Page 50 - ebook HCC

48 PROGRAMME AND ABSTRACTS GENEVA, SWITZERLAND EASL HCC SUMMIT 49
FEBRUARY 13 - 16, 2014

NOTCH1 DRIVES SECRETOME SWITCHING IN CELL CYCLE, METABOLISM AND HCC
RAS-INDUCED SENESCENCE
Matthew Hoare , Michael Weekes , Yoko Ito , Nicholas Matheson , Suraj Menon , Pierre-Damien Denechaud , Lluis Fajas 1
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Rafik Salama , Robin Antrobus , Paul Lehner , Masashi Narita 1 1 Department of Physiology, University of Lausanne, Lausanne, Switzerland
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1 Cambridge Institute, Cancer Research UK, Cambridge Institte for Medical Research,
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University of Cambridge, Cambridge, United Kingdom Corresponding author’s e-mail: Lluis.Fajas@unil.ch
Corresponding author’s e-mail: matt_hoare20@hotmail.com
Introduction: Abnormal metabolic changes are a feature of tumour development and
Introduction: Oncogene-induced senescence (OIS) is a tumour suppressor mechanism cancer. This was already reported early in the last century. Otto Warburg (1928) observed
leading to stable cell-cycle arrest in response to unrestricted oncogene activation. OIS is that tumours have a higher rate of glucose metabolism than normal tissues. De novo
a heterogeneous phenotype involving multiple effector mechanisms, including secretion
of multiple factors such as IL-6 and TGF-β, that have pleiotropic context-dependent fatty acid biosynthesis is another hallmark of tumour cells, including liver cancer cells. We
effects. The secretome of senescent cells has been demonstrated to be important in show here new therapeutic targets in the lipid synthesis pathway in cancer cells, and we
hepatocarcinogenesis. elucidate the molecular mechanisms implicated in the metabolic switch observed during
cancer development and progression. In this context, we show that cell cycle regulators
BASIC SPEAKERS ABSTRACTS the cell surface phenotype of Ras-induced senescence (RIS) in an HDF model. 1504 and/or activity of key proteins implicated in these processes. We prove that cancer cells BASIC SPEAKERS ABSTRACTS
are responsible for triggering the metabolic changes directly regulating the expression
Methodology: We utilised SILAC-based plasma membrane proteomics (PMP) to identify
proteins were identified from 4 independent replicates. From GO annotation, 59% were
change their lipid metabolism. Our data indicates that the cdk4-pRB-E2F1 axis is a
present at the cell surface. The correlation between PMP and transcriptomic changes was
major regulator of lipid synthesis in cancer cells. Furthermore E2F1 directly regulates the
significant (r =0.63). Ingenuity-based network enrichment analysis incorporating PMP
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expression of genes involved in glycolysis and in lipid synthesis pathways. We use models
and transcriptomics identified Notch1 in the top enriched networks during RIS.
of liver cancer and mice deficient for some of these cell cycle regulators to prove that
Results: Notch1, a highly conserved transmembrane receptor that determines cell fate,
oncogenes to sustain tumour growth.
was significantly up-regulated in RIS compared to control cells (3.1 – 3.4 fold, adj p=0.03, inhibition of lipid synthesis results in the abrogation of the ability of these factors and other
FDR=0.1). Upon activation, the cleaved Notch1 intracellular domain (N1ICD) translocates
to the nucleus, binds to cofactors including MAML1 and regulates transcription of target
genes, such as HES1 and HEYL. The up-regulation of plasma membrane Notch1 was
confirmed in both RIS and DNA damage senescence. Up-regulation of Notch1 was
dependent upon p53. However, in contrast to cell surface Notch1, the levels of its
activated form, N1ICD and downstream target genes were transiently up-regulated at an
early phase of RIS, but down-regulated at full senescence.
Inhibition of Notch1 signaling through expression of a dominant-negative MAML1 led to
a reduction in TGF-β, but increased RIS-associated expression of IL-1, IL-6 and IL-8.
Overexpression of N1ICD drove reciprocal secretome changes with reduced IL-1, IL-6
and IL-8 and increased TGF-β. Transcriptional profiling of Notch1- and Ras-expressing
HDFs confirmed the TGF-β-rich Notch1-driven secretome in distinction to the RIS-driven
secretome.
Conclusions: In conclusion, PMP has identified a cell-surface phenotype of RIS. Notch1
cell surface expression is up-regulated, but downstream signaling is dynamically regulated
in RIS. The transition to RIS is correlated with a switch from Notch1-driven TGF-β-rich
secretome to Ras-driven IL-1, -6 and -8 rich secretome. Appropriate regulation of Notch1
signaling is crucial to secretome composition in senescence.