Claudine Bertrand

A Novel Mechanism for JAK2 Activation by a G Protein-coupled Receptor, the CCK2R IMPLICATION OF THIS SIGNALING PATHWAY IN PANCREATIC TUMOR MODELS

To date very few G protein-coupled receptors (GPCRs) have been shown to be connected to the Janus kinase (JAK)/STAT pathway. Thus our understanding of the mechanisms involved in the activation of this signaling pathway by GPCRs remains limited. In addition, little is known about the role of the JAK pathway in the physiological or pathophysiological functions of GPCRs. Here, we described a new mechanism of JAK activation that involves Gαq proteins. Indeed, transfection of a constitutively activated mutant of Gαq (Q209L) in COS-7 cells demonstrated that Gαq is able to associate and activate JAK2. In addition, we showed that this mechanism is used to activate JAK2 by a GPCR principally coupled to Gq, the CCK2 receptor (CCK2R), and involves a highly conserved sequence in GPCRs, the NPXXY motif. In a pancreatic tumor cell line expressing the endogenous CCK2R, we demonstrated the activation of the JAK2/STAT3 pathway by this receptor and the involvement of this signaling pathway in the proliferative effects of the CCK2R. In addition, we showed in vivo that the targeted CCK2R expression in pancreas of Elas-CCK2 mice leads to the activation of JAK2 and STAT3. This process may contribute to the increase of pancreas growth as well as the formation of preneoplastic lesions leading to pancreatic tumor development observed in these transgenic animals.

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.

FGF-2 isoforms of 18 and 22.5 kDa differentially modulate t-PA and PAI-1 expressions on the pancreatic carcinoma cells AR4–2J: Consequences on cell spreading and invasion

Pancreatic tumors overexpress FGF‐2 and t‐PA, but the implication of the growth factor in t‐PA synthesis and t‐PA‐dependent tumor invasion remains unknown. FGF‐2 is present in different isoforms: The 18 kDa FGF‐2 is secreted, while the 22.5 kDa one is nuclearized and exerts intracrine regulations bypassing cell‐surface FGF receptors. Rat pancreatic carcinoma AR4–2J cells producing either the 18 or the 22.5 kDa FGF‐2 after transfection with FGF‐2 cDNAs have been used to analyze the role of FGF‐2 in t‐PA expression and t‐PA‐related cell spreading. The 22.5 kDa FGF‐2 reduced t‐PA and PAI‐1 synthesis 2‐fold. Addition of recombinant 18 kDa FGF‐2 (rFGF‐2) to cell cultures resulted in increased t‐PA and decreased PAI‐1 expression. By contrast, rFGF‐2 did not significantly modify t‐PA synthesis in cells producing the 22.5 kDa FGF‐2. Cell spreading was t‐PA‐dependent. Furthermore, cells producing the 22.5 kDa FGF‐2 migrated less than control cells and cells producing the 18 kDa FGF‐2. Overall, our data show that secretory FGF‐2 is involved in t‐PA synthesis by pancreatic cancer cells and facilitates cell spreading. The 22.5 kDa FGF‐2 exerts opposite effects by decreasing t‐PA expression in basal conditions and during rFGF‐2 stimulation. Since the expression of the 22.5 kDa FGF‐2 is under specific controls, its up‐regulation might have the potential to reduce spreading of pancreatic cancer cells. Int. J. Cancer 85:555–562, 2000.

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