Progastrin production transitions from Bmi1+/Prox1+ to Lgr5high cells during early intestinal tumorigenesis.

Progastrin is an unprocessed soluble peptide precursor with a well-described tumor-promoting role in colorectal cancer. It is expressed at small levels in the healthy intestinal mucosa, and its expression is enhanced at early stages of intestinal tumor development, with high levels of this peptide in hyperplastic intestinal polyps being associated with poor neoplasm-free survival in patients. Yet, the precise type of progastrin-producing cells in the healthy intestinal mucosa and in early adenomas remains unclear. Here, we used a combination of immunostaining, RNAscope labelling and retrospective analysis of single cell RNAseq results to demonstrate that progastrin is produced within intestinal crypts by a subset of Bmi1+/Prox1+/LGR5low endocrine cells, previously shown to act as replacement stem cells in case of mucosal injury. In contrast, our findings indicate that intestinal stem cells, specified by expression of the Wnt signaling target LGR5, become the main source of progastrin production in early mouse and human intestinal adenomas. Collectively our results suggest that the previously identified feed-forward mechanisms between progastrin and Wnt signaling is a hallmark of early neoplastic transformation in mouse and human colonic adenomas.

TRM6/61 connects PKCα with translational control through tRNAi(Met) stabilization: impact on tumorigenesis.

Accumulating evidence suggests that changes of the protein synthesis machinery alter translation of specific mRNAs and participate in malignant transformation. Here we show that protein kinase C α (PKCα) interacts with TRM61, the catalytic subunit of the TRM6/61 tRNA methyltransferase. The TRM6/61 complex is known to methylate the adenosine 58 of the initiator methionine tRNA (tRNAi(Met)), a nuclear post-transcriptional modification associated with the stabilization of this crucial component of the translation-initiation process. Depletion of TRM6/61 reduced proliferation and increased death of C6 glioma cells, effects that can be partially rescued by overexpression of tRNAi(Met). In contrast, elevated TRM6/61 expression regulated the translation of a subset of mRNAs encoding proteins involved in the tumorigenic process and increased the ability of C6 cells to form colonies in soft agar or spheres when grown in suspension. In TRM6/61/tRNAi(Met)-overexpressing cells, PKCα overexpression decreased tRNAi(Met) expression and both colony- and sphere-forming potentials. A concomitant increase in TRM6/TRM61 mRNA and tRNAi(Met) expression with decreased expression of PKCα mRNA was detected in highly aggressive glioblastoma multiforme as compared with Grade II/III glioblastomas, highlighting the clinical relevance of our findings. Altogether, we suggest that PKCα tightly controls TRM6/61 activity to prevent translation deregulation that would favor neoplastic development.

Src family tyrosine kinases-driven colon cancer cell invasion is induced by Csk membrane delocalization.

The nonreceptor tyrosine kinases of the Src family (SFK) are frequently deregulated in human colorectal cancer (CRC), and they have been implicated in tumour growth and metastasis. How SFK are activated in this cancer has not been clearly established. Here, we show that the SFK-dependent invasion is induced by inactivation of the negative regulator C-terminal Src kinase, Csk. While the level of Csk was inconsistent with SFK activity in colon cancer cells, its membrane translocation, needed for efficient regulation of membrane-localized SFK activity, was impaired. Accordingly, Csk downregulation did not affect SFK oncogenic activity in these cells, whereas expression of a membrane-localized form of this kinase affected their invasive activity. Downregulation of the transmembrane and rafts-localized Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomain (PAG), was instrumental for the cytoplasmic accumulation of Csk. Re-expression of PAG in cells from late-stage CRC inhibited SFK invasive activity in a Csk-dependent manner. Conversely, inactivation of its residual expression in early-stage CRC cells promoted SFK invasive activity. Finally, this mechanism was specific to CRC as Csk coupling to SFK was readily detected in breast cancer cells. Therefore, Csk mis-localization defines a novel mechanism for SFK oncogenic activation in CRC cells.

Divergent roles for ferric ions in the biological activity of amidated and non-amidated gastrins.

Amidated forms of the peptide hormone gastrin act via the cholecystokinin-2 receptor to stimulate gastric acid secretion, whereas non-amidated forms stimulate colonic mucosal proliferation via a novel, as yet uncharacterised, receptor. Nuclear magnetic resonance (NMR) and fluorescence spectroscopic studies have revealed that glycine-extended gastrin17 bound two ferric ions, and that ferric ion binding was essential for biological activity. We have therefore investigated the role of ferric ions in the biological activity of amidated gastrin17. As with glycine-extended gastrin17, fluorescence quenching experiments indicated that Glu7 Ala and Glu8,9 Ala mutants of amidated gastrin17 each bound only one ferric ion. The affinity of the mutant peptides for the cholecystokinin-2 receptor on transfected COS-7 cells or on Tlymphoblastoid Jurkat cells, and their potency in stimulation of proliferation in Jurkat cells and inositol phosphate production in transfected COS-7 cells, were similar to the values obtained for amidated gastrin17. In addition, the iron chelator desferrioxamine did not significantly inhibit either binding of amidated gastrin17 to the cholecystokinin-2 receptor, or stimulation of inositol phosphate production by amidated gastrin17 in transfected COS-7 cells. We conclude that, in contrast to glycine-extended gastrin17, binding of ferric ions is not essential for the biological activity of amidated gastrin17. Our results support the concept of distinct modes of action for amidated and non-amidated gastrins, and raise the possibility of developing selective antagonists of the actions of non-amidated and amidated gastrins.

Gastrin induces over-expression of genes involved in human U373 glioblastoma cell migration.

Astrocytic tumors are the most common and the most malignant primary tumors of the central nervous system. We had previously observed that gastrin could significantly modulate both cell proliferation and migration of astrocytoma cells. We have investigated in the present study which genes could be targeted by gastrin in tumor astrocyte migration. Using a subtractive hybridization PCR technique we have cloned genes differentially over-expressed in human astrocytoma U373 cells treated or not with gastrin. We found about 70 genes over-expressed by gastrin. Among the genes overexpressed by gastrin, we paid particular attention to tenascin-C, S100A6 and MLCK genes because their direct involvement in cell migration features. Their gastrin-induced overexpression was quantitatively determined by competitive RT-PCR technique. We also showed by means of a reporter gene system that S100A6 and tenascin-C respective promoters were upregulated after gastrin treatment. These data show that gastrin-mediated effects in glioblastoma cells occur through activation of a number of genes involved in cell migration and suggest that gastrin could be a target in new therapeutic strategies against malignant gliomas.

A synthetic glycine-extended bombesin analogue interacts with the GRP/bombesin receptor.

alpha-amidation of a peptide (which takes place from a glycine-extended precursor) is required to produce biologically active amidated hormones, such as gastrin-releasing peptide (GRP)/Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2) (bombesin). It was shown that glycine-extended gastrin mediates mitogenic effects on various cell lines by interacting with a specific receptor, different from the classical CCK(1) or CCK(2) receptors. On the basis of this observation, we have extended the concept of obtaining active glycine-extended forms of others amidated peptides to produce new active analogues. In this study, we have tested the biological behaviour of a synthetic analogue of the glycine-extended bombesin (para-hydroxy-phenyl-propionyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly-OH or JMV-1458) on various in vitro models. We showed that compound JMV-1458 was able to inhibit specific (3-[125I]iodotyrosyl(15)) GRP ([125I]GRP) binding in rat pancreatic acini and in Swiss 3T3 cells with K(i) values of approximately 10(-8) M. In isolated rat pancreatic acini, we found that JMV-1458 induced inositol phosphates production and amylase secretion in a dose-dependent manner. In Swiss 3T3 cells, the glycine-extended bombesin analogue dose-dependently produced [3H]thymidine incorporation. By using potent GRP/bombesin receptor antagonists, we showed that this synthetic glycine-extended bombesin analogue induces its biological activities via the classical GRP/bombesin receptor.

L-365,260 inhibits in vitro acid secretion by interacting with a PKA pathway.

The aim of this study was to analyse the antisecretory mechanism of L-365,260 in vitro in isolated rabbit gastric glands. We showed that compound L-365,260, described as a non-peptide specific competitive CCK-B receptor antagonist, was able to dose-dependently inhibit [14C]-aminopyrine accumulation induced by histamine (10(-4) M), carbachol (5x10(-5) M), 3-isobutyl-1-methyl-xanthine (IBMX) (5x10(-6) M) and forskolin (5x10(-7) M) with similar IC50 values respectively of 1.1+/-0.6x10(-7) M, 1.9+/-1.2x10(-7) M, 4.2+/-2.0x10(-7) M and 4.0+/-2.8x10(-7) M. We showed that L-365,260 acted beyond receptor activation and production of intracellular second messengers and that it had no action on the H+/K+ -ATPase. We found that L-365,260 inhibited cyclic AMP-induced [14C]-aminopyrine accumulation in digitonin-permeabilized rabbit gastric glands, suggesting that this compound acted, at least in part, as an inhibitor of the cyclic AMP-dependent protein kinase (PKA) pathway.