Identification of DUSP4/6 overexpression as a potential rheostat to NRAS-induced hepatocarcinogenesis.

BACKGROUND: Upregulation of the mitogen-activated protein kinase (MAPK) cascade is common in hepatocellular carcinoma (HCC). Neuroblastoma RAS viral oncogene homolog (NRAS) is mutated in a small percentage of HCC and is hitherto considered insufficient for hepatocarcinogenesis. We aimed to characterize the process of N-Ras-dependent carcinogenesis in the liver and to identify potential therapeutic vulnerabilities. METHODS: NRAS V12 plasmid was delivered into the mouse liver via hydrodynamic tail vein injection (HTVI). The resulting tumours, preneoplastic lesions, and normal tissue were characterized by NanoString® gene expression analysis, Western Blot, and Immunohistochemistry (IHC). The results were further confirmed by in vitro analyses of HCC cell lines. RESULTS: HTVI with NRAS V12 plasmid resulted in the gradual formation of preneoplastic and neoplastic lesions in the liver three months post-injection. These lesions mostly showed characteristics of HCC, with some exceptions of spindle cell/ cholangiocellular differentiation. Progressive upregulation of the RAS/RAF/MEK/ERK signalling was detectable in the lesions by Western Blot and IHC. NanoString® gene expression analysis of preneoplastic and tumorous tissue revealed a gradual overexpression of the cancer stem cell marker CD133 and Dual Specificity Phosphatases 4 and 6 (DUSP4/6). In vitro, transfection of HCC cell lines with NRAS V12 plasmid resulted in a coherent upregulation of DUSP4 and DUSP6. Paradoxically, this upregulation in PLC/PRF/5 cells was accompanied by a downregulation of phosphorylated extracellular-signal-regulated kinase (pERK), suggesting an overshooting compensation. Silencing of DUSP4 and DUSP6 increased proliferation in HCC cell lines. CONCLUSIONS: Contrary to prior assumptions, the G12V NRAS mutant form is sufficient to elicit hepatocarcinogenesis in the mouse. Furthermore, the upregulation of the MAPK cascade was paralleled by the overexpression of DUSP4, DUSP6, and CD133 in vivo and in vitro. Therefore, DUSP4 and DUSP6 might fine-tune the excessive MAPK activation, a mechanism that can potentially be harnessed therapeutically.

FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a neoplasia of the biliary tract driven by genetic, epigenetic and transcriptional mechanisms. Herein, we investigated the role of the transcription factor FOSL1, as well as its downstream transcriptional effectors, in the development and progression of CCA. METHODS: FOSL1 was investigated in human CCA clinical samples. Genetic inhibition of FOSL1 in human and mouse CCA cell lines was performed in in vitro and in vivo models using constitutive and inducible short-hairpin RNAs. Conditional FOSL1 ablation was done using a genetically engineered mouse (GEM) model of CCA (mutant KRAS and Trp53 knockout). Follow-up RNA and chromatin immunoprecipitation (ChIP) sequencing analyses were carried out and downstream targets were validated using genetic and pharmacological inhibition. RESULTS: An inter-species analysis of FOSL1 in CCA was conducted. First, FOSL1 was found to be highly upregulated in human and mouse CCA, and associated with poor patient survival. Pharmacological inhibition of different signalling pathways in CCA cells converged on the regulation of FOSL1 expression. Functional experiments showed that FOSL1 is required for cell proliferation and cell cycle progression in vitro, and for tumour growth and tumour maintenance in both orthotopic and subcutaneous xenograft models. Likewise, FOSL1 genetic abrogation in a GEM model of CCA extended mouse survival by decreasing the oncogenic potential of transformed cholangiocytes. RNA and ChIP sequencing studies identified direct and indirect transcriptional effectors such as HMGCS1 and AURKA, whose genetic and pharmacological inhibition phenocopied FOSL1 loss. CONCLUSIONS: Our data illustrate the functional and clinical relevance of FOSL1 in CCA and unveil potential targets amenable to pharmacological inhibition that could enable the implementation of novel therapeutic strategies. LAY SUMMARY: Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention.