The influence of extracellular - originating signals on THEmTOR / mTORC1 signalling pathway to autophagy induction in HOSCC
Date
2013-07-29
Authors
Nerwich, Ari Nathan
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Abstract
Cell-extracellular matrix (ECM) detachment triggers a cell survival mechanism known as
autophagy. A link between attachment and autophagy suggests a form of adhesion-based
regulation, involving mechanotransduction of extracellular-originating signals to the cellular
machinery controlling autophagy induction. This implies a role for integrin-linked kinase
(ILK), which transmits mechanical stimuli to the mammalian target of rapamycin (mTOR)
signalling pathway. Cells with a propensity for metastasis may negate these adhesive signals,
inducing autophagy inappropriately. Metastasis is a hallmark of transformation frequently
associated with human oesophageal squamous cell carcinoma (HOSCC). Additionally,
hyperactive mTOR/mTORC1 signalling correlates increasingly with HOSCC. Therefore, the
protein expression of significant signal transduction pathway intermediates was investigated
in response to both soluble and ECM-originating stimuli. Measurements by SDS-PAGE and
western-blotting coupled to semi-quantitative densitometry, during standard tissue culture
conditions, revealed that HOSCC’s expressed moderate-to-high levels of mTOR, p-RPS6(Ser
235/236) and mATG-13; indicating elevated levels of autophagy induction despite aberrant
signalling through mTOR/mTORC1. Additionally, an 80 kDa mTORβ isoform was identified
in HOSCC cells with lower mTOR abundance, presumably to maintain aberrant mTORC1
signalling. A canonical role for the PI3K/PKB pathway was also identified; where autophagy
induction accompanied diminished mTORC1 signalling in response to specific PI3K
inhibition with LY294002 and serum withdrawal. However, autophagy induction varied in
response to a dose-dependent decrease in mTORC1 signalling after exposure of HOSCC cells
to rapamycin. Moreover, specific inhibition of p90RSK with BI-D1870, suggests that
mTORC1 phosphorylates RPS6(Ser 235/236) in the absence of MAPK signals. Furthermore,
ectopic ILK expression indicated an enhanced potential for adhesion-based signalling.
Correspondingly, HOSCC cells commonly increased mTOR and p-RPS6(Ser 235/236) expression
following growth on fibronectin or collagen. However, co-immunoprecipitation analysis
revealed that signals transduction to mTOR precludes a direct interaction with ILK or FAK.
Rather, ECM-modulation of mTOR occurs in a integrin-triggered, but PI3K-depedant
manner; since specific inhibition of PI3K negated fibronectin-induced increases of mTOR
concentration and RPS6(Ser 235/236) phosphorylation. Thus, these data strongly suggest mTOR
is a target for adhesion-based signal transduction, where the ECM influences cell survival
through mTORC1. Moreover, exploitation of autophagy induction post cell-ECM detachment
in HOSCC may promote the survival of metastases during dissemination.
Description
A dissertation submitted to the Faculty of Science, University of the
Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree
of Master of Science.
Johannesburg, 2012