I: Initial autophagic vacuole; AVd: degradative autophagic vacuole; M: mitochondrion; Nu: nucleus; NM: nuclear membrane; PM: plasma membrane. Bars: 1 , 200 nm. Original blots see Figure S4.Spautin-1 Autophagy Cancers 2021, 13,14 of3.5. PKC Signaling Interferes with Autophagy Converging on ERK1/2 Pathway To clarify the molecular mechanisms underlying the involvement of PKC within the autophagic course of action, we focused our interest on MTOR, which can be regarded the principle damaging regulator of autophagy also in pancreatic cancer cells [2,14]. Western blot analysis revealed that the phosphorylation of MTOR, too as that of its substrate S6K, evident just after FGF2 stimulation particularly in PANC-1 cells (Figure 6A), have been strongly dampened by PKC knockdown (Figure 6A). Surprisingly, no corresponding effects were observed around the AKT phosphorylation (Figure 6B). Due to the fact AKT is definitely the upstream substrate generally responsible for MTOR activation, our unexpected final results indicated that PKC may possibly activate MTOR through an alternative pathway. This possibility seems to become consistent using the peculiar ability, previously described for PKC in other cellular contexts, to converge on MTOR by way of the activation of Raf/MEK/ERK signaling [25]. Essentially, the significant contribution of ERK1/2 signaling in MTOR activation and consequent autophagy inhibition has been broadly described in pancreatic cancer cells [2]. Determined by these assumptions, we investigated the influence of PKC signaling on ERK1/2 pathway. Biochemical evaluation showed that, in consequence of PKC depletion, the enhance of ERK1/2 phosphorylation in response to FGF2, visible in both pancreatic cell lines (Figure 6C), was decreased in Mia PaCa-2, which maintained a considerable residual ERK phosphorylation (Figure 6C), but absolutely abolished in PANC-1 (Figure 6C). The se final results indicate that the unique expression of FGFR2c displayed by the two PDAC cell lines influence on the dependence on PKC of ERK1/2 signaling. It’s also worth noting that shFGFR2c transduced MiaPaCa-2 cells displayed a higher responsiveness to FGF2 in terms of ERK1/2 phosphorylation when compared with non-transduced ones (see Figure 1B in comparison with Figure 6C), even when this phosphorylation remains significantly decrease than that shown by PANC-1 cells. This variability of MiaPaCa-2 cell response to FGF2 may possibly be the consequence of distinctive AZD4573 MedChemExpress culture conditions. The se benefits indicated that, only in PANC-1 cells, the activation of ERK1/2 pathway upstream is dependent upon PKC activation. Considering that ERK1/2 can also be a wellknown pathway involved in EMT of PDAC cells [4], our results suggest the possibility that, in this tumor context, PKC signaling, when activated in consequence of highly expression of FGFR2c, could simultaneously repress autophagy and orchestrate the EMT plan straight converging on ERK1/2 pathway.Cancers 2021, 13,15 ofFigure 6. PKC signaling shut-off by PKC protein depletion interferes with each MTOR and ERK1/2 signaling pathways. PANC-1 and Mia PaCa-2 cells stably transduced with PKC shRNA or with an unrelated shRNA were left untreated or stimulated with FGF2 as above. (A) Western blot evaluation shows that the enhance of phosphorylation of MTOR and S6K, evident soon after FGF2 stimulation only in PANC-1 cells, are strongly dampened by PKC knockdown. (B) No correspondingCancers 2021, 13,16 ofeffects are observed around the AKT phosphorylation. (C) The raise of ERK1/2 phosphorylation in response to FGF2, visible in both pancreatic cell lines, is significantly greater.
