Abstract:
Background: Cervical cancer although is highly preventable, is still the 4th most common cancer in women globally. World Health Organization estimate 640,000 new cases and 342,000 deaths in 2020, indicating the need for effective treatment at late stages of the disease, when it is usually diagnosed. Fibroblast growth factor (FGF) signaling plays an important role in angiogenesis, cell differentiation, migration, proliferation, wound repair and apoptosis. However, when FGF receptor (FGFR) signaling is deregulated, for example due to activating mutations, gene amplification and oncogenic fusions, it can contribute to the progression of many cancers, including cervical. Interestingly, cancers with aberrant FGFRs face a major challenge which is the development of resistance against the FGFR inhibitors.
To investigate possible mechanisms of drug resistance we have generated three drug resistant (DR) human cervical cancer cell lines (HCCCL; Caski, HeLa and SiHa) to an FGFR inhibitor, PD173074. The main aims of this project are to (1) characterise the PD173074-resistant cancer cervical cell lines and (2) investigate possible mechanism(s) of drug resistance by comparing their transcriptome to their equivalent wild type cell lines.
Methods: Cell proliferation, apoptosis and lateral cell migration were studied using Incucyte zoom system to examine the functional differences between the wild type and DR HCCCL. Western blot analysis were performed to investigate the activation of MAPK, AKT and S6 downstream signaling pathways upon stimulation with recombinant FGF2, FGF4 or FGF7 ligands in the presence and absence of the FGFR inhibitor, PD173074. Transcriptomic analysis were performed to detect differentially expressed genes (DEG) and their protein-protein interactions between DR and wild type HCCCLs. Validation of transcriptome was done by real-time PCR and Western blot using specific primers and antibodies for the identified genes and proteins.
Results: PD173074-resistant HCCCL had higher IC50, were more proliferative and migratory and less apoptotic than the wild type cells with and without PD173074. Biochemical studies revealed Phospho-ERK activation after FGF2, FGF4 and FGF7 stimulation in both the wild type and drug resistant cells. Interestingly, in the presence of the FGFR inhibitor the effect of the phosphorylation was abolished in both cell lines indicating that the DR HCCCL might still have some sensitivity to the drug, despite having higher IC50. However, there was no difference in Phospho AKT expression level at either Ser(473) or Thr (308) sites in the presence or absence of the drug in both wild type and DR cell lines. Transcriptome analysis and subsequent validation via RT-PCR revealed five upregulated and nine downregulated DEGs in DR HCCCL as well as several protein-protein interactions between them. For example PHLDA1 was significantly dowregulated in DR HCCCLs and when overexpressed, DR cells had a less metastatic behavior similar to wild type. In contrast, PLCB4 gene was upregulated in DR HCCCLs.
Conclusion: Our data suggest that DR cell lines have a more metastatic signature compared to wild type HCCCLs. PHLDA1 and PLCB4 were two key genes that were highlighted as potential mechanisms of drug resistance in HCCCLs. Further investigation on the mechanisms of drug resistance will help to design therapeutic strategies to overcome it and improve treatment.
