Thorpe, Hannah
(2016)
Investigating the role of Cten in metastatic colorectal cancer.
PhD thesis, University of Nottingham.
Abstract
Cten is upregulated in a number of tumour types and in colorectal cancer (CRC), expression is associated with advanced stage, poor prognosis and distant metastasis. Cten appears to regulate cell motility and it can confer features of stemness although, the knowledge of underlying signalling mechanisms is sparse. Cten is localised at focal adhesions and in the nucleus. It may promote cell motility through the regulation of cell adhesion and it may confer stemness through gene activation. To determine the biological activity of Cten in CRC, this thesis investigated three areas of Cten signalling.
Nuclear expression of Cten is more prevalent in liver metastasis than the primary tumour. In the nucleus, Cten binds to β-catenin, however, the relevance of this is not known. To investigate the function of Cten in the nucleus, CRC cell lines were transfected with nuclear targeted Cten (NLSCten) and cell proliferation, migration and colony formation efficiency assessed. Expression profiling was performed to identify the underlying molecular mechanisms and additionally, the effect of Cten on β-catenin transcriptional activity was explored. Nuclear localised Cten was associated with increased cell migration and colony formation efficiency however it was found likely that Cten did not induce this activity through the regulation of β-catenin transcriptional activity.
Activation of Epidermal growth factor receptor (EGFR) signalling in breast cell lines stimulates cell motility and upregulates Cten expression whilst simultaneously downregulating the expression of Tensin 3, known as the Tensin switch. Activity is mediated through signalling downstream to Deleted in liver cancer 1 (DLC1). This pathway was tested in CRC. Stimulation of CRC cell lines with recombinant Epidermal growth factor (EGF) and knockdown of Kirsten rat sarcoma viral oncogene homolog
(Kras) resulted in up and downregulation of Cten respectively. A Tensin switch was not observed and, unexpectedly, Tensin 3 was stabilised by Cten signalling. Additionally, DLC1 was not expressed in the majority of cell lines investigated. It is possible that these mechanisms are either tissue dependent or different pathways operate in normal and cancer cell lines.
Since Cten mediated cell migration was not regulated by a Tensin switch mechanism in CRC, other mechanisms of cell migration were sought. In CRC, Cten signalling downregulates E-cadherin expression. This prompted the investigation of Cten’s role in epithelial to mesenchymal transition (EMT) signalling. An EMT marker panel was investigated following the manipulation of Cten expression. Snail was identified as a novel target of Cten signalling and additionally, Cten was shown to promote the stabilisation of Snail protein. Furthermore, this signalling was functionally relevant and contributed to increased cell invasion and migration.
To investigate Cten signalling, expression was manipulated using a dual approach of Cten forced expression using a plasmid expression construct and siRNA mediated knockdown. To develop more refined models, the novel, Clustered regularly interspaced short palindromic repeats – CRISPR associated nuclease 9 (CRISPR-Cas9) genome editing technology was utilised to create a Cten knockout SW620 cell line (SW620CtenKO) thus providing an alternative or supplementary method to interrogate Cten signalling in CRC.
This study adds to the ongoing discussion of the role of Cten in cancer. It was confirmed that Cten was regulated by EGFR and Kras signalling but without a Tensin switch, however Cten did promote cell motility through the stabilisation of Snail suggesting that Cten may play a role in EMT processes. Finally, Cten displays enhanced oncogenic activity in the nucleus which may further promote metastasis. Further refinement of these pathways will increase the understanding of the invasion-metastasis cascade.
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