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1.
Oncogene ; 36(40): 5551-5566, 2017 10 05.
Article in English | MEDLINE | ID: mdl-28581528

ABSTRACT

Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.


Subject(s)
Cancer-Associated Fibroblasts/metabolism , Cell Transformation, Neoplastic , DNA Methylation , Epithelial Cells/pathology , Mesoderm/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Culture Media, Conditioned , Cytosine/metabolism , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA, Neoplasm/metabolism , Gene Expression Regulation, Neoplastic , Humans , Male , Stem Cells/pathology , Transcriptional Activation
2.
Cell Death Dis ; 7: e2108, 2016 Feb 25.
Article in English | MEDLINE | ID: mdl-26913599

ABSTRACT

Immunogenic cell death (ICD) evoked by chemotherapeutic agents implies emission of selected damage-associated molecular patterns (DAMP) such as cell surface exposure of calreticulin, secretion of ATP and HMGB1. We sought to verify whether miR-27a is implicated in ICD, having demonstrated that it directly targets calreticulin. To this goal, we exposed colorectal cancer cell lines, genetically modified to express high or low miR-27a levels, to two bona fide ICD inducers (mitoxantrone and oxaliplatin). Low miR-27a-expressing cells displayed more ecto-calreticulin on the cell surface and increased ATP and HMGB1 secretion than high miR-27a-expressing ones in time-course experiments upon drug exposure. A calreticulin target protector counteracted the miR-27a effects while specific siRNAs mimicked them, confirming the results reported. In addition, miR-27a negatively influenced the PERK-mediated route and the late PI3K-dependent secretory step of the unfolded protein response to endoplasmic reticulum stress, suggesting that miR-27a modulates the entire ICD program. Interestingly, upon chemotherapeutic exposure, low miR-27a levels associated with an earlier and stronger induction of apoptosis and with morphological and molecular features of autophagy. Remarkably, in ex vivo setting, under the same chemotherapeutic induction, the conditioned media from high miR-27a-expressing cells impeded dendritic cell maturation while increased the secretion of specific cytokines (interleukin (IL)-4, IL-6, IL-8) and negatively influenced CD4(+) T-cell interferon γ production and proliferation, all markers of a tumor immunoevasion strategy. In conclusion, we provide the first evidence that miR-27a impairs the cell response to drug-induced ICD through the regulatory axis with calreticulin.


Subject(s)
Apoptosis , Calreticulin/metabolism , MicroRNAs/metabolism , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , Calreticulin/genetics , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Dendritic Cells/cytology , Dendritic Cells/metabolism , Down-Regulation/drug effects , Endoplasmic Reticulum Stress/drug effects , HCT116 Cells , Humans , Interferon-gamma/metabolism , Interleukins/metabolism , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Mitoxantrone/pharmacology , Oligonucleotides, Antisense/metabolism , Organoplatinum Compounds/pharmacology , Oxaliplatin , Phosphatidylinositol 3-Kinases/metabolism , Unfolded Protein Response/drug effects
3.
Cell Death Dis ; 7: e2120, 2016 Feb 25.
Article in English | MEDLINE | ID: mdl-26913609

ABSTRACT

Impairment of the immune response and aberrant expression of microRNAs are emerging hallmarks of tumour initiation/progression, in addition to driver gene mutations and epigenetic modifications. We performed a preliminary survey of independent adenoma and colorectal cancer (CRC) miRnoma data sets and, among the most dysregulated miRNAs, we selected miR-27a and disclosed that it is already upregulated in adenoma and further increases during the evolution to adenocarcinoma. To identify novel genes and pathways regulated by this miRNA, we employed a differential 2DE-DIGE proteome analysis. We showed that miR-27a modulates a group of proteins involved in MHC class I cell surface exposure and, mechanistically, demonstrated that calreticulin is a miR-27a direct target responsible for most downstream effects in epistasis experiments. In vitro miR-27a affected cell proliferation and angiogenesis; mouse xenografts of human CRC cell lines expressing different miR-27a levels confirmed the protein variations and recapitulated the cell growth and apoptosis effects. In vivo miR-27a inversely correlated with MHC class I molecules and calreticulin expression, CD8(+) T cells infiltration and cytotoxic activity (LAMP-1 exposure and perforin release). Tumours with high miR-27a, low calreticulin and CD8(+) T cells' infiltration were associated with distant metastasis and poor prognosis. Our data demonstrate that miR-27a acts as an oncomiRNA, represses MHC class I expression through calreticulin downregulation and affects tumour progression. These results may pave the way for better diagnosis, patient stratification and novel therapeutic approaches.


Subject(s)
Calreticulin/metabolism , MicroRNAs/metabolism , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Apoptosis , Base Sequence , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Calreticulin/chemistry , Calreticulin/genetics , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , Down-Regulation , Female , HCT116 Cells , Histocompatibility Antigens Class I/metabolism , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Neovascularization, Pathologic , Proteomics , RNA Interference , Sequence Alignment , Up-Regulation
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