Dataset Information

MiR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A.

ABSTRACT: BACKGROUND:Development of resistance to inhibitors of BRAF (BRAFi) and MEK (MEKi) remains a great challenge for targeted therapy in patients with BRAF-mutant melanoma. Here, we explored the role of miRNAs in melanoma acquired resistance to BRAFi. METHODS:miRNA expression in two BRAF-mutant melanoma cell lines and their dabrafenib-resistant sublines was determined using Affymetrix GeneChip® miRNA 3.1 microarrays and/or qRT-PCR. The effects of miR-126-3p re-expression on proliferation, apoptosis, cell cycle, ERK1/2 and AKT phosphorylation, dabrafenib sensitivity, invasiveness and VEGF-A secretion were evaluated in the dabrafenib-resistant sublines using MTT assays, flow cytometry, immunoblotting, invasion assays in Boyden chambers and ELISA. ADAM9, PIK3R2, MMP7 and CXCR4 expression in the sensitive and dabrafenib-resistant cells was determined by immunoblotting. Small RNA interference was performed to investigate the consequence of VEGFA or ADAM9 silencing on proliferation, invasiveness or dabrafenib sensitivity of the resistant sublines. Long-term proliferation assays were carried out in dabrafenib-sensitive cells to assess the effects of enforced miR-126-3p expression or ADAM9 silencing on resistance development. VEGF-A serum levels in melanoma patients treated with BRAFi or BRAFi+MEKi were evaluated at baseline (T0), after two months of treatment (T2) and at progression (TP) by ELISA. RESULTS:miR-126-3p was significantly down-regulated in the dabrafenib-resistant sublines as compared with their parental counterparts. miR-126-3p replacement in the drug-resistant cells inhibited proliferation, cell cycle progression, phosphorylation of ERK1/2 and/or AKT, invasiveness, VEGF-A and ADAM9 expression, and increased dabrafenib sensitivity. VEGFA or ADAM9 silencing impaired proliferation and invasiveness of the drug-resistant sublines. ADAM9 knock-down in the resistant cells increased dabrafenib sensitivity, whereas miR-126-3p enforced expression or ADAM9 silencing in the drug-sensitive cells delayed the development of resistance. At T0 and T2, statistically significant differences were observed in VEGF-A serum levels between patients who responded to therapy and patients who did not. In responder patients, a significant increase of VEGF-A levels was observed at TP versus T2. CONCLUSIONS:Strategies restoring miR-126-3p expression or targeting VEGF-A or ADAM9 could restrain growth and metastasis of dabrafenib-resistant melanomas and increase their drug sensitivity. Circulating VEGF-A is a promising biomarker for predicting patients' response to BRAFi or BRAFi+MEKi and for monitoring the onset of resistance.

SUBMITTER: Caporali S

PROVIDER: S-EPMC6588909 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A.

Caporali Simona S Amaro Adriana A Levati Lauretta L Alvino Ester E Lacal Pedro Miguel PM Mastroeni Simona S Ruffini Federica F Bonmassar Laura L Antonini Cappellini Gian Carlo GC Felli Nadia N Carè Alessandra A Pfeffer Ulrich U D'Atri Stefania S

Journal of experimental & clinical cancer research : CR 20190621 1

<h4>Background</h4>Development of resistance to inhibitors of BRAF (BRAFi) and MEK (MEKi) remains a great challenge for targeted therapy in patients with BRAF-mutant melanoma. Here, we explored the role of miRNAs in melanoma acquired resistance to BRAFi.<h4>Methods</h4>miRNA expression in two BRAF-mutant melanoma cell lines and their dabrafenib-resistant sublines was determined using Affymetrix GeneChip® miRNA 3.1 microarrays and/or qRT-PCR. The effects of miR-126-3p re-expression on proliferati ...[more]

PMID: 31227006

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Project description:BackgroundSorafenib can prolong the survival of patients with advanced hepatocellular carcinoma (HCC). However, drug resistance remains the main obstacle to improving its efficiency. This study aimed to explore the likely molecular mechanism of sorafenib resistance.MethodsDifferentially expressed microRNAs (miRNAs) related to sorafenib response were analyzed with the Limma package in R software. The expression levels of miR-126-3p and sprouty-related EVH1 domain-containing protein 1 (SPRED1) in HCC cells were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell viability and proliferation were detected with Cell Counting Kit-8 (CCK-8), EdU proliferation, and clone formation assays. Transwell assays were performed to measure cell migration and invasion. TargetScan, MicroRNA Target Prediction Database (miRDB), and StarBase v2.0 were used to predict the targets of miR-126-3p. SPRED1 was confirmed as a target gene of miR-126-3p by dual-luciferase reporter assay and Western blotting. Finally, the in vivo anti-tumor effect of LV-miR-126-3p inhibitor combined with sorafenib was evaluated via subcutaneous tumor models.ResultsHCC cells with high expression of miR-126-3p exhibited increased resistance to sorafenib. The results of bioinformatics analysis and the dual-luciferase reporter assay showed that miR-126-3p directly targeted SPRED1. The sensitivity of HCC cells to sorafenib was markedly enhanced by SPRED1 upregulation. Gain- and loss-of function experiments verified that miR-126-3p induced sorafenib resistance in HCC through downregulating SPRED1. Furthermore, the inhibition of miR-126-3p markedly increased the effectiveness of sorafenib against HCC in vivo. Mechanistically, our results suggested that miR-126-3p promoted sorafenib resistance via targeting SPRED1 and activating the ERK signaling pathway.ConclusionsOur study demonstrates that regulating the miR-126-3p/SPRED1 axis might be a promising strategy for enhancing the antitumor effect of sorafenib in the treatment of HCC.

Project description:A disintegrin and a metalloprotease (ADAM)-9 is a metzincin cell-surface protease with strongly elevated expression in solid tumors, including pancreatic ductal adenocarcinoma (PDAC). In this study, we performed immunohistochemistry (IHC) of a tissue microarray (TMA) to examine the expression of ADAM9 in a cohort of >100 clinically annotated PDAC cases. We report that ADAM9 is prominently expressed by PDAC tumor cells, and increased ADAM9 expression levels correlate with poor tumor grading (P = 0.027) and the presence of vasculature invasion (P = 0.017). We employed gene expression silencing to generate a loss-of-function system for ADAM9 in two established PDAC cell lines. In vitro analysis showed that loss of ADAM9 does not impede cellular proliferation and invasiveness in basement membrane. However, ADAM9 plays a crucial role in mediating cell migration and adhesion to extracellular matrix substrates such as fibronectin, tenascin, and vitronectin. This effect appears to depend on its catalytic activity. In addition, ADAM9 facilitates anchorage-independent growth. In AsPC1 cells, but not in MiaPaCa-2 cells, we noted a pronounced yet heterogeneous impact of ADAM9 on the abundance of various integrins, a process that we characterized as post-translational regulation. Sprout formation of human umbilical vein endothelial cells (HUVECs) is promoted by ADAM9, as examined by transfer of cancer cell conditioned medium; this finding further supports a pro-angiogenic role of ADAM9 expressed by PDAC cancer cells. Immunoblotting analysis of cancer cell conditioned medium highlighted that ADAM9 regulates the levels of angiogenic factors, including shed heparin-binding EGF-like growth factor (HB-EGF). Finally, we carried out orthotopic seeding of either wild-type AsPC-1 cells or AsPC-1 cells with silenced ADAM9 expression into murine pancreas. In this in vivo setting, ADAM9 was also found to foster angiogenesis without an impact on tumor cell proliferation. In summary, our results characterize ADAM9 as an important regulator in PDAC tumor biology with a strong pro-angiogenic impact.

Dataset Information

MiR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A.

Publications

miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A.

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