Asiri, Abdulaziz
(2018)
Investigations into the role of Cten signalling in colorectal cancer.
PhD thesis, University of Nottingham.
Abstract
C-terminal tensin-like (Cten, also known as Tensin4) is the member of the tensin gene family. Cten functions as an oncogene in a variety of cancer types and its expression is commonly associated with poor prognosis and metastasis in colorectal cancer (CRC). Although several studies have shown that Cten has a critical role in the regulation of cell motility and invasion in different tumour tissues, the underlying signalling mechanisms have not been fully elucidated. This thesis investigated the biological activity of Cten in four different ways in order to further elucidate the mechanisms of Cten signalling in CRC cells.
Potential downstream targets of Cten signalling involved in the regulation of epithelial-to-mesenchymal transition (EMT) induced cell motility i.e. Rho-associated protein kinase1 (ROCK1), Src and Snail were investigated. Cten expression was manipulated in different cell lines using multiple approaches including forced expression, gene knockdown and constitutive depletion (through Crispr/Cas9 gene deletion) to eliminate artefacts of methodology and cell line specific effects. Snail, Src and ROCK1 were identified as novel downstream targets of Cten signalling and additionally, Cten was shown to increase the stabilisation of both Src and Snail proteins. The functional relevance of Cten-Snail, Cten-Src and Cten-ROCK1 signalling was assessed, and the overall findings demonstrated that Cten could promote cell motility and colony formation directly through the positive regulation of the Src/ROCK1/Snail dependent axis.
To gain a deeper insight into the mechanisms of Cten’s biological function, mutations, at two important residues (i.e. arginine 474 and tyrosine 479) in the Src homology 2 (SH2) domain of Cten were introduced into one construct (GFP-CtenR474A+Y479F) using site directed mutagenesis. These two residues in the SH2 domain of Cten were found to not only be important for interacting with Src, ROCK1, or Snail signalling, but also for regulating cell motility and colony formation efficiency.
Numerous Cten regulatory factors have been identified, however, little is known about how Cten is activated and regulated in cancer cells. The relationship between transforming growth factor beta 1 (TGFβ1) and Cten was investigated and stimulation of cells with TGFβ1 or knockdown of TGFβ1 resulted in changes in Cten expression as well as its downstream targets of ROCK1, Src, Snail, and N-cadherin. Furthermore, this positive interaction between TGFβ1 and Cten was functionally relevant and caused changes in cell motility. and the nuclear translocation of ROCK1, Src, and Snail protein increased by TGFβ1 is probably mediated via upregulation of the Cten signalling pathway
The biological function of Cten in the nucleus was further investigated and shown to increase nuclear localisation of Src, ROCK1, and Snail, further promoting the migratory capability and colony formation efficiency in CRC cells. Finally, Cten expression was shown to positively correlate with both ROCK1 and Src expression in a series of primary CRCs. This correlation was consistent with that observed following manipulation of Cten expression in CRC cell lines.
In conclusion, this study has revealed a number of novel findings regarding the biological function of Cten signalling in CRC. However, further validation of the findings may enhance the understanding of the role of Cten in the invasion-metastasis cascade in the future.
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