|dc.description.abstracteng||Lung cancer continuous to be the leading cause of cancer-related death worldwide. A relatively new and effective approach of treating lung cancer is achieved through targeting specific molecular alterations in lung cancer cells. While effective molecular therapies have been developed and approved in adenocarcinoma of the lung (AC), squamous cell lung cancer (SQCLC) and small cell lung cancer (SCLC) still lack any approved molecular targets.
Fibroblast growth factor receptor 1 (FGFR1) is a promising molecular target in squamous cell lung cancer and small cell lung cancer subtypes. Clinical trials proved the principle of targeting FGFR1 in SQCLC patients harboring FGFR1 amplification. However, modest response rates of patients treated with FGFR1 inhibitors suggested presence of different layers of resistance, which are either pre-existed in patients or developed over the course of treatment.
In this thesis, I used a cohort of 421 lung cancer primary patient samples to screen prevalence of FGFR1 gene amplification among SQCLC and SCLC groups using fluorescence in situ hybridization technique (FISH). FGFR1 protein expression was screened among AC, SQCLC and SCLC groups using immunohistochemistry (IHC). The antibody used in IHC analysis was validated through CRISPR-Cas9 knockout system. FGFR1 gene amplification prevalence was compared to protein expression in the same set of patients. In order to understand molecular mechanisms that underline FGFR1 inhibition resistance in lung cancer cells, I established a model of resistant cell lines and compared them to control sensitive cell lines. FGFR1 inhibition resistance model consisted of intrinsic resistant lung cancer cell lines, induced resistant lung cancer single clones and a mutationally resistant lung cancer cell line. Furthermore, mass spectrometric phosphoproteomic analysis was performed to compare FGFR1 inhibition resistant cell lines to a control cell line under control and inhibition status. MTS viability assay together with other cell viability assays were used to validate the co-inhibition effect of FGFR1 alongside with other potential molecular targets in FGFR1 resistant lung cancer cells.
FISH analysis revealed 23% and 8% prevalence of FGFR1 gene amplification among SQCLC and SCLC patients. FGFR1 protein was strongly expressed in 9%, 4% and 35% of SQCLC, SCLC and AC patients, respectively. Statistical analysis showed no correlation between FGFR1 gene amplification and protein expression in lung cancer patients. Comparing activated signaling pathways between FGFR1 inhibition sensitive and resistant cell lines showed Akt activation as a significant difference between the two groups. MTS combination inhibition assays confirmed synergy between FGFR1 inhibition and Akt inhibition in intrinsic, induced and constitutively active Akt resistant lung cancer cell lines. Mass spectrometric phosphoproteomic analysis showed a common resistance pathway in intrinsic and induced resistant cells to FGFR1 inhibition. Over phosphorylated CD44, FAK, PAK1, Paxillin and Afadin proteins formed the core of the resistance pathway. MTS viability assays combined with Chou-Talalay combination index analyses confirmed synergy between FGFR1 and PAK1 or FAK inhibition in intrinsic and induced resistant lung cancer cell lines to FGFR1 inhibition. Finally, CD44 knockdown combined with FGFR1 inhibition showed significant reduction in proliferation of resistant cells compared to single treatments.
To conclude, the current thesis confirmed previously published prevalence of FGFR1 gene amplification (23% in SQCLC and 8% in SCLC) and protein expression (9% in SQCLC, 4% in SCLC and 35% in AC) in lung cancer patients. The thesis revealed that FGFR1 gene amplification seems not to be the most accurate selection criteria of patients who will benefit from FGFR1 inhibition therapy. Molecular and phosphoproteomic analyses alongside with functional validation assays suggested that CD44, PAK1, FAK and Akt are promising potential targets that could synergistically sensitize resistant lung cancer patients to FGFR1 inhibition.||de