Researchers have identified mechanisms of genetic cooperation at the root of liver tumours.

We have applied a genetic method to randomly mutate large numbers of genes in the context of a mouse model with increased RTK levels, predisposed to develop liver cancer. We identified mechanisms that accelerate tumour formation in cooperation with enhanced RTK levels. The wide array of cellular functions among these cooperators illustrates an extraordinary capability of RTKs to render the liver more vulnerable to additional alterations, by priming cells for tumour initiation.

 

Evaluating the landscape of gene cooperativity with RTKs in liver tumorigenesis

(A) The scheme illustrates a remarkable correlation between the expression levels of identified oncogenes and the expression levels of the RTK MET in HCC patients. Each column corresponds to a HCC patient and each line to an oncogene. The intersection highlights if a given oncogene is overexpressed in a patient (red square) or not (white square). HCC patients are subdivided in two groups: those with high levels of MET (109 patients) and those with reduced MET levels (31 patients). (B) The scheme illustrates how the cooperation between altered genes with RTKs leads to liver tumour formation. Slightly increased RTK MET expression levels in the liver (genetically induced in transgenic mice) generate a vulnerable context allowing altered genes belonging to distinct cellular functions to trigger liver tumorigenesis. The alteration of genes (red squares) has been triggered through the mobilisation of transposons (red circles) using the “Sleeping Beauty” system.

(A) The scheme illustrates a remarkable correlation between the expression levels of identified oncogenes and the expression levels of the RTK MET in HCC patients. Each column corresponds to a HCC patient and each line to an oncogene. The intersection highlights if a given oncogene is overexpressed in a patient (red square) or not (white square). HCC patients are subdivided in two groups: those with high levels of MET (109 patients) and those with reduced MET levels (31 patients).
(B) The scheme illustrates how the cooperation between altered genes with RTKs leads to liver tumour formation. Slightly increased RTK MET expression levels in the liver (genetically induced in transgenic mice) generate a vulnerable context allowing altered genes belonging to distinct cellular functions to trigger liver tumorigenesis. The alteration of genes (red squares) has been triggered through the mobilisation of transposons (red circles) using the “Sleeping Beauty” system.

Summary. The variety of alterations found in hepatocellular carcinoma (HCC) challenges the identification of functionally relevant genes and their combinatorial actions in tumorigenesis. Deregulation of Receptor Tyrosine Kinases (RTKs) is frequent in HCC, yet little is known about the molecular events that cooperate with RTKs and whether these cooperative events play an active role at the root of liver tumorigenesis. A forward genetic screen was performed using Sleeping Beauty transposon insertional mutagenesis to accelerate liver tumour formation in a genetic context in which subtly increased MET RTK levels predispose towards tumorigenesis. Systematic sequencing of tumours identified common transposon insertion sites, thus uncovering putative RTK cooperators for liver cancer. Bioinformatic analyses were applied to transposon outcomes and human HCC datasets. In vitro and in vivo functional screens were performed to assess the relevance of distinct cooperative modes to confer tumorigenic properties with RTKs.

We identified 275 genes, most of which are altered in HCC patients. Unexpectedly, these genes are not restricted to a small set of pathway/cellular processes, but cover a large spectrum of cellular functions, including signalling, metabolism, chromatin remodelling, mRNA degradation, proteasome, ubiquitination, cell cycle regulation, and chromatid segregation. We validated 15 tumour suppressor candidates, as shRNA-mediated targeting confers tumorigenicity to RTK-sensitized cells, but not to cells with basal RTK levels. This demonstrates that the context of enhanced RTK levels is essential for their action in tumour initiation. Our study identifies unanticipated genetic interactions underlying gene cooperativity with RTKs in HCC. Moreover, these results show how subtly increased levels of wild-type RTKs provide a permissive context allowing a large spectrum of deregulated mechanisms to initiate liver cancer.

 

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