Correction: Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor ß 1 (TGF-ß 1) Axis in Hepatitis C Virus-Expressing Hepatocytes.

[This corrects the article DOI: 10.1371/journal.pone.0046526.].

Transcriptomics-Based Drug Repurposing Approach Identifies Novel Drugs against Sorafenib-Resistant Hepatocellular Carcinoma.

Objective: Hepatocellular carcinoma (HCC) is frequently diagnosed in patients with late-stage disease who are ineligible for curative surgical therapies. The majority of patients become resistant to sorafenib, the only approved first-line therapy for advanced cancer, underscoring the need for newer, more effective drugs. The purpose of this study is to expedite identification of novel drugs against sorafenib resistant (SR)-HCC. Methods: We employed a transcriptomics-based drug repurposing method termed connectivity mapping using gene signatures from in vitro-derived SR Huh7 HCC cells. For proof of concept validation, we focused on drugs that were FDA-approved or under clinical investigation and prioritized two anti-neoplastic agents (dasatinib and fostamatinib) with targets associated with HCC. We also prospectively validated predicted gene expression changes in drug-treated SR Huh7 cells as well as identified and validated the targets of Fostamatinib in HCC. Results: Dasatinib specifically reduced the viability of SR-HCC cells that correlated with up-regulated activity of SRC family kinases, its targets, in our SR-HCC model. However, fostamatinib was able to inhibit both parental and SR HCC cells in vitro and in xenograft models. Ingenuity pathway analysis of fostamatinib gene expression signature from LINCS predicted JAK/STAT, PI3K/AKT, ERK/MAPK pathways as potential targets of fostamatinib that were validated by Western blot analysis. Fostamatinib treatment reversed the expression of genes that were deregulated in SR HCC. Conclusion: We provide proof of concept evidence for the validity of this drug repurposing approach for SR-HCC with implications for personalized medicine.

Role of B Cell Lymphoma 2 in the Regulation of Liver Fibrosis in miR-122 Knockout Mice.

MicroRNA-122 (miR-122) has been identified as a marker of various liver injuries, including hepatitis- virus-infection-, alcoholic-, and non-alcoholic steatohepatitis (NASH)-induced liver fibrosis. Here, we report that the extracellular miR-122 from hepatic cells can be delivered to hepatic stellate cells (HSCs) to modulate their proliferation and gene expression. Our published Argonaute crosslinking immunoprecipitation (Ago-CLIP) data identified several pro-fibrotic genes, including Ctgf, as miR-122 targets in mice livers. However, treating Ctgf as a therapeutic target failed to rescue the fibrosis developed in the miR-122 knockout livers. Alternatively, we compared the published datasets of human cirrhotic livers and miR-122 KO livers, which revealed upregulation of BCL2, suggesting its potential role in regulating fibrosis. Notably, ectopic miR-122 expression inhibited BCL2 expression in human HSC (LX-2) cells). Publicly available ChIP-seq data in human hepatocellular cancer (HepG2) cells and mice livers suggested miR-122 could regulate BCL2 expression indirectly through c-MYC, which was confirmed by siRNA-mediated depletion of c-MYC in Hepatocellular Carcinoma (HCC) cell lines. Importantly, Venetoclax, a potent BCL2 inhibitor approved for the treatment of leukemia, showed promising anti-fibrotic effects in miR-122 knockout mice. Collectively, our data demonstrate that miR-122 suppresses liver fibrosis and implicates anti-fibrotic potential of Venetoclax.

Ibrutinib Potentiates Antihepatocarcinogenic Efficacy of Sorafenib by Targeting EGFR in Tumor Cells and BTK in Immune Cells in the Stroma.

Hepatocellular carcinoma (HCC), the most prevalent primary liver cancer, is a leading cause of cancer-related death worldwide because of rising incidence and limited therapy. Although treatment with sorafenib or lenvatinib is the standard of care in patients with advanced-stage HCC, the survival benefit from sorafenib is limited due to low response rate and drug resistance. Ibrutinib, an irreversible tyrosine kinase inhibitor (TKI) of the TEC (e.g., BTK) and ErbB (e.g., EGFR) families, is an approved treatment for B-cell malignancies. Here, we demonstrate that ibrutinib inhibits proliferation, spheroid formation, and clonogenic survival of HCC cells, including sorafenib-resistant cells. Mechanistically, ibrutinib inactivated EGFR and its downstream Akt and ERK signaling in HCC cells, and downregulated a set of critical genes involved in cell proliferation, migration, survival, and stemness, and upregulated genes promoting differentiation. Moreover, ibrutinib showed synergy with sorafenib or regorafenib, a sorafenib congener, by inducing apoptosis of HCC cells. In vivo, this TKI combination significantly inhibited HCC growth and prolonged survival of immune-deficient mice bearing human HCCLM3 xenograft tumors and immune-competent mice bearing orthotopic mouse Hepa tumors at a dose that did not exhibit systemic toxicity. In immune-competent mice, the ibrutinib-sorafenib combination reduced the numbers of BTK+ immune cells in the tumor microenvironment. Importantly, we found that the BTK+ immune cells were also enriched in the tumor microenvironment in a subset of primary human HCCs. Collectively, our findings implicate BTK signaling in hepatocarcinogenesis and support clinical trials of the sorafenib-ibrutinib combination for this deadly disease.

CD44 positive and sorafenib insensitive hepatocellular carcinomas respond to the ATP-competitive mTOR inhibitor INK128.

The mTOR pathway is activated in about 50% of patients with hepatocellular carcinoma (HCC). In an effort to identify new pathways and compounds to treat advanced HCC, we considered the ATP-competitive mTOR inhibitor INK128. ATP-competitive mTOR inhibitors attenuate both mTORC1 and mTORC2. INK128 was evaluated in sorafenib sensitive and insensitive HCC cell lines, CD44low and CD44high HCC and those cell lines with acquired sorafenib resistance. CD44 was significantly increased in Huh7 cells made resistant to sorafenib. Forced expression of CD44 enhanced cellular proliferation and migration, and rendered the cells more sensitive to the anti-proliferative effects of INK128. INK128 suppressed CD44 expression in HCC cells while allosteric mTOR inhibitors did not. CD44 inhibition correlated with 4EBP1 phosphorylation status. INK128 showed better anti-proliferative and anti-migration effects on the mesenchymal-like HCC cells, CD44high HCC cells compared to the allosteric mTOR inhibitor everolimus. Moreover, a combination of INK128 and sorafenib showed improved anti-proliferative effects in CD44high HCC cells. INK128 was efficacious at reducing tumor growth in CD44high SK-Hep1 xenografts in mice when given as monotherapy or in combination with sorafenib. Since the clinical response to sorafenib is highly variable, our findings suggest that ATP-competitive mTOR inhibitors may be effective in treating advanced, CD44-expressing HCC patients who are insensitive to sorafenib.

The role of miR-122 in the dysregulation of glucose-6-phosphate dehydrogenase (G6PD) expression in hepatocellular cancer.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Thus, a better understanding of molecular aberrations involved in HCC pathogenesis is necessary for developing effective therapy. It is well established that cancer cells metabolize energy sources differently to rapidly generate biomass. Glucose-6-phosphate-dehydrogenase (G6PD), the rate-limiting enzyme of the Pentose Phosphate Pathway (PPP), is often activated in human malignancies to generate precursors for nucleotide and lipid synthesis. Here, we determined the clinical significance of G6PD in primary human HCC by analyzing RNA-seq and clinical data in The Cancer Genome Atlas. We found that the upregulation of G6PD correlates with higher tumor grade, increased tumor recurrence, and poor patient survival. Notably, liver-specific miR-122, which is essential for metabolic homeostasis, suppresses G6PD expression by directly interacting with its 3'UTR. Luciferase reporter assay confirmed two conserved functional miR-122 binding sites located in the 3'-UTR of G6PD. Furthermore, we show that ectopic expression of miR-122 and miR-1, a known regulator of G6PD expression coordinately repress G6PD expression in HCC cells. These miRNAs also reduced G6PD activity in HepG2 cells that express relatively high activity of this enzyme. Collectively, this study provides evidence that anti-HCC efficacy of miR122 and miR-1 could be mediated, at least in part, through inhibition of PPP by suppressing the expression of G6PD.