Mahmood, Hibe-Tun-Noor Afshan (2020) FGFR signalling in human cervical cancer cell lines. (PhD thesis), Kingston University, .
Abstract
Fibroblast Growth Factors (FGFs) play a vital role in development and tissue regeneration. There are at least 23 distinct members of the FGF family that bind to at least four tyrosine kinase receptors known as FGF Receptors (FGFRs). FGF/FGFR signalling plays an important role in variety of processes and is tightly regulated. However, some cancer cells hijack the control of FGFR pathway, causing deregulation of FGFR signalling. FGF and their receptors, such as FGFR1 and 2, have been involved in susceptibility of breast cancer and progression as well as in a number of other cancers. Cervical cancer (CC) is one of the most commonly diagnosed cancers in women worldwide. One of the main causative agents of CC is the human papillomavirus (HPV). However, not all females infected with HPV develop CC demonstrating that other factors might be important. Although some studies have emphasised the possible involvement of FGFR signalling in CC, the aetiology and the molecular mechanisms related to CC and FGFR signalling remain poorly understood. The main aim of this study was to investigate FGFR signalling in cervical cancer cell lines (HeLa, SiHa and CaSki cell lines; CCCLs). A detailed understanding of the mechanism correlated to CC will make possible to use specific FGFR inhibitors that are currently in clinical trial, for CC treatment either on its own or in conjunction with the existing treatment, in order to increase success rates. In addition, FGFR signalling could be of early prognostic value. In order to examine gene and protein expression of FGFRs and associated ligands in CCCLs, PCR and immunocytochemistry were performed. Downstream extracellular signal-regulated protein kinase (ERK) signalling in CCCLs was investigated using Western blotting. Functional FGFR studies carried out through cell proliferation, migration and apoptosis assays. The above experiments were performed using stimulated and unstimulated CC cells in the presence and absence of FGFR inhibitors PD173074 and SU5402. RNAi mediated FGFR1 and FGFR2 knockdown was also performed to further assess function. The human CCCLs expressed FGFR1, FGFR2, FGFR4, FGF2, FGF4 and FGF7 mRNA and protein, with ‘b’ and ‘c’ isoforms of FGFR1 and FGFR2 confirmed using gene expression analysis. The FGFR1 and FGFR2 protein in CCCLs was localised within the nucleus with a distinct speckled arrangement. Importantly, ELISA revealed that all CCCLs secreted FGF2 ligand only, but not FGF4 and FGF7 suggesting that FGFR was activated in a paracrine manner. Activation of the ERK pathway occurred in response to FGF2, 4 and 7 exposures in all three CCCLs and the activation was eradicated in the presence of PD173074 or SU5402 inhibitor, suggesting that the effect is specific to FGFR signalling. Functional studies revealed that FGFR signalling enhanced cell proliferation and migration. Furthermore, knockdown of FGFR1 and FGFR2 by RNA interference or blockade using PD173074 inhibited cell proliferation, cell migration and also promoted the apoptosis CC cells. Drug resistant CCCLs (HeLa, CaSki and SiHa) were established following long-term exposure to PD17307. Collectively, this work supports an association between CC progression and FGFR signalling, laying the foundations for developing FGFR inhibitors for CC therapy. The drug resistant CCCLs will be valuable for further investigations into the mechanisms involved in acquired drug resistance.
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