Downregulation of SOCS1 increases interferon-induced ISGylation during differentiation of induced-pluripotent stem cells to hepatocytes.

Background & Aims: Increased expression of IFN-stimulated gene 15 (ISG15) and subsequently increased ISGylation are key factors in the host response to viral infection. In this study, we sought to characterize the expression of ISG15, ISGylation, and associated enzymes at each stage of differentiation from induced pluripotent stem cells (iPSCs) to hepatocytes. Methods: To study the regulation of ISGylation, we utilized patient samples and in vitro cell culture models including iPSCs, hepatocytes-like cells, immortalized cell lines, and primary human hepatocytes. Protein/mRNA expression were measured following treatment with poly(I:C), IFNα and HCV infection. Results: When compared to HLCs, we observed several novel aspects of the ISGylation pathway in iPSCs. These include a lower baseline expression of the ISGylation-activating enzyme, UBE1L, a lack of IFN-induced expression of the ISGylation-conjugation enzyme UBE2L6, an attenuated activation of the transcription factor STAT1 and constitutive expression of SOCS1. ISGylation was observed in iPSCs following downregulation of SOCS1, which facilitated STAT1 activation and subsequently increased expression of UBE2L6. Intriguingly, HCV permissive transformed hepatoma cell lines demonstrated higher intrinsic expression of SOCS1 and weaker ISGylation following IFN treatment. SOCS1 downregulation in HCV-infected Huh 7.5.1 cells led to increased ISGylation. Conclusions: Herein, we show that high basal levels of SOCS1 inhibit STAT1 activation and subsequently IFN-induced UBE2L6 and ISGylation in iPSCs. Furthermore, as iPSCs differentiate into hepatocytes, epigenetic mechanisms regulate ISGylation by modifying UBE1L and SOCS1 expression levels. Overall, this study demonstrates that the development of cell-intrinsic innate immunity during the differentiation of iPSCs to hepatocytes provides insight into cell type-specific regulation of host defense responses and related oncogenic processes. Impact and implications: To elucidate the mechanism underlying regulation of ISGylation, a key process in the innate immune response, we studied changes in ISGylation-associated genes at the different stages of differentiation from iPSCs to hepatocytes. We found that high basal levels of SOCS1 inhibit STAT1 activation and subsequently IFN-induced UBE2L6 and ISGylation in iPSCs. Importantly, epigenetic regulation of SOCS1 and subsequently ISGylation may be important factors in the development of cell type-specific host defense responses in hepatocytes that should be considered when studying chronic infections and oncogenic processes in the liver.

GABC: A comprehensive resource and Genome Atlas for Breast Cancer.

We developed the Genome Atlas of Breast Cancer (GABC), a global map of noncoding events in the human genome associated with breast cancer that provides a valuable reference resource for users to investigate the underlying genome abnormalities in breast cancer patients. Although significant progress has been made in breast cancer treatment, its morbidity and recurrence rates in women are still high worldwide. Curation and integration of breast cancer-related dysregulations from multiple aspects is essential for disease prevention and diagnosis. In this study, we developed the GABC, which contains 10 172 aberrant noncoding events occurring at multiomics levels, including the genome (single nucleotide polymorphism and somatic mutation), transcriptome (long noncoding RNA and microRNA) and epigenome (DNA methylation, enhancer and superenhancer). Each event entry provides descriptions of detailed biological mechanisms specific to the region or element. Users can also check the genome locations and relationships of functional regulators. The GABC provides a flexible and user-friendly interface for users to search, browse and download data. In addition, the GABC provides an interface to submit newly discovered noncoding events that can be included in the database. Therefore, the GABC aims to constantly enhance our understanding of noncoding genomic events in breast cancer.

LnCeVar: a comprehensive database of genomic variations that disturb ceRNA network regulation.

LnCeVar (http://www.bio-bigdata.net/LnCeVar/) is a comprehensive database that aims to provide genomic variations that disturb lncRNA-associated competing endogenous RNA (ceRNA) network regulation curated from the published literature and high-throughput data sets. LnCeVar curated 119 501 variation-ceRNA events from thousands of samples and cell lines, including: (i) more than 2000 experimentally supported circulating, drug-resistant and prognosis-related lncRNA biomarkers; (ii) 11 418 somatic mutation-ceRNA events from TCGA and COSMIC; (iii) 112 674 CNV-ceRNA events from TCGA; (iv) 67 066 SNP-ceRNA events from the 1000 Genomes Project. LnCeVar provides a user-friendly searching and browsing interface. In addition, as an important supplement of the database, several flexible tools have been developed to aid retrieval and analysis of the data. The LnCeVar-BLAST interface is a convenient way for users to search ceRNAs by interesting sequences. LnCeVar-Function is a tool for performing functional enrichment analysis. LnCeVar-Hallmark identifies dysregulated cancer hallmarks of variation-ceRNA events. LnCeVar-Survival performs COX regression analyses and produces survival curves for variation-ceRNA events. LnCeVar-Network identifies and creates a visualization of dysregulated variation-ceRNA networks. Collectively, LnCeVar will serve as an important resource for investigating the functions and mechanisms of personalized genomic variations that disturb ceRNA network regulation in human diseases.

Comprehensive Characterization of Somatic Mutations Impacting lncRNA Expression for Pan-Cancer.

Somatic mutations have long been recognized as an important feature of cancer. However, analysis of somatic mutations, to date, has focused almost entirely on the protein coding regions of the genome. The potential roles of somatic mutations in human long noncoding RNAs (lncRNAs) are therefore largely unknown, particularly their functional significance across different cancer types. In this study, we characterized some lncRNAs whose expression was affected by somatic mutations (defined as MutLncs) and constructed global MutLnc landscapes across 17 cancer types by systematically integrating multiple levels of data. MutLncs were commonly downregulated and carried low mutation frequencies and non-silent mutations in most cancer types. Co-occurrence analysis in pan-cancer highlighted combined patterns of specific MutLncs, suggesting that a number of MutLncs influence diverse cancer types through combination effects. Several conserved and cancer-specific functions of MutLncs were determined. We further explored the somatic mutations affecting lncRNA expression via mixed and unmixed effects, which led to specific functions in pan-cancer. Survival analysis indicated that MutLncs and co-occurrence pairs can potentially serve as cancer biomarkers. Clarification of the specific roles of MutLncs in human cancers could be beneficial for understanding the molecular pathogenesis of different cancer types and developing the appropriate treatments.

Obstacles to successful treatment of hepatitis C in uninsured patients from a minority population.

BACKGROUND: Hepatitis C virus (HCV) treatment regimens (DAAs) are well tolerated, efficacious but costly. Their high cost and restricted availability, raises concerns about the outcome of treatment in uninsured patients. This study investigated sustained virologic response (SVR) outcomes in a predominately uninsured patient population and completion of four steps along the HCV treatment cascade. METHODS: A retrospective chart review was conducted to characterize the patient population and analyze covariates to determine association with insurance status, attainment of SVR and progression through the HCV treatment cascade. RESULTS: Out of a total of 216 patients, 154 (71%) were uninsured. Approximately 50% of patients (109 of 216 patients) were male and 57% were Hispanic (123 of 216 patients). Sex, race, ethnicity, treatment compliance, and rates of complications were not associated with insurance status. Insured patients were older (median 60 years vs 57 years, p-value < 0.001) and had higher rates of cirrhosis: 32 out of 62 patients (52%) vs 48 out of 154 patients (31%) (p-value = 0.005). Insured patients were tested for SVR at similar rates as uninsured patients: 84% (52 of 62 patients) vs 81% (125 of 154 patients), respectively. Of those tested for SVR, the cure rate for insured patients was 98% (51 out of 52 patients) compared to 97% (121 out of 125 patients) in the uninsured. Out of those who completed treatment, 177 of 189 (94%) were tested for attainment of SVR. Compliance rates were significantly different between tested and untested patients: 88% (156 of 177 patients) vs 0% (0 of 12 patients), respectively (p-value < 0.001). However, insurance status, race ethnicity, cirrhosis, and complications were not associated with being tested for SVR. CONCLUSIONS: These results demonstrate that insured and uninsured patients with chronic HCV infection, with access to patient assistance programs, can be treated and have comparable clinical outcomes. In addition, testing for SVR remains an important obstacle in completion of the HCV treatment cascade. Nevertheless, patient assistance programs remove a significant barrier for treatment access in real-world HCV infected populations.

NFκB in Pancreatic Stellate Cells Reduces Infiltration of Tumors by Cytotoxic T Cells and Killing of Cancer Cells, via Up-regulation of CXCL12.

BACKGROUND & AIMS: Immunotherapies are ineffective against pancreatic cancer. We investigated whether the activity of nuclear factor (NF)κB in pancreatic stromal cells contributes to an environment that suppresses antitumor immune response. METHODS: Pancreata of C57BL/6 or Rag1-/- mice were given pancreatic injections of a combination of KrasG12D/+; Trp53 R172H/+; Pdx-1cre (KPC) pancreatic cancer cells and pancreatic stellate cells (PSCs) extracted from C57BL/6 (control) or mice with disruption of the gene encoding the NFκB p50 subunit (Nfkb1 or p50-/- mice). Tumor growth was measured as an endpoint. Other mice were given injections of Lewis lung carcinoma (LLC) lung cancer cells or B16-F10 melanoma cells with control or p50-/- fibroblasts. Cytotoxic T cells were depleted from C57BL/6 mice by administration of antibodies against CD8 (anti-CD8), and growth of tumors from KPC cells, with or without control or p50-/- PSCs, was measured. Some mice were given an inhibitor of CXCL12 (AMD3100) and tumor growth was measured. T-cell migration toward cancer cells was measured using the Boyden chamber assay. RESULTS: C57BL/6 mice coinjected with KPC cells (or LLC or B16-F10 cells) and p50-/- PSCs developed smaller tumors than mice given injections of the cancer cells along with control PSCs. Tumors that formed when KPC cells were injected along with p50-/- PSCs had increased infiltration by activated cytotoxic T cells along with decreased levels of CXCL12, compared with tumors grown from KPC cells injected along with control PSCs. KPC cells, when coinjected with control or p50-/- PSCs, developed the same-size tumors when CD8+ T cells were depleted from C57BL/6 mice or in Rag1-/- mice. The CXCL12 inhibitor slowed tumor growth and increased tumor infiltration by cytotoxic T cells. In vitro expression of p50 by PSCs reduced T-cell migration toward and killing of cancer cells. When cultured with cancer cells, control PSCs expressed 10-fold higher levels of CXCL12 than p50-/- PSCs. The CXCL12 inhibitor increased migration of T cells toward KPC cells in culture. CONCLUSIONS: In studies of mice and cell lines, we found that NFκB activity in PSCs promotes tumor growth by increasing expression of CXCL12, which prevents cytotoxic T cells from infiltrating the tumor and killing cancer cells. Strategies to block CXCL12 in pancreatic tumor cells might increase antitumor immunity.

Thioredoxin-1 protects against androgen receptor-induced redox vulnerability in castration-resistant prostate cancer.

Androgen deprivation (AD) therapy failure leads to terminal and incurable castration-resistant prostate cancer (CRPC). We show that the redox-protective protein thioredoxin-1 (TRX1) increases with prostate cancer progression and in androgen-deprived CRPC cells, suggesting that CRPC possesses an enhanced dependency on TRX1. TRX1 inhibition via shRNA or a phase I-approved inhibitor, PX-12 (untested in prostate cancer), impedes the growth of CRPC cells to a greater extent than their androgen-dependent counterparts. TRX1 inhibition elevates reactive oxygen species (ROS), p53 levels and cell death in androgen-deprived CRPC cells. Unexpectedly, TRX1 inhibition also elevates androgen receptor (AR) levels under AD, and AR depletion mitigates both TRX1 inhibition-mediated ROS production and cell death, suggesting that AD-resistant AR expression in CRPC induces redox vulnerability. In vivo TRX1 inhibition via shRNA or PX-12 reverses the castration-resistant phenotype of CRPC cells, significantly inhibiting tumor formation under systemic AD. Thus, TRX1 is an actionable CRPC therapeutic target through its protection against AR-induced redox stress.