Audrey Ferrand

Direct binding of p85 to sst2 somatostatin receptor reveals a novel mechanism for inhibiting PI3K pathway

Phosphatidylinositol 3‐kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein‐coupled receptor (GPCR) sst2, has potent proapoptotic and anti‐invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a pre‐existing complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated‐Y71 residue of a p85‐binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH‐terminal SH2 as direct interacting domains. Somatostatin‐mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y71 residue. Mutating sst2‐Y71 disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2s ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand‐activated GPCR of PI3K‐dependent survival pathways, which may be an important molecular target for antineoplastic therapy.

A gastrin precursor, gastrin-gly, upregulates VEGF expression in colonic epithelial cells through an HIF-1-independent mechanism.

One of the major angiogenic factor released by tumor cells is VEGF. Its high expression is correlated with poor prognosis in colorectal tumors. In colon cancer, gastrin gene expression is also upregulated. In these tumors, gastrin precursors are mainly produced and act as growth factors. Recently, a study has also shown that the gastrin precursor, G‐gly induced in vitro tubules formation by vascular endothelial cells suggesting a potential proangiogenic role. Here, we demonstrate that stimulation of human colorectal cancer cell lines with G‐gly increases the expression of the proangiogenic factor VEGF at the mRNA and protein levels. In addition, blocking the progastrin autocrine loop leads to a downregulation of VEGF. Although HIF‐1 is a major transcriptional activator for VEGF our results suggest an alternative mechanism for VEGF regulation in normoxic conditions, independent of HIF‐1 that involves the PI3K/AKT pathway. Indeed we show that G‐gly does not lead to HIF‐1 accumulation in colon cancer cells. Moreover, we found that G‐gly activates the PI3K/AKT pathway and inhibition of this pathway reverses the effects of G‐gly observed on VEGF mRNA and protein levels. In correlation with these results, we observed in vivo, on colon tissue sections from transgenic mice overexpressing G‐gly, an increase in VEGF expression in absence of HIF‐1 accumulation. In conclusion, our study demonstrates that gastrin precursors, known to promote colon epithelial cells proliferation and survival can also contribute to the angiogenesis process by stimulating the expression of the proangiogenic factor VEGF via the PI3K pathway and independently of hypoxia conditions.

The G-protein-coupled CCK2 receptor associates with phospholipase Cγ1

In ElasCCK2 transgenic mice expressing cholecystokinin (CCK2) receptor in acinar cells, pancreatic phenotypic alterations and preneoplastic lesions are observed. We determined whether activation of phospholipase C gamma1 (PLCγ1), known to contribute to the tumorigenesis pathophysiology, could take place as a new signaling pathway induced by the CCK2 receptor. Overexpression and activation of the PLCγ1 in response to gastrin was observed in acinar cells. The possibility that the C‐terminal tyrosine 438 of the CCK2 receptor associates with the SH2 domains of PLCγ1 was examined. A specific interaction was demonstrated using surface plasmon resonance, confirmed in a cellular system and by molecular modeling.

PAR2-dependent activation of GSK3β regulates the survival of colon stem/progenitor cells

Protease-activated receptors PAR1 and PAR2 play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor cells promoted by proinflammatory mediators may be critical in oncogenesis. The glycogen synthase kinase-3β (GSK3β) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR1 and PAR2 effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3β. First, PAR1 and PAR2 were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR2 activation decreased numbers and size of normal or cancerous spheroids, and PAR2-deficient spheroids showed increased proliferation, indicating that PAR2 represses proliferation. PAR2-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR2 Accordingly, active caspase-3 was strongly increased in PAR2-deficient normal spheroids. PAR2 but not PAR1 triggered GSK3β activation through serine-9 dephosphorylation in normal and tumor cells. The PAR2-triggered GSK3β activation implicates an arrestin/PP2A/GSK3β complex that is dependent on the Rho kinase activity. Loss of PAR2 was associated with high levels of GSK3β nonactive form, strengthening the role of PAR2 in GSK3β activation. GSK3 pharmacological inhibition impaired the survival of PAR2-stimulated spheroids and serum-starved cells. Altogether our data identify PAR2/GSK3β as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer.

Cell surface F1-ATPase binds the Gastrin precursor, G-gly, and mediates its proliferative effects on colorectal cancer cells and vascular endothelial cells.

incubation of peptides with each cell line and analysis of catabolites by mass spectrometry, it was found that bradykininwas highly labile and each antagonist was highly stable under the conditions employed. Bradykinin signalling pathways are thus worthy of further investigation in human prostate cancer cell lines and the evidence presented here would suggest the testing of efficacy in animal models of prostate cancer as a positive outcome could lead to a drug development programme for the treatment of this disease.

Abstract B16: Progastrin activates colon fibroblasts and participates to the dialogue between tumor epithelial cells and stromal fibroblasts in colorectal cancer

Colorectal cancer (CRC) represents the second leading cause of cancer-related death worldwide. One of the main reasons for this high mortality rate is because CRC develops into invasive lesions with high metastasis potential. The cancer-associated fibroblasts (CAFs) present in the tumour reactive stroma promote epithelial cancer cell motility and aggressiveness. However, whereas the role of tumour-stroma crosstalks in cancer progression is increasingly documented, how “normal” resident fibroblasts are converted into pro-invasive CAFs and how CAFs participate to the epithelial-stroma dialogue remains an open question. Precursor of the hormone gastrin, progastrin, now established as a growth factor for colon cells, is detected in colorectal polyps and tumours while it is absent in healthy colon. Using immunohistochemical and biochemical approaches we found that progastrin induces the expression of fibroblast activation markers (alphaSMA, FAPalpha) in the normal colon mucosa of progastrin-overexpressing mice as well as in the human colon fibroblast cell line CCD18Co. To investigate the role of progastrin-induced fibroblast activation, we compared the migration of colon epithelial cancer cells expressing or not the hormone in presence or absence of fibroblasts in Boyden chamber. Progastrin-expressing epithelial cells have increased migration capacities in presence of fibroblasts while not difference between the two epithelial cell lines was observed in absence of fibroblasts. Moreover, we identified the implication of different chemokines in this process. Thus, here we demonstrate that progastrin, secreted by tumour epithelial cells, contributes to the activation of the fibroblasts present in the cancer-associated stroma and participates to the epithelial-stroma dialogue. Citation Format: Nicolas Fenie, Claudine Bertrand, Aurelien Lacombe, Serge Roche, Corinne Bousquet, Valerie Gouaze-Andersson, Christine Toulas, Elizabeth Moyal, Audrey Ferrand. Progastrin activates colon fibroblasts and participates to the dialogue between tumor epithelial cells and stromal fibroblasts in colorectal cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B16. doi:10.1158/1538-7445.CHTME14-B16

Abstract 1073: Cell surface F1-ATPase, a new potential target in colorectal cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL High concentrations of gastrin precursors have been observed in colon tumors and in blood of patients with colorectal cancer while they are absent in healthy subjects. These precursors are now established as growth factors for colonic epithelial cells and play an important role in colon carcinogenesis. The glycine-extended form of gastrin (G-gly) is the first gastrin precursor for which growth factor properties have been demonstrated. More recently, an in vitro study has also shown that G-gly induces the tubule formation by human vascular endothelial cells (HUVEC) in a similar manner to what is observed with VEGF. Several publications are in favor of the existence of high affinity binding sites for G-gly at the cell surface of gastrointestinal cells. However, the cell surface protein mediating G-gly effects remains unidentified to-date. In the present study, using SPR technology coupled to mass spectrometry, we identify in the solubilized plasma membranes from human colorectal cancer cells, the F1-ATPase as a binding protein for G-gly. This mitochondrial protein was recently found located at the cell surface of lung and prostate tumor cells and vascular endothelial cells, where it plays a role in cell proliferation and angiogenesis. Using purified F1-ATPase we confirmed by SPR a direct interaction between G-gly and this multi-subunits transmembrane protein (Kd in the nanomolar range). In addition by molecular modeling, we identified the motif in the G-gly sequence (EE/DxY) which directly interacts with F1-ATPase as well as the amino-acid residues in the alpha-and beta-subunits of the F1-ATPase which bind this motif. We confirmed this molecular model by mutation of the EE/DxY motif that resulted in a strong decrease of G-gly binding to the F1-ATPase as well as a loss of the proliferative activity of the peptide. Using immunofluorescent approaches and confocal microscopy, we demonstrate for the first time the presence of the F1-ATPase at the cell surface of normal or cancerous colonic epithelial cells in vitro and in vivo. We also found that cell surface F1-ATPase activity is modulated by G-gly on endothelial and colorectal cancer cells. Furthermore, the proliferative effects of G-gly on both cell types, was blocked by a specific inhibitor of the F1-ATPase (IF1) or antibodies targeting the beta- subunit of the enzyme. These results suggest an important contribution of cell surface ATPase in the pro-tumoral action of the gastrin precursor, G-gly. Interestingly, an anti-tumor strategy using monoclonal antibodies against the beta-subunit of ATPase has been previously proposed for hepatocarcinoma since these antibodies have been shown to reduce growth of these tumors. In view of our results, targeting cell surface ATPase might also be a new therapeutic approach for colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1073. doi:1538-7445.AM2012-1073