Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis

Yannan Fan, Sehrish K. Bazai, Fabrice Daian, Maria Arechederra, Sylvie Richelme, Nuri A Temiz, Annie Yim, Bianca H. Habermann, Rosanna Dono, David A Largaespada, Flavio Maina

Research output: Contribution to journalArticle

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Abstract

Background & Aims: The variety of alterations found in hepatocellular carcinoma (HCC) makes the identification of functionally relevant genes and their combinatorial actions in tumorigenesis challenging. 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. Methods: 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 mice to 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 (through xenografts) functional screens were performed to assess the relevance of distinct cooperative modes to the tumorigenic properties conferred by RTKs. Results: We identified 275 genes, most of which are altered in patients with HCC. 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. Conclusion: 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 tumour permissive cellular environment allowing a large spectrum of deregulated mechanisms to initiate liver cancer. Lay summary: Receptor tyrosine kinases (RTKs) are among signals frequently deregulated in patients with hepatocellular carcinoma and their deregulation confers essential biological properties to cancer cells. 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.

Original languageEnglish (US)
Pages (from-to)470-482
Number of pages13
JournalJournal of Hepatology
Volume70
Issue number3
DOIs
StatePublished - Mar 1 2019

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Receptor Protein-Tyrosine Kinases
Mutagenesis
Carcinogenesis
Liver
Genes
Hepatocellular Carcinoma
Neoplasms
Liver Neoplasms
Proto-Oncogene Proteins c-met
Beauty
Chromatids
Chromatin Assembly and Disassembly
Insertional Mutagenesis
Ubiquitination
RNA Stability
Proteasome Endopeptidase Complex
Computational Biology
Heterografts
Small Interfering RNA
Cell Cycle

Keywords

  • Functional screening
  • HCC
  • Hepatocellular carcinoma
  • Liver cancer
  • Mouse model
  • Oncogenes
  • Receptor tyrosine kinases
  • Signalling
  • Tumour suppressors

PubMed: MeSH publication types

  • Journal Article

Cite this

Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis. / Fan, Yannan; Bazai, Sehrish K.; Daian, Fabrice; Arechederra, Maria; Richelme, Sylvie; Temiz, Nuri A; Yim, Annie; Habermann, Bianca H.; Dono, Rosanna; Largaespada, David A; Maina, Flavio.

In: Journal of Hepatology, Vol. 70, No. 3, 01.03.2019, p. 470-482.

Research output: Contribution to journalArticle

Fan, Yannan ; Bazai, Sehrish K. ; Daian, Fabrice ; Arechederra, Maria ; Richelme, Sylvie ; Temiz, Nuri A ; Yim, Annie ; Habermann, Bianca H. ; Dono, Rosanna ; Largaespada, David A ; Maina, Flavio. / Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis. In: Journal of Hepatology. 2019 ; Vol. 70, No. 3. pp. 470-482.
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T1 - Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis

AU - Fan, Yannan

AU - Bazai, Sehrish K.

AU - Daian, Fabrice

AU - Arechederra, Maria

AU - Richelme, Sylvie

AU - Temiz, Nuri A

AU - Yim, Annie

AU - Habermann, Bianca H.

AU - Dono, Rosanna

AU - Largaespada, David A

AU - Maina, Flavio

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N2 - Background & Aims: The variety of alterations found in hepatocellular carcinoma (HCC) makes the identification of functionally relevant genes and their combinatorial actions in tumorigenesis challenging. 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. Methods: 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 mice to 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 (through xenografts) functional screens were performed to assess the relevance of distinct cooperative modes to the tumorigenic properties conferred by RTKs. Results: We identified 275 genes, most of which are altered in patients with HCC. 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. Conclusion: 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 tumour permissive cellular environment allowing a large spectrum of deregulated mechanisms to initiate liver cancer. Lay summary: Receptor tyrosine kinases (RTKs) are among signals frequently deregulated in patients with hepatocellular carcinoma and their deregulation confers essential biological properties to cancer cells. 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.

AB - Background & Aims: The variety of alterations found in hepatocellular carcinoma (HCC) makes the identification of functionally relevant genes and their combinatorial actions in tumorigenesis challenging. 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. Methods: 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 mice to 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 (through xenografts) functional screens were performed to assess the relevance of distinct cooperative modes to the tumorigenic properties conferred by RTKs. Results: We identified 275 genes, most of which are altered in patients with HCC. 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. Conclusion: 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 tumour permissive cellular environment allowing a large spectrum of deregulated mechanisms to initiate liver cancer. Lay summary: Receptor tyrosine kinases (RTKs) are among signals frequently deregulated in patients with hepatocellular carcinoma and their deregulation confers essential biological properties to cancer cells. 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.

KW - Functional screening

KW - HCC

KW - Hepatocellular carcinoma

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KW - Receptor tyrosine kinases

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