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.

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.

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.

Inhibiting Integrin ß8 to Differentiate and Radiosensitize Glioblastoma-Initiating Cells.

Glioblastomas (GB) are malignant brain tumors with poor prognosis despite treatment with surgery and radio/chemotherapy. These tumors are defined by an important cellular heterogeneity and notably contain a subpopulation of GB-initiating cells (GIC), which contribute to tumor aggressiveness, resistance, and recurrence. Some integrins are specifically expressed by GICs and could be actionable targets to improve GB treatment. Here, integrin ß8 (ITGB8) was identified as a potential selective target in this highly tumorigenic GIC subpopulation. Using several patient-derived primocultures, it was demonstrated that ITGB8 is overexpressed in GICs compared with their differentiated progeny. Furthermore, ITGB8 is also overexpressed in GB, and its overexpression is correlated with poor prognosis and with the expression of several other classic stem cell markers. Moreover, inhibiting ITGB8 diminished several main GIC characteristics and features, including self-renewal ability, stemness, migration potential, and tumor formation capacity. Blockade of ITGB8 significantly impaired GIC cell viability via apoptosis induction. Finally, the combination of radiotherapy and ITGB8 targeting radiosensitized GICs through postmitotic cell death. IMPLICATIONS: This study identifies ITGB8 as a new selective marker for GICs and as a promising therapeutic target in combination with chemo/radiotherapy for the treatment of highly aggressive brain tumors.

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.

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.

Interest of integrins targeting in glioblastoma according to tumor heterogeneity and cancer stem cell paradigm: an update.

Glioblastomas are malignant brain tumors with dismal prognosis despite standard treatment with surgery and radio/chemotherapy. These tumors are defined by an important cellular heterogeneity and notably contain a particular subpopulation of Glioblastoma-initiating cells, which recapitulate the heterogeneity of the original Glioblastoma. In order to classify these heterogeneous tumors, genomic profiling has also been undertaken to classify these heterogeneous tumors into several subtypes. Current research focuses on developing therapies, which could take into account this cellular and genomic heterogeneity. Among these targets, integrins are the subject of numerous studies since these extracellular matrix transmembrane receptors notably controls tumor invasion and progression. Moreover, some of these integrins are considered as membrane markers for the Glioblastoma-initiating cells subpopulation. We reviewed here integrin expression according to glioblastoma molecular subtypes and cell heterogeneity. We discussed their roles in glioblastoma invasion, angiogenesis, therapeutic resistance, stemness and microenvironment modulations, and provide an overview of clinical trials investigating integrins in glioblastomas. This review highlights that specific integrins could be identified as selective glioblastoma cells markers and that their targeting represents new diagnostic and/or therapeutic strategies.

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. .

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.

αv integrin: a new gastrin target in human pancreatic cancer cells.

AIM: To analyse αv integrin expression induced by gastrin in pancreatic cancer models. METHODS: αv integrin mRNA expression in human pancreatic cancer cells was analysed using a "cancer genes" array and confirmed by real-time reverse transcription-polymerase chain reaction (PCR). Western blotting and semi-quantitative immunohistochemistry were used to examine protein levels in human pancreatic cancer cell lines and pancreatic tissues, respectively. The role of αv integrin on gastrin-induced cell adhesion was examined using blocking anti-αv integrin monoclonal antibodies. Adherent cells were quantified by staining with crystal violet. RESULTS: Using a "cancer genes" array we identified αv integrin as a new gastrin target gene in human pancreatic cancer cells. A quantitative real-time PCR approach was used to confirm αv integrin gene expression. We also demonstrate that Src family kinases and the PI 3-kinase, two signalling pathways specifically activated by the CCK-2 receptor (CCK2R), are involved in gastrin-mediated αv integrin expression. In contrast, inhibition of the ERK pathway was without any effect on αv integrin expression induced by gastrin. Our results also show that gastrin modulates cell adhesion via αv integrins. Indeed, in vitro adhesion assays performed on fibronectin show that gastrin significantly increases adhesion of pancreatic cancer cells. The use of blocking anti-αv integrin monoclonal antibodies completely reversed the increase in cell-substrate adhesion induced by gastrin. In addition, we showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, leads to the overexpression of αv integrin. This process may contribute to pancreatic tumour development observed in these transgenic animals. CONCLUSION: αv integrin is a new gastrin target in pancreatic cancer models and contributes to gastrin effects on cell adhesion.

Phosphorylation of spinal N-methyl-d-aspartate receptor NR1 subunits by extracellular signal-regulated kinase in dorsal horn neurons and microglia contributes to diabetes-induced painful neuropathy.

The N-methyl-d-aspartate receptor (NMDAR) contributes to central sensitization in the spinal cord, a phenomenon which comprises various pathophysiological mechanisms responsible for neuropathic pain-like signs in animal models. NMDAR function is modulated by post-translational modifications including phosphorylation, and this is proposed to underlie its involvement in the production of pain hypersensitivity. As in diabetic patients, streptozotocin-induced diabetic rats exhibit or not somatic mechanical hyperalgesia; these rats were named DH and DNH respectively. At three weeks of diabetes, we present evidence that somatic mechanical hyperalgesia was correlated with an enhanced phosphorylation of the NMDAR NR1 subunit (pNR1) in the rat spinal cord. This increase was not found in normal and DNH rats, suggesting that this regulation was specific to hyperalgesia. Double immunofluorescence studies revealed that the numbers of pNR1-immunoreactive neurons and microglial cells were significantly increased in all laminae (I-II and III-VI) of the dorsal horn from DH animals. Western-blots analysis showed no change in NR1 protein levels, whatever the behavioural and glycemic status of the animals. Chronic intrathecal treatment (5µg/rat/day for 7days) by U0126 and MK801, which blocked MEK (an upstream kinase of extracellular signal-regulated protein kinase: ERK) and the NMDAR respectively, simultaneously suppressed somatic mechanical hyperalgesia developed by diabetic rats and decreased pNR1. These results indicate for the first time that increased expression of pNR1 is regulated by ERK and the NMDAR via a feedforward mechanism in spinal neurons and microglia and represents one mechanism involved in central sensitization and somatic mechanical hyperalgesia after diabetes.

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.

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.

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.

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 Galpha(q) proteins. Indeed, transfection of a constitutively activated mutant of Galpha(q) (Q209L) in COS-7 cells demonstrated that Galpha(q) 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 G(q), 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.

Involvement of JAK2 upstream of the PI 3-kinase in cell-cell adhesion regulation by gastrin.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway has been implicated in cell transformation and proliferation. Besides aberrant cell proliferation, loss of cell-cell adhesion during epithelial-mesenchymal transition (EMT) is an important event which occurs during development of epithelial cancers. However, the role of JAK-dependent pathways in this process is not known. We analyzed the involvement of these pathways in the regulation of E-cadherin-dependent cell-cell adhesion by gastrin, a mitogenic factor for gastrointestinal (GI) tract. We identified JAK2/STAT3 as a new pathway in gastrin signaling. We demonstrated that JAK2 functions as an upstream mediator of the phosphatidylinositol 3 (PI 3)-kinase activity in gastrin signaling. Indeed, we observed a coprecipitation of both kinases and an inhibition of gastrin-induced PI 3-kinase activation when JAK2 activity is blocked. We also demonstrated that loss of cell-cell adhesion and the increase in cell motility induced by gastrin required the activation of JAK2 and the PI 3-kinase. Indeed, the modifications in localization of adherens junctions proteins and the migration, observed in gastrin-stimulated cells, were reversed by inhibition of both kinases. These results described the involvement of JAK2 in the modulation of cell-cell adhesion in epithelial cells. They support a possible role of JAK2 in the epithelial-mesenchymal transition which occurs during malignant development.

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.

c-Jun NH(2)-terminal kinase pathway in growth-promoting effect of the G protein-coupled receptor cholecystokinin B receptor: a protein kinase C/Src-dependent-mechanism.

The proliferative effects of gastrin on normal and malignant gastrointestinal tissues have been shown to be mediated by a G protein-coupled receptor (GPCR), the cholecystokinin B receptor. The c-Jun NH(2)-terminal kinase (JNK) pathway has been implicated in the regulation of mitogenesis by growth factors or cytokines. However, the contribution of this signaling cascade to the proliferative effects of GPCR remains largely unknown. Here, we show that cholecystokinin B receptor occupancy by gastrin leads to the activation of the JNK pathway. The mechanism involves certain protein kinase C isoforms and Src family kinases other than p60Src. The complex p130Cas/CrkII, known to be involved in JNK activation, is also activated in response to gastrin by a protein kinase C- and Src-dependent mechanism. However, gastrin-induced CrkII and JNK pathways are independent. Using a dominant negative mutant of c-Jun, we blocked the ability of gastrin to induce DNA synthesis, demonstrating a major role of the JNK pathway in the growth-promoting effect of a GPCR agonist.