Pancreatic ductal adenocarcinoma (PDAC) is a treatment-resistant malignancy driven by the "undruggable", constitutively active KRAS, which made the search for new therapeutic strategies bypassing this oncogenic substrate very urgent. Since the FGFR2 altered splicing and the triggering of PKCepsilon downstream signaling have been recently described by us as involved in the early steps of epidermal carcinogenesis, aim of this research project will be to assess if and to what extent these events could contribute to the induction of EMT, to the deregulation of autophagy and possibly to the control of their interplay in the context of pancreatic cancer cells. Biochemical, molecular and immunofluorescence approaches will be applied in different PDAC cell lines, selected for different expression of mesenchymal FGFR2c isoform and poor levels of its epithelial counterpart FGFR2b, in order to check the possible correlation between FGFR2c expression and the induction of EMT-related transcription factors in response to FGF2, as well as the modulation of epithelial/mesenchymal markers and changes in cell morphology compatible with the pathological EMT. Specific inhibitors for FGFR2 tyrosine kinase activity, such as SU5402, and receptor depletion via shRNA will be used to confirm the direct dependence of the events possibly observed on FGFR2c expression and signaling. Parallel experiments of gene silencing by shRNA will be also performed to assess the contribution of PKCepsilon and to dissect the signaling network downstream this PKC isoform possibly responsible for FGFR2c-dependent modulation of EMT and autophagy.
Overall, our research project aims to contribute to the identification of new molecular players at the crossroad between FGF/FGFR aberrant signaling, EMT and dysregulated autophagy that would represent new key biomarkers and targets for innovative therapeutic strategies aimed to counteract tumor development and/or to overcome RAS-dependent "undruggability" in PDAC.
PDAC is an aggressive carcinoma whose KRAS constitutive activation is the main hallmark for malignancy (Mollinedo and Gajate, 2019). However, considering the evidence that, in specific conditions, KRAS could be dispensable (Will et al., 2014; Muzumdar et al., 2017), research efforts have been recently focused on the identification of new signaling molecules and pathways, acting bypassing RAS, whose inhibition might significantly impact on PDAC cell malignant phenotype.
FGFR2 isoform switch is an additional oncogenic event occurring during pancreatic carcinogenesis, whose contribution in EMT induction and cell invasion still appears controversial (Ishiwata et al., 2012; Ueda et al., 2014; Ishiwata 2018). Therefore, with the aim to further clarify this topic, in this research proposal we firstly plan to verify if the extent of the expression of the EMT-related transcription factors activated downstream FGFR2c in other epithelial contexts (Ranieri et al., 2020), as well as a modulation of epithelial/mesenchymal markers compatible with a pathological EMT, will correlate with FGFR2c levels. In addition, a detailed biochemical analysis will highlight if the responsiveness to FGF2 in terms of intracellular signaling activation, improvement of EMT expression profile, as well as acquisition of a mesenchymal morphology could correlate to FGFR2c expression. Again, the FGFR2c signaling shut-off by SU5402 or the specific receptor depletion by shRNA will permit us to confirm the dependence of observed effects on receptor expression and signaling. The expected results could help to assess to what extent, in the in vivo context of pancreatic cancer, the levels of aberrant FGFR2c could affect tumor cell sensitivity to paracrine factors secreted by other microenvironmental cells, such as cancer associated fibroblasts (CAFs). Similar results would be in line with previous evidences, pointing on the relevance of CAFs and CAF-released factors, such as FGF2, in establishing more aggressive behaviors of pancreatic cancer cells (Ahrens et al., 2017; Awaji et al., 2019).
In this project we also aim to investigate the signaling pathways and substrates downstream FGFR2c possibly responsible for EMT-related phenotype, paying particular attention to PKCepsilon, whose role in FGFR2c-mediated EMT has been recently described by us in the context of human keratinocytes (Ranieri et al., 2020; Ranieri et al., 2021). In addition, PKCs are considered RAS-independent substrates of FGFRs (Touat et al., 2015). Therefore, the identification of one of a PKC family member as pivotal signaling effector in establishing of PDAC aggressive phenotype could represent a fundamental advance towards new therapeutic strategies in this specific cancer type in which constitutively active RAS has long been considered quite the exclusive, but unfortunately ¿undruggable¿ target. We also plan to investigate the possible role of PKCepsilon downstream FGFR2c in the modulation of autophagy: an additional process contributing to PDAC development and progression and exerting alternative, oncogenic or tumor suppressive function, depending on tumor stages and microenvironmental conditions (Piffoux et al., 2019; Mollinedo and Gajate, 2019; New and Tooze, 2020). The study of the involvement of PKCepsilon downstream FGFR2c could therefore help to clarify the articulated role played by this process in PDAC.
We also propose to investigate the molecular mechanisms downstream PKCepsilon, paying particular attention on the canonical autophagic negative regulator MTOR. In fact, in the light of what recently proposed in breast cancer (Basu, 2020), we have reason to believe that, also in PDAC cells, PKCepsilon could act on autophagy activating this pathway. The contribution of AKT, which is the canonical upstream activator of MTOR, will be also checked, together to that of ERK1/2 signaling which has been proposed to directly activate MTOR in PDAC tumor cells (Mollinedo and Gajate, 2019). Since ERK1/2 pathway is also a well-known signaling regulating EMT in PDAC (Huang et al., 2020; Safa, 2020), its involvement downstream PKCepsilon could evidence a key hub function of this PKC isoform at the crossroad between autophagy and EMT, via a direct convergence on ERK1/2. If so, the specific inhibition of PKCepsilon could revert EMT and simultaneously enhance autophagy, enhancing tumor cell dependence on this process for survival and consequently their susceptibility to autophagic inhibitors.
Overall, our research project could significantly help to the identification of new molecular players at the crossroad between FGF/FGFR aberrant signaling, EMT and dysregulated autophagy that would be new biomarkers and targets for innovative therapeutic strategies aimed to counteract the early steps of carcinogenesis and/or to overcome RAS-dependent ¿undruggability¿ of PDAC.