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Inactivation of p53 contributes significantly to the dismal prognosis of breast tumors, most notably triple-negative breast cancers (TNBCs). How the relief from p53 tumor suppressive functions results in tumor cell aggressive behavior is only partially elucidated. In an attempt to shed light on the implication of microRNAs in this context, we discovered a new signaling axis involving p53, miR-30a and ZEB2. By an in silico approach we identified miR-30a as a putative p53 target and observed that in breast tumors reduced miR-30a expression correlated with p53 inactivation, lymph node positivity and poor prognosis. We demonstrate that p53 binds the MIR30A promoter and induces the transcription of both miRNA strands 5p and 3p. Both miR-30a-5p and -3p showed the capacity of targeting ZEB2, a transcription factor involved in epithelial-mesenchymal transition (EMT), tumor cell migration and drug resistance. Intriguingly, we found that p53 does restrain ZEB2 expression via miR-30a. Finally, we provide evidence that the new p53/miR-30a/ZEB2 axis controls tumor cell invasion and distal spreading and impinges upon miR-200c expression. Overall, this study highlights the existence of a novel axis linking p53 to EMT via miR-30a, and adds support to the notion that miRNAs represent key elements of the complex network whereby p53 inactivation affects TNBC clinical behavior.

Loss of expression of miR-200 family members has been implicated in cellular plasticity, a phenomenon that accounts for epithelial-to-mesenchymal transition (EMT) and stem-like features of many carcinomas and is considered a major cause of tumor aggressiveness and drug resistance. Nevertheless, the mechanisms of miR-200 downregulation in breast cancer are still largely unknown. Here we show that miR-200c expression inversely correlates with miR-200c/miR-141 locus methylation in triple-negative breast tumors (TNBC). Importantly, low levels of miR-200c expression and high levels of miR-200c/miR-141 locus methylation associated with lymph node metastasis. Moreover, miR-200c/miR-141 locus methylation was significantly related to high expression of ZEB1 in two independent TNBC series. Silencing of ZEB1 in vitro reduced miR-200c/miR-141 DNA methylation and, concurrently, decreased histone H3K9 trimethylation. This chromatin modifications were paralleled by an increase in the expression of both miR-200c and E-cadherin. Similar effects were achieved by treatment with a demethylating agent. Our data suggest that gene methylation is an important element in the regulation of the miR-200c/ZEB1 axis and that chromatin remodeling of the miR-200c/miR-141 locus is affected by ZEB1 and, thus, contributes to ZEB1-induced cellular plasticity. © 2016 Wiley Periodicals, Inc.

Since publication of the article, the authors were notified by ATCC that the cell line HCC1395 (ATCC® CRL-2324™ Lot 62235652) suffered a “low level of cell line cross-contamination” with another cell line.

Inactivation of p53 plays a major role in the poor prognosis of basal-like /triple-negative breast cancers (TNBC). However, how p53 inactivation impinges upon TNBC aggressive phenotype is only partially defined. Here we report the identification of a novel axis involving p53, miR30a and ZEB2. We provide evidence that p53 affects the expression of ZEB2, a transcription factor involved in epithelial-mesenchymal transition (EMT), by relying on miR-30a. In particular, we found that p53 regulates miR-30a expression by direct promoter binding and that miR30a targets ZEB2. Finally, we provide evidence that the new p53/miR-30a/ZEB2 axis controls tumor cell invasion and migration and that reduced miR-30a expression correlates with p53 inactivation and dismal prognosis in human breast cancers. Overall, this study highlights the existence of a novel axis linking p53 to EMT via miR-30a, and adds a novel element to the complex network whereby p53 inactivation impinge upon tumor aggressiveness. Citation Format: Valentina Damiano, Alessandra di Gennaro, Giulia Brisotto, Michela Armellin, Tiziana Perin, Antonella Zucchetto, Michela Guardascione, Herman P. Spaink, Claudio Doglioni, Ewa Snaar-Jagalska, Manuela Santarosa, Roberta Maestro. A p53/miR-30a/ZEB2 axis controls basal-like/triple-negative breast cancer aggressiveness [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 500.

Background DNA methylation is an important epigenetic mechanism of transcriptional control that plays an essential role in several cellular functions. Aberrant DNA methylation in cancer has been frequently associated with downregulation of microRNAs and protein coding genes, such as miR-200c/miR-141 cluster and E-cadherin. Current strategies to assess DNA methylation, including bisulfite treatment-based assays, tend to be time-consuming and may be quite expensive when a precise appraisal is required. The Sanger-sequencing of the amplified bisulfite-treated DNA (BSP) might represent a practical option to measure DNA methylation at single CpG resolution. However, this strategy often produces noisy data, which affects accurate quantification. Here we propose an improved, reliable and cost-effective BSP-based protocol that allows proper DNA methylation assessment. Methods Our strategy, named normalized-BSP (NBSP), takes advantage of tailed C-balanced primers and a normalization procedure based on C/T ratio to overcome BSP-associated noise problems and nucleotide signal unbalance. NBSP was applied to estimate miR-200c/miR-141 locus methylation in serial dilution experiments and was compared to conventional methods. Besides, it was applied in the analysis of FFPE breast cancer samples and further validated in the context of the E-cadherin promoter. Results NBSP strategy outperformed conventional BSP in the estimate of the fraction of methylated cytosine in serial dilution experiments, providing data in agreement with the widely used but cumbersome cloning-based protocol. This held true for both miR-200c/miR-141 locus and E-cadherin promoter analyses. Moreover, the miR-200c/miR-141 locus methylation reflected the decrease in miRNA expression both in breast cancer cell lines and in the FFPE samples. Conclusions NBSP is a rapid and economical method to estimate the extent of methylation at each CpG of a given locus. Notably, NBSP works efficiently on FFPE samples, thus disclosing the perspective of its application also in the diagnostic setting. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1646-6) contains supplementary material, which is available to authorized users.