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| 086P London, UK Pharmacology 2017 |
Itch e3 ubiquitin ligase influences downstream receptor tyrosine kinase signalling in the oe33 cell line
Introduction: Deregulation of the receptor tyrosine kinases (RTKs) EGFR and Met is an important driver of cancer cell development and progression to metastasis. Trafficking regulates ERK1/2 and Akt signalling pathways; that have been implicated in cell survival, invasion and metastasis. Cell migration, metastasis and tumorigenesis have been shown to be promoted where mutations affect endocytic trafficking due to increased endosomal signalling (1). Advancements in our understanding of regulation of RTK signalling are essential to understand their role in cancer progression. Itch, an E3 ubiquitin ligase, has been shown to increase tumourgenicity in breast cancer (2) and affect EGFR signalling (3) with both receptor degradation and membrane trafficking regulated by ubiquitination.
Hypothesis: Here we investigated the role of the ubiquitin E3 ligase Itch in endocytosis and EGFR and Met downstream signalling in the oesophageal cancer cell line OE33.
Methods: Stable Itch shRNA knockdown cell lines were generated by transfection of oesophageal adenocarcinoma OE33 cells with lentivirus. For receptor tyrosine kinase (RTK) signalling experiments, cells were starved for 24 hours in RPMI with 0.1% sera before incubated with 2ng or 40ng/ml recombinant EGF or HGF, respectively, at given time points and lysates analysed by Western blotting and quantified by densitometry. The statistical significance was measured by paired Student’s t-test, with a p value of < 0.05 considered to be significant.
Results: Our results show Itch knockdown in OE33 cells affected ERK1/2 signalling downstream of the EGFR receptor, and not the Met receptor. Cell signalling assays demonstrated enhanced pERK signalling (p=0.0478, n=4) compared to CTRL Sh cells. Itch knockdown reduced proliferation when grown in 10% sera (p=0.0458, n=4), however under restricted mitogen conditions, Itch depletion enhanced proliferation (p=0.0155, n=4) and viability (p=0.009, n=4) compared to control. Itch was shown to bind to Intersectin-1 (Itsn1) in GST pull-down assays, a protein involved in clathrin-mediated endocytosis. Expression levels of Intersectin-1 have been shown to influence RTK internalisation (4), thus implicating a novel Itch-mediated regulatory mechanism for membrane trafficking.
Conclusion: Our study highlights the importance of the regulation of receptor signalling and membrane trafficking in oesophageal cancer to further understand their roles in cell survival, invasion and metastasis.
References:
1. Joffre, C. et al. (2011) Nat. Cell Bio., 13, 827-837.
2. Salah, Z. et al. (2014) Oncotarget, 5(11), 10886-10900.
3. Azakir, B.A. et al. (2009) Cell Signal., 21(8), 1326-1336
4. Martin, N.P. et al. (2006) Molecular Pharmacology., 70(5), 1643-1653.