The epigenetically regulated miR-494 associates with stem-cell phenotype and induces sorafenib resistance in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, especially in patients not amenable for curative treatments. The multi-kinase inhibitor sorafenib represents the first-line treatment option for advanced HCC; nevertheless, its effectiveness is limited due to tumor heterogeneity as well as innate or acquired drug resistance, raising the need for new therapeutic strategies. MicroRNAs (miRNAs) involvement in treatment response as well as their safety and efficacy in preclinical models and clinical trials have been widely documented in the oncologic field, including HCC. Here, we identified miR-494 upregulation in a subgroup of human and rat HCCs with stem cell-like characteristics, as well as multiple epigenetic mechanisms involved in its aberrant expression in HCC cell lines and patients. Moreover, we identified p27, puma and pten among miR-494 targets, contributing to speed up cell cycle progression, enhance survival potential in stressful conditions and increase invasive and clonogenic capabilities. MiR-494 overexpression increased sorafenib resistance via mTOR pathway activation in HCC cell lines and, in line, high miR-494 levels associated with decreased sorafenib response in two HCC animal models. A sorafenib-combined anti-miR-494-based strategy revealed an enhanced anti-tumor potential with respect to sorafenib-only treatment in our HCC rat model. In conclusion, our findings suggested miR-494 as a possible therapeutic target as well as a candidate biomarker for patient stratification in advanced HCC.

MicroRNAs in Animal Models of HCC

“…Intratumoral injection of adenoviral particles Ad5-AlncRNA in a sorafenib-resistant xenograft mouse model induced only a slight decrease of tumor growth but strongly synergized sorafenib antitumor activity, half-reducing tumor weight, decreasing Ki-67 staining, and increasing terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells with respect to control animals [68]. In agreement, in our experience, the only overexpression of miR-494 in HCC xenografts does not impact on tumor growth, whereas in the setting of sorafenib treatment, overexpression of miR-494 determines resistance, suggesting the central role for miR-494/PTEN/AKT/mTOR/p27 axis in sorafenib sensitization in HCC [15]. He and coworkers described similar findings reporting the synergistic effect of anti-miR-21 oligonucleotides coupled with sorafenib in a xenograft model obtained by subcutaneous inoculation of sorafenib resistant Huh-7 cells.…”

“…Notably, in vivo gene targeting of Dlk1-Dio3 locus by AAV vectors causing its overexpression led to HCC development in 100% of mice and, in line, overexpression of this miRNA cluster associated with an aggressive stem-cell-like phenotype in HCC [40]. Others and our group reported the upregulation of miR-494, a member of the Dlk1-Dio3 miRNA cluster, in 25-30% of HCCs with stemness features and demonstrated its involvement in tumor progression and sorafenib resistance through the direct targeting of mutated in colorectal cancer (MCC) and the phosphatase and tensin homolog (PTEN) tumor suppressor gene [15,16]. Regarding IGF2/483 oncogenic locus, an increase of miR-483-3p was found in 30% of human HCCs and Bcl-2 binding component 3 (BBC3/PUMA), a Bcl-2 family member with pro-apoptotic functions, was identified among its target genes, suggesting miR-122 KO models as representative of a subgroup of HCCs with stem-cell-like characteristics and epigenetic deregulation [41].…”

“…Since miR-494 showed the highest activity in a colony forming unit assay and increased cell cycle progression through MCC targeting, the antitumor potential of miR-494 antagonist molecules was tested in Myc-driven HCC bearing mice showing decreased tumor burden associated with increased p27 and MCC protein levels [16]. In line, we demonstrated p27 as a miR-494 direct target gene in HCC cell lines and xenograft mice playing a role in cell cycle fastening and cell proliferation in miR-494 overexpressing Huh-7 cells, suggesting that both animal models might be suitable for identification of miR-494 downstream molecular mechanisms [15]. Similarly, miR-206 was commonly downregulated among c-Myc and AKT/Ras-oncogene induced HCC mouse models, HCC cell lines, and human HCCs contributing to tumor development and progression through cell cycle regulator cyclin D1 (CCND1), c-MET, and cyclin-dependent kinase 6 (CDK6) direct targeting, as demonstrated by in vitro and in vivo experiments [21].…”

“…Interestingly, compound Astragalus and Salvia miltiorrhiza extract (CASE) containing active principles extracted from Astragalus membranaceus and Salvia miltiorrhiza provided anticancer therapy more likely through miR-145 and miR-21 upregulation and downregulation, respectively [166]. miR-494 is overexpressed in the great majority of rat DEN-induced HCCs, and its expression correlates with stem-cell features, representing an appropriate model for studying molecular alterations occurring in the stem-cell-like HCC subgroup with miR-494 aberrant expression and poor survival (25% of human HCCs) [15]. Pollutri et al also demonstrated an antitumor effect of a sorafenib-combined anti-miR-494 strategy in DEN HCC rats, showing decreased tumor progression with respect to sorafenib monotherapy.…”

Sestrin 2 confers primary resistance to sorafenib by simultaneously activating AKT and AMPK in hepatocellular carcinoma

“…However, the upstream regulatory network of PI3K/AKT in sorafenib primary resistance is partially understood. It has already been confirmed that overexpression of miR‐494, as well as increased insulin‐like growth factor 1 receptor (IGF1R) expression was responsible for triggering AKT signaling activation to mediate sorafenib primary resistance in HCC. In addition, the combination of a histone deacetylase inhibitor valproic acid (VPA) with sorafenib was capable to inhibit AKT activation, thus helping to increase sensitivity to short‐term sorafenib exposure .…”