two of these microRNAs have been predicted as regulators of chemokine receptors CXCR4 and CCR7. Both receptors direct lymphocyte migration to lymphoid organs, and their role has been also implicated in cancer. Our results suggest that down regulation of specific microRNAs, and up regulation of their targets, may induce a metastatic behavior of primary breast tumor cells, promoting invasion and colonization of lymph nodes. In this sense, changes in the expression of microRNAs may serve as biomarkers of breast cancer prognosis in patients. Citation Format: Elisa Pérez-Moreno, Valentina Zavala, Gabriela Valarezo, Wanda Fernández, Pilar Carvallo. microRNAs targeting EMT transcription factors in breast cancer and their relation to lymph node metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2013.

Breast cancer is the leading cause of cancer-associated deaths in women. Lymph node near to the primary breast tumor have a high chance of developing a secondary tumor, representing one of the first signs of metastasis in breast cancer. Metastasis is promoted by epithelial-mesenchymal transition (EMT), process leaded by the transcription factors SNAIL, SLUG, ZEB and TWIST. MicroRNAs are small non-coding RNAs, whose expression has been demonstrated to be altered in different cancer types. Because of their ability to regulate large sets of genes involved in cancer growth and metastasis, microRNAs have emerged as candidate molecular biomarkers and novel therapeutic targets. The aim of this study is to identify microRNAs differentially expressed in breast tumors in relation to EMT-transcription factor expression and lymph node metastasis, and that are involved in epithelial-mesenchymal transition. For this purpose, we used microRNA microarray data from 50 fresh frozen breast tumors with different tumor grades (1 to 3), 28 from patients with lymph node metastasis. Microarray data analysis using RankProd (R package) revealed approximately 40 microRNAS down-regulated in breast tumors with expression of EMT-transcription factors (p<0.05). Several of the identified microRNAs have been previously described to regulate early steps of metastasis in different cancer types, like members of the miR-200 family (miR-200c and miR-141), miR-205 and miR-30a. In addition we found other microRNAs not previously associated to metastasis. Our results suggest that down regulation of specific microRNAs, and up regulation of their targets, may induce a metastatic behavior of tumor cells of the primary breast tumors, promoting invasion and colonization of the lymph nodes. In this sense, the changes in the expression of microRNAs may serve as biomarkers of breast cancer prognosis in patients. Citation Format: Elisa Pérez-Moreno, Gabriela Valarezo, Valentina Zavala, Wanda Fernández, Pilar Carvallo. microRNAs targeting EMT transcription factors may be associated to lymph node metastasis in breast cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5810. doi:10.1158/1538-7445.AM2017-5810