Browsing by Author "Reshkin, Stephan Joel"

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease with a very poor prognosis, due to the influence of the tumor stroma, which promotes tumor growth, early invasion and chemoradiation resistance. Efforts to develop models for identifying novel anticancer therapeutic compounds have been hampered by the limited ability of in vitro models to mimic these in vivo tumor-stroma interactions. This has led to the development of various three-dimensional (3D) culture platforms recapitulating the in vivo tumor-stroma crosstalk and designed to better understand basic cancer processes and screen drug action. However, a consensus for different experimental 3D platforms is still missing in PDAC. We compared four PDAC cell lines of different malignancy grown in 2D monolayers to three of the more commonly used 3D techniques (ultralow adhesion concave microwells, Matrigel inclusion and organotypic systems) and to tumors derived from their orthotopic implantation in mice. In these 3D platforms, we observed that cells grow with very different tumor morphologies and the organotypic setting most closely resembles the tumor cytoarchitecture obtained by orthotopically implanting the four cell lines in mice. We then analyzed the molecular and cellular responses of one of these cell lines to epidermal growth factor receptor (EGFR) stimulation with EGF and inhibition with erlotinib and found that only in the 3D platforms, and especially the organotypic, cells: i) responded to EGF by changing the expression of signalling components underlying cell-stroma crosstalk and tissue architecture, growth, invasion and drug resistance (E-cadherin, EGFR, ezrin, beta 1 integrin, NHERF1 and HIF-1 alpha) similar to those reported in vivo; ii) had stimulated growth and increased erlotinib sensitivity in response to EGF, more faithfully mimicking their known in vivo behaviour. Altogether, these results, indicate the organotypic as the most relevant physiological 3D system to study the complex tumor stroma interactions driving progression and determining chemio-resistance.

As potential autocrine or paracrine factors, extracellular nucleotides are known to be important regulators of renal ion transporters by activating cell surface receptors and intracellular signaling pathways. We investigated the influence of extracellular adenine nucleotides on Na+/H+ exchanger isoform 3 (NHE3) activity in A6-NHE3 cells. This is a polarized cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3, which is expressed on the apical membrane. Basolateral addition of the P2Y(1) agonist, 2-Me-SADP, induced an inhibition of NHE3 activity, which was prevented by preincubation with selective P2Y(1) antagonists, MRS 2179 (N-6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS 2286 (2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane- 1,3-bisoxy(diammoniumphosphate)). NHE3 activity was also significantly inhibited by ATP and ATP-gamma-S but not by UTP. 2-Me-SADP induced a P2Y(1) antagonist-sensitive increase in both [Ca2+]i and cAMP production. Pre-incubation with a PKC inhibitor, Calphostin C, or the calcium chelator BAPTA-AM, had no effect on the 2-MeSADP-dependent inhibition of NHE3 activity, whereas this inhibition was reversed by either incubation with the PKA inhibitor H89 or by mutation of two PKA target serines (S552 and S605) on NHE3. Pre-incubation of the A6-NHE3 cells with the synthetic peptide, Ht31, which prevents the binding between AKAPs and the regulatory PKA subunits RII, also prevented the 2-MeSADP-induced inhibition of NHE3. We conclude that only the cAMP/PKA pathway is involved in the inhibition of NHE3 activity.

Although Cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to regulate the activity of NHE3, the potential reciprocal interaction of NHE3 to modulate the protein kinase A (PKA)-dependent regulation of CFTR in epithelial cells is still unknown. In the present work, we describe experiments to define the interactions between CFTR and NHE3 with the regulatory, scaffolding protein, NHERF that organize their PKA-dependent regulation in a renal epithelial cell line that expresses endogenous CFTR. The expression of rat NHE3 significantly decreased PKA-dependent activation of CFTR without altering CFTR expression, and this decrease was prevented by mutation of either of the two rat NHE3 PKA target serines to alanine (S552A or S605A). Inhibition of CFTR expression by antisense treatment resulted in an acute decrease in PKA-dependent regulation of NHE3 activity. CFTR, NHE3, and ezrin were recognized by NHERF-2 but not NHERF-1 in glutathione S-transferase pull-down experiments. Ezrin may function as a protein kinase A anchoring protein (AKAP) in this signaling complex, because blocking the binding of PKA to an AKAP by incubation with the S-Ht31 peptide inhibited the PKA-dependent regulation of CFTR in the absence of NHE3. In the A6-NHE3 cells S-Ht31 blocked the PKA regulation of NHE3 whereas it now failed to affect the regulation of CFTR. We conclude that CFTR and NHE3 reciprocally interact via a shared regulatory complex comprised of NHERF-2, ezrin, and PKA.