Dottorato in MEDICINA SPERIMENTALE

Titolo della tesi: Role of Lipid Rafts in the FGFR2c-mediated oncogenic signaling by involvement of TRPA1 channel in pancreatic ductal adenocarcinoma cells

The fibroblast growth factor receptor (FGFR) signaling pathway represents a key regulator of numerous cellular processes frequently deregulated in cancer. In pancreatic ductal adenocarcinoma (PDAC)-derived cells, the aberrant expression of the mesenchymal FGFR2 isoform, FGFR2c, promotes epithelial-to-mesenchymal transition (EMT), cell survival, and invasiveness through the activation of the PKCε-mediated signaling cascade. Recent studies have also demonstrated that other membrane proteins, including members of the transient receptor potential (TRP) channel family, can modulate receptor tyrosine kinase signaling. In the context of PDAC, it has recently been suggested that both TRPA1 and FGFR2 contribute to the regulation of cell motility and invasion. Moreover, some studies indicate the possible existence of a functional link between FGFR2 and TRPA1, which would also involve a direct interaction between the two and the consequent ligand-independent amplification of FGFR2 downstream signaling, as described in lung adenocarcinoma. However, another study proposed that the oncogenic role of TRPA1 primarily depends on its ion channel function and would be completely independent of any interaction with FGFR2; this mechanism has been reported in breast and lung cancers. Therefore, in this research project we focused our attention in PDAC context, investigating the contribution of TRPA1 to the FGFR2c/PKCε oncogenic axis in PDAC-derived cell lines and its possible involvement in the regulation of FGFR2c recruitment in plasma membrane lipid rafts, cholesterol-enriched membrane microdomains that function as signaling platforms in physiological and pathological conditions, including cancer. Biochemical and immunofluorescence analyses revealed that either the inhibition and the sustained ligand-dependent activation of the FGFR2c signaling modulates TRPA1 protein expression, suggesting a possible functional dependence between them. In addition, the transient silencing of TRPA1 was sufficient to reduce FGFR2c downstream ERK1/2/mTOR and MCL1/SRC oncogenic signaling, resulting in decreased EMT marker expression and cell invasive behaviour: all phenotypic traits displayed by PDAC-derived cells and significantly exacerbated by FGF2 stimulation only in those PDAC cell lines highly expressing FGFR2c. Focusing our attention on lipid rafts function, immunofluorescence and biochemical approaches revealed that FGF2 stimulation promoted the recruitment of the activated FGFR2c into lipid rafts, while raft disruption by methyl-β-cyclodextrin (MβCD) treatment markedly inhibited FGFR2c-mediated signaling and the enhancement of both EMT and cell invasive behavior. Finally, co-immunoprecipitation experiments combined with gene-silencing approaches demonstrated that, independently of its channel function, TRPA1 is required for the recruitment of the activated FGFR2c in the lipid rafts, ensuring a proper spatial organization and an efficient oncogenic signal transduction. Overall, these findings suggest that the tumorigenic activity of FGFR2c in PDAC is strictly dependent on both lipid raft integrity and TRPA1 presence. The functional cooperation among FGFR2c, TRPA1, and lipid rafts enables the coordinated activation of signaling pathways that promote and enhances EMT and cellular invasiveness. These results identify FGFR2c, TRPA1, and lipid rafts as potential synergistic therapeutic targets, whose modulation could represent a promising strategy for the development of innovative and effective treatments against PDAC progression and resistance.