Role of FGFR2c and Its PKCε Downstream Signaling in the Control of EMT and Autophagy in Pancreatic Ductal Adenocarcinoma Cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a treatment-resistant malignancy characterized by a high malignant phenotype including acquired EMT signature and deregulated autophagy. Since we have previously described that the aberrant expression of the mesenchymal FGFR2c and the triggering of the downstream PKCε signaling are involved in epidermal carcinogenesis, the aim of this work has been to assess the contribution of these oncogenic events also in the pancreatic context. Biochemical, molecular and immunofluorescence approaches showed that FGFR2c expression impacts on PDAC cell responsiveness to FGF2 in terms of intracellular signaling activation, upregulation of EMT-related transcription factors and modulation of epithelial and mesenchymal markers compatible with the pathological EMT. Moreover, shut-off via specific protein depletion of PKCε signaling, activated by high expression of FGFR2c resulted in a reversion of EMT profile, as well as in a recovery of the autophagic process. The detailed biochemical analysis of the intracellular signaling indicated that PKCε, bypassing AKT and directly converging on ERK1/2, could be a signaling molecule downstream FGFR2c whose inhibition could be considered as possible effective therapeutic approach in counteracting aggressive phenotype in cancer.

The FGFR2c/PKCε Axis Controls MCL-1-Mediated Invasion in Pancreatic Ductal Adenocarcinoma Cells: Perspectives for Innovative Target Therapies

“…To analyze the contribution of FGFR2c and of its downstream PKCε signaling in the establishment of late oncogenic features, such as cell invasion, in PDAC tumor cells, we first confirmed the relationship between FGF2-mediate activation of FGFR2c and PKCε signaling and their dependence on FGF2c expression levels. To this aim, stable protein depletion of FGFR2 or PKCε by short hairpin RNA (shRNA) was performed in PANC-1 and MIAPaCa-2: two PDAC cell lines expressing opposite levels of the mesenchymal FGFR2c isoform, and very poor levels of the epithelial FGFR2b variant [ 14 , 15 , 16 ]. Previous experiments performed by us using both FGFR2 shRNA- and FGFR2 isoform-specific FGFR2b and FGFR2c small interfering RNAs (siRNA), have assessed that, especially in PANC-1 cells, the generic FGFR2 shRNA efficiently caused a specific depletion of the FGFR2c variant [ 14 ].…”

“…To this aim, stable protein depletion of FGFR2 or PKCε by short hairpin RNA (shRNA) was performed in PANC-1 and MIAPaCa-2: two PDAC cell lines expressing opposite levels of the mesenchymal FGFR2c isoform, and very poor levels of the epithelial FGFR2b variant [ 14 , 15 , 16 ]. Previous experiments performed by us using both FGFR2 shRNA- and FGFR2 isoform-specific FGFR2b and FGFR2c small interfering RNAs (siRNA), have assessed that, especially in PANC-1 cells, the generic FGFR2 shRNA efficiently caused a specific depletion of the FGFR2c variant [ 14 ]. The efficiency of the gene silencing performed by FGFR2 and PKCε shRNAs was assessed by Western blot analysis ( Figure 1 A), which also confirmed the evident divergence of the two cell lines in terms of FGFR2c expression ( Figure 1 A, left panel).…”

“…PDAC-derived tissues and cell lines display different patterns and amounts of the four FGFR family members, with FGFR1 and FGFR2 appearing the most involved in oncogenesis and FGFR4 playing an opposite tumor-suppressive function [ 12 ]. Interestingly, the involvement of FGFR1 has been specifically described in the MIAPaCa-2 cell line, which poorly expresses FGFR2 [ 13 ], while we recently highlighted a crucial involvement of the mesenchymal FGFR2c isoform in Pancreatic Adenocarcinoma 1 cells (PANC-1), which highly express this receptor [ 14 ]. FGFR1 and FGFR2c enhance alternative oncogenic pathways (Protein Kinase B (Akt)/Myelocytomastosis Oncogene (MYC) and PKCε/Extracellular Signaling-Regulated Kinase 1-2 (ERK1-2), respectively), both potentially bypassing RAS signaling, and resulting in different oncogenic outcomes (increased cell proliferation and enhanced EMT, coupled with dysregulated autophagy, respectively) [ 13 , 14 ].…”

“…Interestingly, the involvement of FGFR1 has been specifically described in the MIAPaCa-2 cell line, which poorly expresses FGFR2 [ 13 ], while we recently highlighted a crucial involvement of the mesenchymal FGFR2c isoform in Pancreatic Adenocarcinoma 1 cells (PANC-1), which highly express this receptor [ 14 ]. FGFR1 and FGFR2c enhance alternative oncogenic pathways (Protein Kinase B (Akt)/Myelocytomastosis Oncogene (MYC) and PKCε/Extracellular Signaling-Regulated Kinase 1-2 (ERK1-2), respectively), both potentially bypassing RAS signaling, and resulting in different oncogenic outcomes (increased cell proliferation and enhanced EMT, coupled with dysregulated autophagy, respectively) [ 13 , 14 ]. These observations strongly suggest that the FGFR expression profile could significantly contribute to PDAC tumor cell plasticity, determining the response to paracrine FGFs and consequently the efficacy of the FGF/FGFR axis-based target therapies.…”

Functional mechanism of hsa-miR-128-3p in epithelial-mesenchymal transition of pancreatic cancer cells via ZEB1 regulation

“…We will further investigate the expression levels of miR-128-3p and ZEB1 and their correlation in clinical samples in future studies. Apart from ZEB1, there are too many genes such as ACTR 3 and FGFR2c ( Hu et al., 2021 ; Ranieri et al., 2021 ) that play regulatory roles in EMT in PC to list. These are not the downstream targets of miR-128-3p and are associated with other regulatory signaling pathways that fall outside of this study.…”