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.

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.

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.

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 new biomarker that predicts colonic neoplasia outcome in patients with hyperplastic colonic polyps.

The most frequently occurring lesions in the colon are the hyperplastic polyps. Hyperplastic polyps have long been considered as lesions with no malignant potential and colonoscopy for these patients is not recommended. However, recent works suggest that hyperplastic polyps may represent precursor lesions of some sporadic colorectal cancers. Until now, no biomarker allows to identify the subset of hyperplastic polyps that may have a malignant potential. Because the hormone precursor progastrin has been involved in colon carcinogenesis, we investigated whether its expression in hyperplastic polyps predicts the occurrence of colonic neoplasm after resection of hyperplastic polyps. We retrospectively analyzed progastrin expression in hyperplastic polyps from 74 patients without history of colorectal pathology. In our study, 41% of patients presenting an initial hyperplastic polyp subsequently developed adenomatous polyps, recognized as precursor lesions for colorectal adenocarcinomas. Progastrin was overexpressed in the hyperplastic polyps in 40% of the patients. We showed a significant association between progastrin overexpression and shortened neoplasm-free survival (P = 0.001). Patients with high overexpression of progastrin had a 5-year neoplasm-free survival rate of 38% as compared with 100% for the patients with low progastrin expression. In addition, we established a predictive test on the basis of progastrin staining and patients' age that predicts occurrence of neoplasm after developing a first hyperplastic polyp with a sensitivity of 100% [95% confidence interval (CI), 79%-100%] and a specificity of 74% (51%-90%). We show that progastrin expression evaluation in hyperplastic polyps is an efficient prognostic tool to determine patients with higher risk of metachronous neoplasms who could benefit from an adapted follow-up.

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.

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.

Inhibitory role of the somatostatin receptor SST2 on the intracrine-regulated cell proliferation induced by the 210-amino acid fibroblast growth factor-2 isoform: implication of JAK2.

The fibroblast growth factor (FGF)-2 isoform of 210 amino acids (HMW FGF-2) contains a nuclear localization sequence (NLS) and is targeted to the nucleus. This FGF-2 isoform allows cells to grow in low serum concentrations through still unknown mechanisms called intracrine regulations. Different peptide hormones and cytokines have been found to be nuclearized through NLS and to induce cell proliferation. The existence of molecules acting as negative regulators of the intracrine-induced cell growth has not been explored. Pancreatic cells AR4-2J were stably transfected to express selectively the HMW FGF-2. We demonstrated that activation of the somatostatin receptor subtype SST2 by the somatostatin analogue RC-160 in serum-deprived medium inhibits the mitogenic effect of the HMW FGF-2, without affecting growth of control cells. The signaling pathway implicates Galphai/JAK2/SHP-1. The Galphai inhibitor pertussis toxin and the JAK2 inhibitor AG490 abrogate the inhibitory effect of RC-160 on HMW FGF-2-induced cell growth. Co-immunoprecipitation studies demonstrate the constitutive association of JAK2 and SHP-1, and RC-160 induces a rapid activation of both proteins followed by the dissociation of the complex. AG490 prevents the RC-160 induced SHP-1 activation indicating the implication of JAK2 in this process. JAK2 and SHP-1 are immunoprecipitated with SST2 in basal conditions indicating the existence of a functional signaling complex at the receptor level. In summary, these data provide the following evidence: 1) the intracrine-induced proliferation can be reversed by extracellular acting polypeptides; 2) SST2 inhibitory signaling may involve the JAK2/SHP-1 pathway.

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.