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.].

Adenosine A2A receptor null chondrocyte transcriptome resembles that of human osteoarthritic chondrocytes.

Adenosine signaling plays a critical role in the maintenance of articular cartilage and may serve as a novel therapeutic for osteoarthritis (OA), a highly prevalent and morbid disease without effective therapeutics in the current market. Mice lacking adenosine A2A receptors (A2AR) develop spontaneous OA by 16 weeks of age, a finding relevant to human OA since loss of adenosine signaling due to diminished adenosine production (NT5E deficiency) also leads to development of OA in mice and humans. To better understand the mechanism by which A2AR and adenosine generation protect from OA development, we examined differential gene expression in neonatal chondrocytes from WT and A2AR null mice. Analysis of differentially expressed genes was analyzed by KEGG pathway analysis, and oPOSSUM and the flatiron database were used to identify transcription factor binding enrichment, and tissue-specific network analyses and patterns were compared to gene expression patterns in chondrocytes from patients with OA. There was a differential expression of 2211 genes (padj<0.05). Pathway enrichment analysis revealed that pro-inflammatory changes, increased metalloprotease, reduced matrix organization, and homeostasis are upregulated in A2AR null chondrocytes. Moreover, stress responses, including autophagy and HIF-1 signaling, seem to be important drivers of OA and bear marked resemblance to the human OA transcriptome. Although A2AR null mice are born with grossly intact articular cartilage, we identify here the molecular foundations for early-onset OA in these mice, further establishing their role as models for human disease and the potential use of adenosine as a treatment for human disease.

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.

Intraarticular injection of liposomal adenosine reduces cartilage damage in established murine and rat models of osteoarthritis.

Osteoarthritis (OA) affects nearly 10% of the population of the United States and other industrialized countries and, at present, short of surgical joint replacement, there is no therapy available that can reverse the progression of the disease. Adenosine, acting at its A2A receptor (A2AR), is a critical autocrine factor for maintenance of cartilage homeostasis and here we report that injection of liposomal suspensions of either adenosine or a selective A2AR agonist, CGS21680, significantly reduced OA cartilage damage in a murine model of obesity-induced OA. The same treatment also improved swelling and preserved cartilage in the affected knees in a rat model of established post-traumatic OA (PTOA). Differential expression analysis of mRNA from chondrocytes harvested from knees of rats with PTOA treated with liposomal A2AR agonist revealed downregulation of genes associated with matrix degradation and upregulation of genes associated with cell proliferation as compared to liposomes alone. Studies in vitro and in affected joints demonstrated that A2AR ligation increased the nuclear P-SMAD2/3/P-SMAD1/5/8 ratio, a change associated with repression of terminal chondrocyte differentiation. These results strongly suggest that targeting the A2AR is an effective approach to treat OA.

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.

Polyubiquitin Chains Linked by Lysine Residue 48 (K48) Selectively Target Oxidized Proteins In Vivo.

Aims: Ubiquitin is a highly conserved protein modifier that heavily accumulates during the oxidative stress response. Here, we investigated the role of the ubiquitination system, particularly at the linkage level, in the degradation of oxidized proteins. The function of ubiquitin in the removal of oxidized proteins remains elusive because of the wide range of potential targets and different roles that polyubiquitin chains play. Therefore, we describe in detail the dynamics of the K48 ubiquitin response as the canonical signal for protein degradation. We identified ubiquitin targets and defined the relationship between protein ubiquitination and oxidation during the stress response. Results: Combining oxidized protein isolation, linkage-specific ubiquitination screens, and quantitative proteomics, we found that K48 ubiquitin accumulated at both the early and late phases of the stress response. We further showed that a fraction of oxidized proteins are conjugated with K48 ubiquitin. We identified ∼750 ubiquitinated proteins and ∼400 oxidized proteins that were modified during oxidative stress, and around half of which contain both modifications. These proteins were highly abundant and function in translation and energy metabolism. Innovation and Conclusion: Our work showed for the first time that K48 ubiquitin modifies a large fraction of oxidized proteins, demonstrating that oxidized proteins can be targeted by the ubiquitin/proteasome system. We suggest that oxidized proteins that rapidly accumulate during stress are subsequently ubiquitinated and degraded during the late phase of the response. This delay between oxidation and ubiquitination may be necessary for reprogramming protein dynamics, restoring proteostasis, and resuming cell growth.

Macrophage-derived netrin-1 promotes abdominal aortic aneurysm formation by activating MMP3 in vascular smooth muscle cells.

Abdominal aortic aneurysms (AAA) are characterized by extensive extracellular matrix (ECM) fragmentation and inflammation. However, the mechanisms by which these events are coupled thereby fueling focal vascular damage are undefined. Here we report through single-cell RNA-sequencing of diseased aorta that the neuronal guidance cue netrin-1 can act at the interface of macrophage-driven injury and ECM degradation. Netrin-1 expression peaks in human and murine aneurysmal macrophages. Targeted deletion of netrin-1 in macrophages protects mice from developing AAA. Through its receptor neogenin-1, netrin-1 induces a robust intracellular calcium flux necessary for the transcriptional regulation and persistent catalytic activation of matrix metalloproteinase-3 (MMP3) by vascular smooth muscle cells. Deficiency in MMP3 reduces ECM damage and the susceptibility of mice to develop AAA. Here, we establish netrin-1 as a major signal that mediates the dynamic crosstalk between inflammation and chronic erosion of the ECM in AAA.

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.

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.