Identification of membrane associated proteins as potential markers for the DLKP clonal subpopulations
McCann, Andrew (2016) Identification of membrane associated proteins as potential markers for the DLKP clonal subpopulations. PhD thesis, Dublin City University.
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Lung cancer is one of the major causes of cancer death worldwide, it represents a heterogeneous group of tumours with distinct morphological, histological and molecular
features reflected by varied clinical outcome and response to treatment. DLKP is a cell line originating from a lymph node metastasis of a primary lung tumour histologically described as a “poorly differentiated squamous cell carcinoma”. DLKPSQ, DLKPI and DLKPM are three distinct subpopulations derived from DLKP, which are morphologically and phenotypically different from each other. The research outlined in this thesis aims to identify membrane associated proteins that could be used as potential markers for the DLKP clones, but also to identify potentially novel proteins associated
with lung cancer.
Cell surface protein isolation and label-free mass spectrometry analysis were performed on proteins isolated from DLKP and the DLKP clones. Eight proteins were successfully validated in the DLKP clones; AHNAK, HDGF, ROBO2, SLIT2, ALCAM, IQGAP1, INA and SPR, using Western blot analysis, Immunofluorescence and Immunocytochemistry. Their expression was also investigated in various tumour types
and representative cell lines. SPR is associated with poor survival of patients with Luminal A and basal-like breast cancers and also shows high expression across triple negative breast cancer subtypes. siRNA mediated knockdown studies of three candidate proteins. ALCAM, INA and SPR knockdown led to a significant reduction in the invasion and migration of DLKPM cells, indicating a potential functional role for these proteins in
lung cancer invasion/migration.
A pilot in vivo study using severe combined immune-deficient (SCID) mice to investigate the growth of DLKP and its clones resulted in all four cell lines forming tumours following implantation. Tumours derived from DLKP and DLKPI were the fastest growing and largest tumours, DLKPM formed smaller solid tumours following an initial lag phase, while DLKPSQ formed large, highly vascularised tumours. Immunohistochemical analysis of the xenografts derived from all four cell lines revealed; strong Ki67 immunoreactivity, negligible CD31 immunoreactivity and strong N-cadherin immunoreactivity. Explant culture revealed a significant reduction in migratory capacity
of DLKPSQ cells, but also a significant reduction in invasion/migration of DLKPM cells. Western blot analysis and immunohistochemical analysis indicated that expression of SLIT2, ALCAM, IQGAP1, INA and SPR were maintained in vivo.
The research presented demonstrates that proteomic analysis of cell surface isolates from the DLKP variants can lead to the identification of proteins with potential functional roles in cancer. AHNAK, SLIT2, ALCAM, and SPR were identified as potential markers capable of distinguishing between each of the DLKP clones. In addition, SPR may represent a novel target for triple negative breast cancer. The DLKP cell line model could
also prove to be an invaluable model to study the effects of potential novel therapeutics in lung cancer, in vivo.
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