Heparanase-1 is upregulated by hepatitis C virus and favors its replication.

BACKGROUND & AIMS: Over time, chronic HCV infection can lead to hepatocellular carcinoma (HCC), a process that involves changes to the liver extracellular matrix (ECM). However, the exact mechanisms by which HCV induces HCC remain unclear. Therefore, we sought to investigate the impact of HCV on the liver ECM, with a focus on heparanase-1 (HPSE). METHODS: HPSE expression was assessed by quantitative reverse-transcription PCR, immunoblotting and immunofluorescence in liver biopsies infected or not with HCV, and in 10-day-infected hepatoma Huh7.5 cells. Cell lines deficient for or overexpressing HPSE were established to study its role during infection. RESULTS: HCV propagation led to significant HPSE induction, in vivo and in vitro. HPSE enhanced infection when exogenously expressed or supplemented as a recombinant protein. Conversely, when HPSE expression was downregulated or its activity blocked, HCV infection dropped, suggesting a role of HPSE in the HCV life cycle. We further studied the underlying mechanisms of such observations and found that HPSE favored HCV release by enhancing CD63 synthesis and exosome secretion, but not by stimulating HCV entry or genome replication. We also showed that virus-induced oxidative stress was involved in HPSE induction, most likely through NF-κB activation. CONCLUSIONS: We report for the first time that HCV infection is favored by HPSE, and upregulates HPSE expression and secretion, which may result in pathogenic alterations of the ECM. LAY SUMMARY: Chronic hepatitis C virus (HCV) infection can lead to hepatocellular carcinoma development in a process that involves derangement of the extracellular matrix (ECM). Herein, we show that heparanase-1, a protein involved in ECM degradation and remodeling, favors HCV infection and is upregulated by HCV infection; this upregulation may result in pathogenic alterations of the ECM.

The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent.

INTRODUCTION: Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. METHODS: Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. RESULTS: We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. CONCLUSION: Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

Genome-scale CRISPR/Cas9 screen determines factors modulating sensitivity to ProTide NUC-1031.

Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to NUC-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with NUC-1031.