RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death, with a 5-year survival of <10% and severely limited treatment options. PDAC hallmarks include profound metabolic acid production and aggressive local proliferation and invasiveness. This phenotype is supported by upregulated net acid extrusion and epithelial-to-mesenchymal transition (EMT), the latter typically induced by aberrant transforming growth factor-ß (TGFß) signaling. It is, however, unknown whether TGFß-induced EMT and upregulation of acid extrusion are causally related. Here, we show that mRNA and protein expression of the net acid extruding transporters Na+/H+ exchanger 1 (NHE1, SLC9A1) and Na+, HCO 3 - cotransporter 1 (NBCn1, SLC4A7) are increased in a panel of human PDAC cell lines compared to immortalized human pancreatic ductal epithelial (HPDE) cells. Treatment of Panc-1 cells (which express SMAD4, required for canonical TGFß signaling) with TGFß-1 for 48 h elicited classical EMT with down- and upregulation of epithelial and mesenchymal markers, respectively, in a manner inhibited by SMAD4 knockdown. Accordingly, less pronounced EMT was induced in BxPC-3 cells, which do not express SMAD4. TGFß-1 treatment elicited a SMAD4-dependent increase in NHE1 expression, and a smaller, SMAD4-independent increase in NBCn1 in Panc-1 cells. Consistent with this, TGFß-1 treatment led to elevated intracellular pH and increased net acid extrusion capacity in Panc-1 cells, but not in BxPC-3 cells, in an NHE1-dependent manner. Proliferation was increased in Panc-1 cells and decreased in BxPC-3 cells, upon TGFß-1 treatment, and this, as well as EMT per se, was unaffected by NHE1- or NBCn1 inhibition. TGFß-1-induced EMT was associated with a 4-fold increase in Panc-1 cell invasiveness, which further increased ~10-fold upon knockdown of the tumor suppressor Merlin (Neurofibromatosis type 2). Knockdown of NHE1 or NBCn1 abolished Merlin-induced invasiveness, but not that induced by TGFß-1 alone. In conclusion, NHE1 and NBCn1 expression and NHE-dependent acid extrusion are upregulated during TGFß-1-induced EMT of Panc-1 cells. NHE1 upregulation is SMAD4-dependent, and SMAD4-deficient BxPC-3 cells show no change in pHi regulation. NHE1 and NBCn1 are not required for EMT per se or EMT-associated proliferation changes, but are essential for the potentiation of invasiveness induced by Merlin knockdown.
RESUMO
Primary cilia are microtubule-based sensory organelles projecting from most quiescent mammalian cells, which disassemble in cells cultured in serum-deprived conditions upon re-addition of serum or growth factors. Platelet-derived growth factors (PDGF) are implicated in deciliation, but the specific receptor isoforms and mechanisms involved are unclear. We report that PDGFRß promotes deciliation in cultured cells and provide evidence implicating PLCγ and intracellular Ca(2+) release in this process. Activation of wild-type PDGFRα alone did not elicit deciliation. However, expression of constitutively active PDGFRα D842V mutant receptor, which potently activates PLCγ (also known as PLCG1), caused significant deciliation, and this phenotype was rescued by inhibiting PDGFRα D842V kinase activity or AURKA. We propose that PDGFRß and PDGFRα D842V promote deciliation through PLCγ-mediated Ca(2+) release from intracellular stores, causing activation of calmodulin and AURKA-triggered deciliation.
