The Autophagy Machinery Contributes to E-cadherin Turnover in Breast Cancer.

Autophagy is an intracellular catabolic process that is increasingly being recognized as a crucial factor in several human diseases including cancers. Mounting evidence suggests that autophagy allows tumor cells to overcome otherwise fatal stresses and to increase dissemination. Nevertheless, how autophagy controls these processes and in particular how it impinges on cell-cell adhesion is still poorly understood. Here, we investigate the role of autophagy in the turnover of the epithelial adhesion molecule E-cadherin in the context of breast cancer. We demonstrated in breast cancer cell lines that autophagy impinges on E-cadherin expression and in the configuration of adherens junctions. Besides, we showed that E-cadherin colocalizes with LC3B and SQSTM1/p62, two components of the autophagosome machinery. Pull down and immunoprecipitation analyses provided evidence that E-cadherin and SQSTM1/p62 physically interact. Moreover, the physical closeness of E-cadherin and SQSTM1/p62 was demonstrated by proximity ligation assays in breast cancer cell lines and primary tumors. Finally, we proved that the silencing of SQSTM1/p62 diminished the E-cadherin/LC3B colocalization, further supporting the role of SQSTM1/p62 in E-cadherin delivery to autophagosomes. These findings suggest that the activation of autophagy, reported in breast cancers with poor prognosis and in dormant breast cancer cells, may contribute to the control of tumor progression via downmodulation of E-cadherin protein levels.

Correction to: A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness.

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.

A p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness.

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.

Epigenetic silencing of miR-200c in breast cancer is associated with aggressiveness and is modulated by ZEB1.

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.

An improved sequencing-based strategy to estimate locus-specific DNA methylation.

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.

Intracellular inactivation of thyroid hormone is a survival mechanism for muscle stem cell proliferation and lineage progression.

Precise control of the thyroid hormone (T3)-dependent transcriptional program is required by multiple cell systems, including muscle stem cells. Deciphering how this is achieved and how the T3 signal is controlled in stem cell niches is essentially unknown. We report that in response to proliferative stimuli such as acute skeletal muscle injury, type 3 deiodinase (D3), the thyroid hormone-inactivating enzyme, is induced in satellite cells where it reduces intracellular thyroid signaling. Satellite cell-specific genetic ablation of dio3 severely impairs skeletal muscle regeneration. This impairment is due to massive satellite cell apoptosis caused by exposure of activated satellite cells to the circulating TH. The execution of this proapoptotic program requires an intact FoxO3/MyoD axis, both genes positively regulated by intracellular TH. Thus, D3 is dynamically exploited in vivo to chronically attenuate TH signaling under basal conditions while also being available to acutely increase gene programs required for satellite cell lineage progression.

Epigenetic control of type 2 and 3 deiodinases in myogenesis: role of Lysine-specific Demethylase enzyme and FoxO3.

The proliferation and differentiation of muscle precursor cells require myogenic regulatory factors and chromatin modifiers whose concerted action dynamically regulates access to DNA and allows reprogramming of cells towards terminal differentiation. Type 2 deiodinase (D2), the thyroid hormone (TH)-activating enzyme, is sharply upregulated during myoblast differentiation, whereas type 3 deiodinase (D3), the TH-inactivating enzyme, is downregulated. The molecular determinants controlling synchronized D2 and D3 expression in muscle differentiation are completely unknown. Here, we report that the histone H3 demethylating enzyme (LSD-1) is essential for transcriptional induction of D2 and repression of D3. LSD-1 relieves the repressive marks (H3-K9me2-3) on the Dio2 promoter and the activation marks (H3-K4me2-3) on the Dio3 promoter. LSD-1 silencing impairs the D2 surge in skeletal muscle differentiation while inducing D3 expression thereby leading to a global decrease in intracellular TH production. Furthermore, endogenous LSD-1 interacts with FoxO3a, and abrogation of FoxO3-DNA binding compromises the ability of LSD-1 to induce D2. Our data reveal a novel epigenetic control of reciprocal deiodinases expression and provide a molecular mechanism by which LSD-1, through the opposite regulation of D2 and D3 expression, acts as a molecular switch that dynamically finely tunes the cellular needs of active TH during myogenesis.