A high level of liver-specific expression of oncogenic KrasV12 drives robust liver tumorigenesis in transgenic zebrafish

Abstract

SUMMARYHuman liver cancer is one of the deadliest cancers worldwide, with hepatocellular carcinoma (HCC) being the most common type. Aberrant Ras signaling has been implicated in the development and progression of human HCC, but a complete understanding of the molecular mechanisms of this protein in hepatocarcinogenesis remains elusive. In this study, a stable in vivo liver cancer model using transgenic zebrafish was generated to elucidate Ras-driven tumorigenesis in HCC. Using the liver-specific fabp10 (fatty acid binding protein 10) promoter, we overexpressed oncogenic krasV12 specifically in the transgenic zebrafish liver. Only a high level of krasV12 expression initiated liver tumorigenesis, which progressed from hyperplasia to benign and malignant tumors with activation of the Ras-Raf-MEK-ERK and Wnt–β-catenin pathways. Histological diagnosis of zebrafish tumors identified HCC as the main lesion. The tumors were invasive and transplantable, indicating malignancy of these HCC cells. Oncogenic krasV12 was also found to trigger p53-dependent senescence as a tumor suppressive barrier in the pre-neoplastic stage. Microarray analysis of zebrafish liver hyperplasia and HCC uncovered the deregulation of several stage-specific and common biological processes and signaling pathways responsible for krasV12-driven liver tumorigenesis that recapitulated the molecular hallmarks of human liver cancer. Cross-species comparisons of cancer transcriptomes further defined a HCC-specific gene signature as well as a liver cancer progression gene signature that are evolutionarily conserved between human and zebrafish. Collectively, our study presents a comprehensive portrait of molecular mechanisms during progressive Ras-induced HCC. These observations indicate the validity of our transgenic zebrafish to model human liver cancer, and this model might act as a useful platform for drug screening and identifying new therapeutic targets.

Males develop faster and more severe hepatocellular carcinoma than females in krasV12 transgenic zebrafish

“…E-cadherin, a binding partner of β-catenin, also plays critical roles in liver tumorigenesis as a suppressor of tumor and invasion20. Our previous study has shown the gradual activation of WNT/β-catenin pathway through nuclear localization of β-catenin and loss of E-cadherin during kras -induced liver tumorigenesis21. In normal livers, β-catenin was localized in the cell membrane.…”

Conserved IL-2Rγc Signaling Mediates Lymphopoiesis in Zebrafish

“…This was subjected to semiquantitative RT-PCR with previously published primers for TCR-b chains (Vb1.5/17.5, Vb12) and Ig heavy chains (igVH1, igVH4) (46,47). Total RNA was also subjected to quantitative RT-PCR (QRT-PCR) with the following primers: il-2rgc.a 59-GTCACTGGTCTTGTATGCTGT and 59-GCTCTCACTATCACTGCTGG, il-2rgc.b 59-AGAAAGACCCAAGCCA-GT and 59-ATCTTTTTCCTCTCACAGTACC, jak3 59-AACAGAGC-GAGCAGCAGAGAG and 59-GTGTGACCACCCTTCCTTCC, pik3cg 59-AGGGGCACTTGTGATTGAG and 59-CTTCACTATTTCAATCATTC-CAA, b-actin 59-TGGCATCACACCTTCTAC and 59-AGACCATCACCA-GAGTCC and map2k1 and map2k2 (48). Data were normalized to b-actin, and fold change was calculated using the DDCt method.…”

Zebrafish Models of Human Liver Development and Disease

“…Numerous studies in zebrafish have shown that constitutive or inducible transgenic expression of oncogenic forms of the Ras/mitogen-activated protein kinase signaling cascade, often activated in human HCC, can induce zebrafish liver tumors. Expression of k-ras (V12), human Δ RAF1, and constitutively active xmrk (zebrafish homolog of epidermal growth factor receptor) lead to the development of HCC that is treatable with MEK/ERK and PI3K/AKT inhibitors (71, 108, 129, 130). Comparative gene expression studies have shown that human and zebrafish HCCs have generally conserved expression profiles, including similar markers of tumor progression (71, 99, 100, 129).…”