Xintong Chen

Epidermal growth factor receptor inhibition attenuates liver fibrosis and development of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most rapidly increasing cause of cancer‐related mortality in the United States. Because of the lack of viable treatment options for HCC, prevention in high‐risk patients has been proposed as an alternative strategy. The main risk factor for HCC is cirrhosis and several lines of evidence implicate epidermal growth factor (EGF) in the progression of cirrhosis and development of HCC. We therefore examined the effects of the EGF receptor (EGFR) inhibitor erlotinib on liver fibrogenesis and hepatocellular transformation in three different animal models of progressive cirrhosis: a rat model induced by repeated, low‐dose injections of diethylnitrosamine (DEN), a mouse model induced by carbon tetrachloride (CCl4), and a rat model induced by bile duct ligation (BDL). Erlotinib reduced EGFR phosphorylation in hepatic stellate cells (HSC) and reduced the total number of activated HSC. Erlotinib also decreased hepatocyte proliferation and liver injury. Consistent with all these findings, pharmacological inhibition of EGFR signaling effectively prevented the progression of cirrhosis and regressed fibrosis in some animals. Moreover, by alleviating the underlying liver disease, erlotinib blocked the development of HCC and its therapeutic efficacy could be monitored with a previously reported gene expression signature predictive of HCC risk in human cirrhosis patients. Conclusion: These data suggest that EGFR inhibition using Food and Drug Administration‐approved inhibitors provides a promising therapeutic approach for reduction of fibrogenesis and prevention of HCC in high‐risk cirrhosis patients who can be identified and monitored by gene expression signatures. (Hepatology 2014;59:1577‐1590)

UHRF1 overexpression drives DNA hypomethylation and hepatocellular carcinoma.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential regulator of DNA methylation that is highly expressed in many cancers. Here, we use transgenic zebrafish, cultured cells, and human tumors to demonstrate that UHRF1 is an oncogene. UHRF1 overexpression in zebrafish hepatocytes destabilizes and delocalizes Dnmt1 and causes DNA hypomethylation and Tp53-mediated senescence. Hepatocellular carcinoma (HCC) emerges when senescence is bypassed. tp53 mutation both alleviates senescence and accelerates tumor onset. Human HCCs recapitulate this paradigm, as UHRF1 overexpression defines a subclass of aggressive HCCs characterized by genomic instability, TP53 mutation, and abrogation of the TP53-mediated senescence program. We propose that UHRF1 overexpression is a mechanism underlying DNA hypomethylation in cancer cells and that senescence is a primary means of restricting tumorigenesis due to epigenetic disruption.

A genomic and clinical prognostic index for hepatitis C-related early-stage cirrhosis that predicts clinical deterioration

Objective The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. Design We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1 mg/dL) and platelet count (<100 000/mm3), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8 years). Results In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child–Turcotte–Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001). Conclusions A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions.

Transcriptome Profiling of Archived Sectioned Formalin-Fixed Paraffin-Embedded (AS-FFPE) Tissue for Disease Classification

Background Archived tissues from previously completed prospective trials represent invaluable resource for biomarker development. However, such specimens are often stored as sections on glass slides, in which RNA is severely degraded due to prolonged air exposure. We evaluated whether a proportion of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissues yield transcriptome profiles comparable to freshly cut (FC) FFPE tissues, which can be used for retrospective class prediction analysis. Methods Genome-wide transcriptome profiles of 6 to 7-year-old AS-FFPE tissue sections (generated from 5 to 16-year-old blocks) of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina) and digital transcript counting (nCounter) assay (NanoString), and gene signature-based prediction of HCC subclasses and prognosis was compared with previously generated FC-FFPE profiles from the same tissue blocks. Results RNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate to poor quality FC-FFPE samples (RNA Integrity Number: up to 2.50, R-square for technical replicates: up to 0.93). Analyzable transcriptome profiles were obtained in 64 (77%) HCC and 36 (77%) cirrhosis samples. Statistically more confident predictions based on random resampling-based method (nearest template prediction) were obtained in 37 (58%) HCC and 13 (36%) cirrhosis samples. Predictions made in FC-FFPE profiles were reproduced in 36 (97%) HCC and 11 (85%) cirrhosis AS-FFPE profiles. nCounter assay was tested in 24 cirrhosis samples, which yielded confident prediction in 15 samples (63%), of which 10 samples (67%) showed concordant predictions with FC-FFPE profiles. Conclusions AS-FFPE tissues yielded poorer quality RNA and transcriptome profiles compared to FC-FFPE tissues. Statistically more confident class prediction was feasible in 37 of 83 HCC samples and 13 of 47 cirrhosis samples. These results suggest that AS-FFPE tissues can be regarded as a resource for retrospective transcriptome-based class prediction analysis when they are the only available materials.

DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the re-replicating and arrested hepatocytes. Importantly, the DNA re-replication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis. Summary: Uhrf1 recruits DNA methyltransferase 1; upon its mutation, cells show enhanced DNA replication but fail to proliferate and ultimately die - leading to reduced organ size.

Interleukin-15 receptor α on hepatic stellate cells regulates hepatic fibrogenesis in mice

BACKGROUND & AIMS Interleukin-15 (IL-15) and its high affinity receptor interleukin-15 receptor alpha (IL-15Rα) are widely expressed in immune cells and hepatic resident cells. IL-15 signaling has important functions in homeostasis of natural killer (NK), natural killer T (NKT) and cytotoxic T (CD8(+) T) cells, and in liver regeneration. We hypothesized that IL-15 has a protective role in liver fibrosis progression by maintaining NK cell homeostasis. METHODS Fibrosis was induced using two mechanistically distinct models. Congenic bone marrow transplantation was used to evaluate the contribution of IL-15 signaling from various compartments to NK, CD8(+) T and NKT cell homeostasis and fibrogenesis. The gene expression profile of hepatic stellate cell (HSC) from IL-15Rα knockout (IL-15RαKO) mice and wild-type mice were captured using microarray analysis and validated in isolated HSC. Quantitative real-time PCR was used to assess repressors of collagen transcription. RESULTS IL-15RαKO mice exhibited more fibrosis in both models. IL-15 signaling from specific types of hepatic cells had divergent roles in maintaining liver NK, CD8(+) T and NKT cells, with a direct and protective role on radio-resistant non-parenchymal cells beyond the control of NK homeostasis. HSCs isolated from IL-15RαKO mice demonstrated upregulation of collagen production. Finally, IL-15RαKO HSC with or without transforming growth factor beta (TGF-β) stimulation exhibited increased expression of fibrosis markers and decreased collagen transcription repressors expression. CONCLUSIONS IL-15Rα signaling has a direct anti-fibrotic effect independent of preserving NK homeostasis. These findings establish a rationale to further explore the anti-fibrotic potential of enhancing IL-15 signaling in HSCs. LAY SUMMARY We investigated how a cellular protein, Interleukin-15 (IL-15), decreases the amount of scar tissue that is formed upon liver injury. We found that IL-15 and its receptor decrease the amount of scar tissue that is created by specialized liver cells (called stellate cells) and increase the number of a specific subgroup of immune cells (natural killer cells) that are known to eliminate stellate cells. TRANSCRIPT PROFILING ACCESSION NUMBER GSE45612, GSE 68001 and GSE 25097.

Prothymosin α Variants Isolated From CD8+ T Cells and Cervicovaginal Fluid Suppress HIV-1 Replication Through Type I Interferon Induction

Soluble factors from CD8(+) T cells and cervicovaginal mucosa of women are recognized as important in controlling human immunodeficiency virus type 1 (HIV-1) infection and transmission. Previously, we have shown the strong anti-HIV-1 activity of prothymosin α (ProTα) derived from CD8(+) T cells. ProTα is a small acidic protein with wide cell distribution, to which several functions have been ascribed, depending on its intracellular or extracellular localization. To date, activities of ProTα have been attributed to a single protein known as isoform 2. Here we report the isolation and identification of 2 new ProTα variants from CD8(+) T cells and cervicovaginal lavage with potent anti-HIV-1 activity. The first is a splice variant of the ProTα gene, known as isoform CRA_b, and the second is the product of a ProTα gene, thus far classified as a pseudogene 7. Native or recombinant ProTα variants potently restrict HIV-1 replication in macrophages through the induction of type I interferon. The baseline expression of interferon-responsive genes in primary human cervical tissues positively correlate with high levels of intracellular ProTα, and the knockdown of ProTα variants by small interfering RNA leads to downregulation of interferon target genes. Overall, these findings suggest that ProTα variants are innate immune mediators involved in immune surveillance.

Survival analysis tools in genomics research.

There is an increasing demand to determine the clinical implication of experimental findings in molecular biomedical research. Survival (or failure time) analysis methodologies have been adapted to the analysis of genomics data to link molecular information with clinical outcomes of interest. Genome-wide molecular profiles have served as sources for discovery of predictive/prognostic biomarkers as well as therapeutic targets in the past decade. In this review, we overview currently available software, web applications, and databases specifically developed for survival analysis in genomics research and discuss issues in assessing clinical utility of molecular features derived from genomic profiling.

Characterization of drug-induced splicing complexity in prostate cancer cell line using long read technology

We characterize the transcriptional splicing landscape of a prostate cancer cell line treated with a previously identified synergistic drug combination. We use a combination of third generation long-read RNA sequencing technology and short-read RNAseq to create a high-fidelity map of expressed isoforms and fusions to quantify splicing events triggered by treatment. We find strong evidence for drug-induced, coherent splicing changes which disrupt the function of oncogenic proteins, and detect novel transcripts arising from previously unreported fusion events.

Abstract 790: Coupled dynamics of drug synergy, gene expression, and alternative splicing in combination therapies in breast cancer

Drug combination therapies in the cancer setting often succeed where mono-therapies fail, facilitating durable and robust responses that may curtail metastases and even be accompanied by milder side-effects. Predicting synergistic and antagonistic combinations based on the gene expression data of mono-therapy drug-tumor response is an important open problem wherein the role of transcriptional splicing dynamics is often ignored or too poorly correlated with phenotypes to be useful. In this work we leverage the inherent transcript/exon level resolution of RNA-seq data to infer combination-specific splicing signatures associated with additive and synergistic (subadditive) drug combinations as defined by canonical viability measurements in a time-course experiment. We integrate splicing information into a gene regulatory network (GRN) to compute leading order effects of splicing on the GRN by adaptive network refinement. Briefly, we use RNA-seq to study the transcriptional response over time (0, 3, 6, 9, 12, and 24 h) for three drugs (A, B and C) and their combinations (AB, AC and BC) in MCF-7 (ER+) breast cancer cells lines. Cell viability measurements show that one of the combinations (AB) is strongly synergistic, whereas the other two (AC and BC) are merely additive. We show via rigorous linear modeling of RNA-seq count data at the exon level that in addition to a novel transcriptional signature driven by differential expression, the combination AB transcriptional landscape is characterized by persistent alternative splicing signatures mostly comprised of genes which are not differentially expressed with respect to A or B but whose functional role has been dramatically changed by the addition (deletion) of a key regulatory protein domain encoded by the extra(missing) exon. We construct an isoform-level co-expression network (IRN) to probe the regulatory changes this dynamical splicing induces and show that it crucially contributes to the emergence of extensive transcriptional cascades by creating and removing key gene-gene correlations and altering the modular structure of the network. Using this approach we show that a key isoform of Protein beta-arrestin-1 (ARBB1), which participates in desensitization of G-protein coupled receptors through intracellular response to Estrogen, is enriched in the calcium ion-dependent exocytosis module of IRN, while ARRB1 remains functionally uninformative in the regular GRN. Our results suggest that any gene-signature based drug synergy prediction algorithm must take into account alternative splicing in order to effectively characterize the novel pathways being activated in the synergistic drug-tumor interaction. Citation Format: Xintong Chen, Gustavo Stolovitzky, Bojan Losic. Coupled dynamics of drug synergy, gene expression, and alternative splicing in combination therapies in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 790.