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The molecular background of hepatocellular carcinoma (HCC) is highly heterogeneous, and biomarkers predicting response to treatments are an unmet clinical need. We investigated miR-30e-3p contribution to HCC phenotype and response to sorafenib, as well as the mutual modulation of TP53/MDM2 pathway, in HCC tissues and preclinical models. MiR-30e-3p was downregulated in human and rat HCCs, and its downregulation associated with TP53 mutations. TP53 contributed to miR-30e-3p biogenesis, and MDM2 was identified among its target genes, establishing an miR-30e-3p/TP53/MDM2 feedforward loop and accounting for miR-30e-3p dual role based on TP53 status. EpCAM, PTEN, and p27 were demonstrated as miR-30e-3p additional targets mediating its contribution to stemness and malignant features. In a preliminary cohort of patients with HCC treated with sorafenib, increased miR-30e-3p circulating levels predicted the development of resistance. In conclusion, molecular background dictates miR-30e-3p dual behavior in HCC. Mdm2 targeting plays a predominant tumor suppressor function in wild-type TP53 contexts, whereas other targets such as PTEN, p27, and EpCAM gain relevance and mediate miR-30e-3p oncogenic role in nonfunctional TP53 backgrounds. Increased circulating levels of miR-30e-3p predict the development of sorafenib resistance in a preliminary series of patients with HCC and deserve future investigations. Significance: The dual role of miR-30e-3p in HCC clarifies how the molecular context dictates the tumor suppressor or oncogenic function played by miRNAs.

The only first-line treatment approved for advanced hepatocellular carcinoma (HCC) is sorafenib. Since many patients experience drug resistance, the discovery of more effective therapeutic strategies represents an unmet clinical need. MicroRNA (MiR)-122 is downregulated in most HCCs, while oncogenic SerpinB3 is upregulated. Here, we assessed the relationship between miR-122 and SerpinB3 and their influence on cell phenotype and sorafenib resistance in HCC. A bioinformatics analysis identified SerpinB3 among hypothetical miR-122 targets. In SerpinB3-overexpressing HepG2 cells, miR-122 transfection decreased SerpinB3 mRNA and protein levels, whereas miR-122 inhibition increased SerpinB3 expression. Luciferase assay demonstrated the interaction between miR-122 and SerpinB3 mRNA. In an HCC rat model, high miR-122 levels were associated with negative SerpinB3 expression, while low miR-122 levels correlated with SerpinB3 positivity. A negative correlation between miR-122 and SerpinB3 or stem cell markers was found in HCC patients. Anti-miR-122 transfection increased cell viability in sorafenib-treated Huh-7 cells, while miR-122 overexpression increased sorafenib sensitivity in treated cells, but not in those overexpressing SerpinB3. In conclusion, we demonstrated that miR-122 targets SerpinB3, and its low levels are associated with SerpinB3 positivity and a stem-like phenotype in HCC. MiR-122 replacement therapy in combination with sorafenib deserves attention as a possible therapeutic strategy in SerpinB3-negative HCCs.

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.

Purpose: The aberrant expression of miR-221 is a hallmark of human cancers, including hepatocellular carcinoma (HCC), and its involvement in drug resistance, together with a proved in vivo efficacy of anti-miR-221 molecules, strengthen its role as an attractive target candidate in the oncologic field. The discovery of biomarkers predicting the response to treatments represents a clinical challenge in the personalized treatment era. This study aimed to investigate the possible role of miR-221 as a circulating biomarker in HCC patients undergoing sorafenib treatment as well as to evaluate its contribution to sorafenib resistance in advanced HCC. Experimental Design: A chemically induced HCC rat model and a xenograft mouse model, together with HCC-derived cell lines were employed to analyze miR-221 modulation by Sorafenib treatment. Data from the functional analysis were validated in tissue samples from surgically resected HCCs. The variation of circulating miR-221 levels in relation to Sorafenib treatment were assayed in the animal models and in two independent cohorts of patients with advanced HCC. Results: MiR-221 over-expression was associated with Sorafenib resistance in two HCC animal models and caspase-3 was identified as its target gene, driving miR-221 anti-apoptotic activity following Sorafenib administration. Lower pre-treatment miR-221 serum levels were found in patients subsequently experiencing response to Sorafenib and an increase of circulating miR-221 at the two months assessment was observed in responder patients. Conclusions: MiR-221 might represent a candidate biomarker of likelihood of response to Sorafenib in HCC patients to be tested in future studies. Caspase-3 modulation by miR-221 participates to Sorafenib resistance. Clin Cancer Res; 23(14); 3953–65. ©2017 AACR.

The role of microRNAs as oncogenes and tumor suppressor genes has emerged in several cancers, including hepatocellular carcinoma (HCC). The pivotal tumor suppressive role of p53-axis is indicated by the presence of inactivating mutations in TP53 gene in nearly all cancers. A close interaction between these two players, as well as the establishment of complex p53/miRNAs loops demonstrated the strong contribution of p53-effector miRNAs in enhancing the p53-mediated tumor suppression program. On the other hand, the direct and indirect targeting of p53, as well as the regulation of its stability and activity by specific microRNAs, underlie the importance of the fine-tuning of p53 pathway, affecting the cell fate of damaged/transformed cells. The promising results of miRNAs-based therapeutic approaches in preclinical studies and their entrance in clinical trials demonstrate the feasibility of this strategy in several diseases, including cancer. Molecularly targeted drugs approved so far for HCC treatment show intrinsic or acquired resistances with disease progression in many cases, therefore the identification of effective and non-toxic agents for the treatment of HCC is actually an unmet clinical need. The knowledge of p53/miRNA inter-relations in HCC may provide useful elements for the identification of novel combined approaches in the context of the “personalized-medicine” era.