Association of Radiochemotherapy to Immunotherapy in unresectable locally advanced Oesophageal carciNoma-randomized phase 2 trial ARION UCGI 33/PRODIGE 67: the study protocol.

BACKGROUND: In case of locally advanced and/or non-metastatic unresectable esophageal cancer, definitive chemoradiotherapy (CRT) delivering 50 Gy in 25 daily fractions in combination with platinum-based regimen remains the standard of care resulting in a 2-year disease-free survival of 25% which deserves to be associated with new systemic strategies. In recent years, several immune checkpoint inhibitors (anti-PD1/anti-PD-L1, anti-Program-Death 1/anti-Program-Death ligand 1) have been approved for the treatment of various solid malignancies including metastatic esophageal cancer. As such, we hypothesized that the addition of an anti-PD-L1 to CRT would provide clinical benefit for patients with locally advanced oesophageal cancer. To assess the efficacy of the anti-PD-L1 durvalumab in combination with CRT and then as maintenance therapy we designed the randomized phase II ARION (Association of Radiochemotherapy with Immunotherapy in unresectable Oesophageal carciNoma- UCGI 33/PRODIGE 67). METHODS: ARION is a multicenter, open-label, randomized, comparative phase II trial. Patients are randomly assigned in a 1:1 ratio in each arm with a stratification according to tumor stage, histology and centre. Experimental arm relies on CRT with 50 Gy in 25 daily fractions in combination with FOLFOX regimen administrated during and after radiotherapy every two weeks for a total of 6 cycles and durvalumab starting with CRT for a total of 12 infusions. Standard arm is CRT alone. Use of Intensity Modulated radiotherapy is mandatory. The primary endpoint is to increase progression-free survival at 12 months from 50 to 68% (HR = 0.55) (power 90%; one-sided alpha-risk, 10%). Progression will be defined with central external review of imaging. ANCILLARY STUDIES ARE PLANNED: PD-L1 Combined Positivity Score on carcinoma cells and stromal immune cells of diagnostic biopsy specimen will be correlated to disease free survival. The study of gut microbiota will aim to determine if baseline intestinal bacteria correlates with tumor response. Proteomic analysis on blood samples will compare long-term responder after CRT with durvalumab to non-responder to identify biomarkers. CONCLUSION: Results of the present study will be of great importance to evaluate the impact of immunotherapy in combination with CRT and decipher immune response in this unmet need clinical situation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT: 03777813.Trial registration date: 5th December 2018.

By modulating α2ß1 integrin signalling, gastrin increases adhesion oF AGS-GR gastric cancer cells.

Peritoneal metastasis is a major cause of recurrence of gastric cancer and integrins are key molecules involved in gastric cancer cells attachment to the peritoneum. The peptide hormone, gastrin, initially identified for its role in gastric acid secretion is also a growth factor for gastric mucosa. Gastrin has also been shown to contribute to gastric cancers progression. Here, we provide the first evidence that gastrin increases the adhesion of gastric cancer cells. Gastrin treatment induces the expression of α2 integrin subunit through a mechanism that involves the ERK pathway. We also observed in response to gastrin an increase in the amount of α2 integrin associated with ß1subunit. In addition, gastrin-stimulated cell adhesion was blocked with an anti-α2ß1 integrin neutralizing antibody. We also show that gastrin activates the integrin pathway via the phosphorylation of ß1 integrin by a Src family kinase. This mechanism may contribute to the enhancement of cell adhesion observed in response to gastrin since we found an inhibition of gastrin-mediated cell adhesion when cells were treated with a Src inhibitor. By regulating one of the key step of the metastatic process gastrin might contribute to increase the aggressive behaviour of human gastric tumours.

Identification of the F1-ATPase at the cell surface of colonic epithelial cells: role in mediating cell proliferation.

F1 domain of F(1)F(o)-ATPase was initially believed to be strictly expressed in the mitochondrial membrane. Interestingly, recent reports have shown that the F1 complex can serve as a cell surface receptor for apparently unrelated ligands. Here we show for the first time the presence of the F(1)-ATPase at the cell surface of normal or cancerous colonic epithelial cells. Using surface plasmon resonance technology and mass spectrometry, we identified a peptide hormone product of the gastrin gene (glycine-extended gastrin (G-gly)) as a new ligand for the F(1)-ATPase. By molecular modeling, we identified the motif in the peptide sequence (E(E/D)XY), that directly interacts with the F(1)-ATPase and the amino acids in the F(1)-ATPase that bind this motif. Replacement of the Glu-9 residue by an alanine in the E(E/D)XY motif resulted in a strong decrease of G-gly binding to the F(1)-ATPase and the loss of its biological activity. In addition we demonstrated that F(1)-ATPase mediates the growth effects of the peptide. Indeed, blocking F(1)-ATPase activity decreases G-gly-induced cell growth. The mechanism likely involves ADP production by the membrane F(1)-ATPase, which is induced by G-gly. These results suggest an important contribution of cell surface F(1)-ATPase in the pro-proliferative action of this gastrointestinal peptide.

Activation of pro-oncogenic pathways in colorectal hyperplastic polyps.

BACKGROUND: In contrast to sessile serrated adenomas and traditional serrated adenomas which are associated with a significant cancer risk, the role of hyperplastic polyps (HP) in colorectal carcinogenesis as well as the molecular mechanisms underlying their development remain controversial and still need to be clarified. Several reports suggest that a subset of HP may represent precursor lesions of some colorectal cancers. However, biomarkers are needed to identify the subset of HP that may have a malignant potential. The hormone precursor, progastrin (PG) has been involved in colon carcinogenesis and is known to activate pro-oncogenic pathways such as the ERK or the STAT3 pathway. We therefore analyzed PG expression and the activation of these signaling factors in HP. METHODS: We retrospectively analyzed PG expression as well as the phosphorylation of ERK and STAT3 by immunohistochemistry in HP from 48 patients. RESULTS: Mean percentages of epithelial cells positive for PG or phospho-ERK were respectively, 31% and 33% in HP and were significantly higher in these lesions compared to normal colon (3%, p=0.0021 and 7%, p=0.0008, respectively). We found a significant correlation between PG and phospho-ERK expression in HP with ERK activation significantly stronger in lesions with high progastrin expression (p=0.015). In contrast, STAT3 was not significantly activated in HP compared to normal colon and we did not observe a significant correlation with PG expression. CONCLUSIONS: HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.

Overexpression of Growth Differentiation Factor 15 in Glioblastoma Stem Cells Promotes Their Radioresistance.

GSCs play an important role in GBM recurrence. Understanding the resistance mechanisms in these cells is therefore crucial for radiation therapy optimization. In this study, using patient-derived GSCs, we demonstrate that GDF15, a cytokine belonging to the TGF-ß superfamily, is regulated by irradiation (IR) and the transcription factor WWTR1/TAZ. Blocking WWTR1/TAZ using specific siRNAs significantly reduces GDF15 basal expression and reverses the upregulation of this cytokine induced by IR. Furthermore, we demonstrate that GDF15 plays an important role in GSC radioresistance. Targeting GDF15 expression by siRNA in GSCs expressing high levels of GDF15 sensitizes the cells to IR. In addition, we also found that GDF15 expression is critical for GSC spheroid formation, as GDF15 knockdown significantly reduces the number of GSC neurospheres. This study suggests that GDF15 targeting in combination with radiotherapy may be a feasible approach in patients with GBM.

The Glycoprotein M6a Is Associated with Invasiveness and Radioresistance of Glioblastoma Stem Cells.

Systematic recurrence of glioblastoma (GB) despite surgery and chemo-radiotherapy is due to GB stem cells (GBSC), which are particularly invasive and radioresistant. Therefore, there is a need to identify new factors that might be targeted to decrease GBSC invasive capabilities as well as radioresistance. Patient-derived GBSC were used in this study to demonstrate a higher expression of the glycoprotein M6a (GPM6A) in invasive GBSC compared to non-invasive cells. In 3D invasion assays performed on primary neurospheres of GBSC, we showed that blocking GPM6A expression by siRNA significantly reduced cell invasion. We also demonstrated a high correlation of GPM6A with the oncogenic protein tyrosine phosphatase, PTPRZ1, which regulates GPM6A expression and cell invasion. The results of our study also show that GPM6A and PTPRZ1 are crucial for GBSC sphere formation. Finally, we demonstrated that targeting GPM6A or PTPRZ1 in GBSC increases the radiosensitivity of GBSC. Our results suggest that blocking GPM6A or PTPRZ1 could represent an interesting approach in the treatment of glioblastoma since it would simultaneously target proliferation, invasion, and radioresistance.

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 m6A RNA Demethylase ALKBH5 Promotes Radioresistance and Invasion Capability of Glioma Stem Cells.

Recurrence of GBM is thought to be due to GBMSCs, which are particularly chemo-radioresistant and characterized by a high capacity to invade normal brain. Evidence is emerging that modulation of m6A RNA methylation plays an important role in tumor progression. However, the impact of this mRNA modification in GBM is poorly studied. We used patient-derived GBMSCs to demonstrate that high expression of the RNA demethylase, ALKBH5, increases radioresistance by regulating homologous recombination (HR). In cells downregulated for ALKBH5, we observed a decrease in GBMSC survival after irradiation likely due to a defect in DNA-damage repair. Indeed, we observed a decrease in the expression of several genes involved in the HR, including CHK1 and RAD51, as well as a persistence of γ-H2AX staining after IR. We also demonstrated in this study that ALKBH5 contributes to the aggressiveness of GBM by favoring the invasion of GBMSCs. Indeed, GBMSCs deficient for ALKBH5 exhibited a significant reduced invasion capability relative to control cells. Our data suggest that ALKBH5 is an attractive therapeutic target to overcome radioresistance and invasiveness of GBMSCs.

Translation reprogramming by eIF3 linked to glioblastoma resistance.

Intrinsic resistance to current therapies, leading to dismal clinical outcomes, is a hallmark of glioblastoma multiforme (GBM), the most common and aggressive brain tumor. Understanding the underlying mechanisms of such malignancy is, therefore, an urgent medical need. Deregulation of the protein translation machinery has been shown to contribute to cancer initiation and progression, in part by driving selective translational control of specific mRNA transcripts involved in distinct cancer cell behaviors. Here, we focus on eIF3, a multimeric complex with a known role in the initiation of translation and that is frequently deregulated in cancer. Our results show that the deregulated expression of eIF3e, the e subunit of eIF3, in specific GBM regions could impinge on selective protein synthesis impacting the GBM outcome. In particular, eIF3e restricts the expression of proteins involved in the response to cellular stress and increases the expression of key functional regulators of cell stemness. Such a translation program can therefore serve as a double-edged sword promoting GBM tumor growth and resistance to radiation.

Alpha6-Integrin Regulates FGFR1 Expression through the ZEB1/YAP1 Transcription Complex in Glioblastoma Stem Cells Resulting in Enhanced Proliferation and Stemness.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults and is known to be particularly aggressive and resistant to anti-cancer therapies, mainly due to the presence of GBM stem cells (GBMSC). By in vitro approaches supported by analysis from patients' databases, we determined how α6-integrin and Fibroblast Growth Factor Receptor 1 (FGFR1) work in concert to regulate proliferation and stemness of GBMSC. We showed that α6-integrin regulates the expression of FGFR1 and its target gene Fokhead Box M1 (FOXM1) via the ZEB1/YAP1 transcription complex. These results were in accordance with the positive correlation observed in GBM between α6-integrin expression and its target genes ZEB1/YAP1, FGFR1, and FOXM1 in the databases, TCGA and Rembrandt. In addition, the clinical data demonstrate that GBM patients with high levels of the five genes signature, including α6-integrin, ZEB1/YAP1, FGFR1 and FOXM1, have a significantly shorter overall survival. In vitro, we observed a similar decrease in the expression of stemness-related factors, neurospheres forming capacity, as well as spheroids growth when α6-integrin or FGFR1 was blocked individually with specific siRNA, whereas the combination of both siRNA led to a significantly higher inhibition of spheres formation. These data suggest that co-administration of anti-FGFR1 and anti-α6-integrin could provide an improved therapeutic response in GBMSC.

Alpha-6 integrin promotes radioresistance of glioblastoma by modulating DNA damage response and the transcription factor Zeb1.

Radiotherapy is the cornerstone of glioblastoma (GBM) standard treatment. However, radioresistance of cancer cells leads to an inevitable recurrence. In the present study, we showed that blocking α6-integrin in cells derived from GBM biopsy specimens cultured as neurospheres, sensitized cells to radiation. In cells downregulated for α6-integrin expression, we observed a decrease in cell survival after irradiation and an increase in radio-induced cell death. We also demonstrated that inhibition of α6-integrin expression affects DNA damage checkpoint and repair. Indeed, we observed a persistence of γ-H2AX staining after IR and the abrogation of the DNA damage-induced G2/M checkpoint, likely through the downregulation of the checkpoint kinase CHK1 and its downstream target Cdc25c. We also showed that α6-integrin contributes to GBM radioresistance by controlling the expression of the transcriptional network ZEB1/OLIG2/SOX2. Finally, the clinical data from TCGA and Rembrandt databases demonstrate that GBM patients with high levels of the five genes signature, including α6-integrin and its targets, CHK1, ZEB1, OLIG2 and SOX2, have a significantly shorter overall survival. Our study suggest that α6-integrin is an attractive therapeutic target to overcome radioresistance of GBM cancer cells.

An ITIM-like motif within the CCK2 receptor sequence required for interaction with SHP-2 and the activation of the AKT pathway.

SHP-2 is a tyrosine phosphatase which functions as a positive regulator downstream of RTKs, activating growth-stimulatory signalling pathways. To date, very few G protein-coupled receptors (GPCRs) have been shown to be connected to SHP-2 and very little is known about the positive role of SHP-2 in GPCR signalling. The CCK2 receptor (CCK2R), a GPCR, is now recognized to mediate mitogenic effects of gastrin on gastrointestinal cells. In the present study, we demonstrate the role of SHP-2 in the activation of the AKT pathway by the CCK2R in COS-7 cells transfected with the CCK2R and in a pancreatic cancer cell line expressing the endogenous receptor. Using surface plasmon resonance analysis, we identified a highly conserved ITIM motif, containing the tyrosine residue 438, located in the C-terminal intracellular tail of the CCK2R which directly interacts with the SHP-2 SH2 domains. The interaction was confirmed by pull down assays and co-immunoprecipitation of the receptor with SHP-2. This interaction was transiently increased following gastrin stimulation of the CCK2R and correlated with the tyrosine phosphorylation of SHP-2. Mutational analysis of the key ITIM residue 438 confirmed that the CCK2R ITIM sequence is required for interaction with SHP-2 and the activation of the AKT pathway.

Involvement of cholecystokinin 2 receptor in food intake regulation: hyperphagia and increased fat deposition in cholecystokinin 2 receptor-deficient mice.

The role of cholecystokinin (CCK) as a satiety factor has been extensively documented. Although most work implies that CCK1 receptor mediates the control of food intake, a contributing role for CCK2 receptor (CCK2R) in the CCK-induced satiety cannot be totally excluded. The hypothesis that CCK2R invalidation disrupts regulatory pathways with impact on feeding behavior was examined in CCK2R(-/-) mice. CCK2R(-/-) mice developed obesity that was associated with hyperphagia. Obesity was related with increased fat deposition resulting from adipocyte hypertrophy. Expression of several adipokines was dysregulated consistently with obesity. Moreover, obesity was associated with disturbed glucose homeostasis as revealed by increased fasting glycemia and insulinemia, impaired glucose tolerance, and hepatic insulin resistance in CCK2R(-/-) mice. In vitro analysis of isolated adipocytes metabolism was consistent with increased storage but preserved insulin sensitivity. Suppression of feeding and concomitant increased expression of hypothalamic proopiomelanocortin after intracerebroventricular injection of gastrin into control mice demonstrates that hypothalamic CCK2 receptors mediate inhibition of food intake. Comparative analysis of hypothalamic mediator gene expression in fed knockout and control mice demonstrated overexpression of ghrelin receptors in CCK2R(-/-) mice, indicating up-regulation of orexigenic pathways. This effect was also observed after body weight normalization, indicating a causative role in the development of hyperphagia and obesity of CCK2R(-/-) mice. Our results give evidence that CCK2 receptor activity plays a contributing regulatory role in the control of food intake.

Signaling pathways associated with colonic mucosa hyperproliferation in mice overexpressing gastrin precursors.

MTI/G-Gly mice and hGAS mice, overexpressing glycine-extended gastrin (G-Gly) and progastrin, respectively, display colonic mucosa hyperplasia, hyperproliferation, and an increased susceptibility to intestinal neoplasia. Here, we have used these transgenic mice to analyze in vivo the modulation of intracellular signaling pathways that may be responsible for the proliferative effects of gastrin precursors. The expression, activation, and localization of signaling and cell-to-cell adhesion molecules were studied using immunofluorescence and Western blot techniques on colonic tissues derived from MTI/G-Gly, hGAS, or wild-type FVB/N mice. These analyses revealed an up-regulation of Src tyrosine kinase and related signaling pathways [phosphatidyl inositol 3'-kinase (PI3K)/Akt, Janus-activated kinase (JAK) 2, signal transducer and activator of transcription (STAT) 3, and extracellular-signal regulated kinases (ERK)] in both MTI/G-Gly and hGAS mice compared with the wild-type control animals as well as an overexpression of transforming growth factor-alpha (TGF-alpha). In contrast, overexpression of the gastrin precursors did not affect the activation status of STAT1 nor the expression and the distribution of adhesion proteins (focal adhesion kinase, cadherins, and catenins). We report for the first time that the transition from a normal colonic epithelium to a hyperproliferative epithelium in MTI/G-Gly and hGAS mice may be a consequence of the up-regulation of Src, PI3K/Akt, JAK2, STAT3, ERKs, and TGF-alpha. Deregulation of cell adhesion, a late event in tumor progression, does not occur in these transgenic models.

Targeting progastrin enhances radiosensitization of colorectal cancer cells.

A high percentage of advanced rectal cancers are resistant to radiation. Therefore, increasing the efficacy of radiotherapy by targeting factors involved in radioresistance seems to be an attractive strategy. Here we demonstrated that the pro-hormone progastrin (PG), known to be over-expressed in CRC, and recognized as a pro-oncogenic factor, is a radioresistance factor that can be targeted to sensitize resistant rectal cancers to radiations. First, we observed an increase in PG mRNA expression under irradiation. Our results also demonstrated that down-regulating PG mRNA expression using a shRNA strategy, significantly increases the sensitivity to irradiation (IR) in a clonogenic assay of different colorectal cancer cell lines. We also showed that the combination of PG gene down-regulation and IR strongly inhibits tumours progression in vivo. Then, we demonstrated that targeting PG gene radiosensitizes cancer cells by increasing radio-induced apoptosis shown by an increase in annexin V positive cells, caspases activation and PARP cleavage. We also observed the up-regulation of the pro-apoptotic pathway, JNK and the induction of the expression of pro-apoptotic factors such as BIM. In addition, we demonstrated in this study that inhibition of PG gene expression enhances radiation-induced DNA damage. Our data also suggest that, in addition to increase radio-induced apoptosis, targeting PG gene also leads to the inhibition of the survival pathways, AKT and ERK induced by IR. Taken together, our results highlight the role of PG in radioresistance and provide a preclinical proof of concept that PG represents an attractive target for sensitizing resistant rectal tumours to irradiation. .

Mechanism for Src activation by the CCK2 receptor: Patho-physiological functions of this receptor in pancreas.

AIM: To investigate in vivo, whether CCK2 receptors (CCK2R) regulate proteins known to play a crucial role in cell proliferation and cancer development and analyse in vitro the molecular mechanisms that lead to Src activation; in particular, to identify the domains within the CCK2R sequence that are implicated in this activation. METHODS: The expression and activation of Src and ERK were studied in vivo using immuno-fluorescence and western-blot techniques. We used pancreatic tissues derived from wild type or Elas-CCK2 mice that expressed the CCK2R in pancreatic acini, displayed an increased pancreatic growth and developed preneoplastic lesions. The pancreatic tumor cell line AR4-2J expressing the endogenous CCK2R or COS-7 cells transiently transfected with wild type or mutant CCK2R were used as in vitro models to study the mechanism of Src activation. Src activation was measured by in vitro kinase assays, ERK activation by western blot using anti-phospho-ERK antibodies and the involvement of Src in gastrin-induced cell proliferation by MTT test. RESULTS: We showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, led to the activation of Src and the ERK pathway. Src was activated upstream of the ERK pathway by the CCK2R in pancreatic tumoral cells and contributed to the proliferative effects mediated by this receptor. In vitro results demonstrated that activation of the Src/ERK pathway by the CCK2R required the NPXXY motif, located within the CCK2R sequence at the end of the 7th transmembrane domain, and suggested the putative role of Gq in this mechanism. CONCLUSION: Deregulation of the Src/ERK pathway by the CCK2R might represent an early step that contributes to cell proliferation, formation of preneoplastic lesions and pancreatic tumor development.

Glycine-extended gastrin activates two independent tyrosine-kinases in upstream of p85/p110 phosphatidylinositol 3-kinase in human colonic tumour cells.

AIM: To investigate whether Src, JAK2 and phosphatidylinositol 3-kinase (PI3K) pathways are involved in the proliferation of human colonic tumour cells induced by glycine-extended gastrin (G-gly), the precursor of the mature amidated gastrin and to elucidate the molecular interaction between these three kinases in response to this peptide. METHODS: Using the human colonic tumour cell line HCT116 as a model, we first measured the activation of PI3K, p60-Src and JAK2 in response to G-gly by in vitro kinase assays. Then we investigated the involvement of these kinases in G-gly-induced cell proliferation by MTT test. RESULTS: G-gly stimulation induced p60-Src, JAK2 and PI3K activation in HCT116. The different pathways were involved in proliferation of human colon cancer cells induced by G-gly. Furthermore, we found that both Src and JAK2 were necessary to PI3K regulation by this peptide. However, we did not find any cross-talk between the two tyrosine kinases. CONCLUSION: Our results suggest that the p60-Src/PI3K and JAK2/PI3K pathways act independently to mediate G-gly proliferative effect on human colonic tumour cells.

Gastrin mediated cholecystokinin-2 receptor activation induces loss of cell adhesion and scattering in epithelial MDCK cells.

The presence of gastrin and CCK-2/gastrin receptors in human preneoplastic and neoplastic lesions of pancreas and colon suggests a role in cancer development. Gastrin's growth-promoting action has been established, but a role in cellular morphogenetic processes promoting tumor invasion has been elusive. Our aim was (i) to investigate whether activation of the CCK-2R affects cellular morphology, intercellular adhesion and motility, as crucial parameters of epithelial differentiation, and (ii) to identify the signaling pathways and mechanisms implicated. Madin-Darby Canine Kidney (MDCK) cells were chosen to generate an epithelial non-tumorigenic model system expressing human CCK-2R. Epithelial differentiation and motility were analysed upon CCK-2R activation using immunocytochemistry and invasion assays. The functionality of adhesion complexes and activity of signaling proteins was determined with biochemical techniques. CCK-2R activation induced cell dissociation and enhanced invasion, preceded by decreased membrane localization of adherens junction molecules and nuclear accumulation of beta-catenin. Concomitantly, and requiring the activation of several signaling pathways, catenins were shifted from the cytoskeletal to the cytoplasmic fraction, suggesting the detachment of the cytoskeleton from the adherens complex. These data represent the first evidence for the CCK-2R, regulating cell-cell and cell-substrate adhesion and support a role for CCK-2R in the progression of carcinoma.

Modeled structure of a G-protein-coupled receptor: the cholecystokinin-1 receptor.

The Cholecystokinin-1 receptor (CCK1R) mediates actions of CCK in areas of the central nervous system and of the gut. It is a potential target to treat a number of diseases. As for all G-protein-coupled receptors, docking of ligands into modeled CCK1R binding site should greatly help to understand intrinsic mechanisms of activation. Here, we describe the procedure we used to progressively build a structural model for the CCK1R, to integrated, and on the basis of site-directed mutagenesis data on its binding site. Reliability of the CCK1R model was confirmed by interaction networks that involved conserved and functionally crucial motifs in G-protein-coupled receptors, such as Glu/Asp-Arg-Tyr and Asn-Pro-Xaa-Xaa-Tyr motifs. In addition, the 3-D structure of CCK1R-bound CCK resembled that determined by NMR in a lipid environment. The derived computational model was also used for revealing binding modes of several nonpeptide ligands and for rationalizing ligand structure-activity relationships known from experiments. Our findings indeed support that our "validated CCK1R model" could be used to study the intrinsic mechanism of CCK1R activation and design new ligands.

The G-protein-coupled CCK2 receptor associates with phospholipase Cgamma1.

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 (PLCgamma1), 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 PLCgamma1 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 PLCgamma1 was examined. A specific interaction was demonstrated using surface plasmon resonance, confirmed in a cellular system and by molecular modeling.