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Background & Aims Gastric carcinoma is related mostly to CagA+-Helicobacter pylori infection, which disrupts the gastric mucosa turnover and elicits an epithelial-mesenchymal transition (EMT) and preneoplastic transdifferentiation. The tumor suppressor Hippo pathway controls stem cell homeostasis; its core, constituted by the large tumor suppressor 2 (LATS2) kinase and its substrate Yes-associated protein 1 (YAP1), was investigated in this context. Methods Hippo, EMT, and intestinal metaplasia marker expression were investigated by transcriptomic and immunostaining analyses in human gastric AGS and MKN74 and nongastric immortalized RPE1 and HMLE epithelial cell lines challenged by H pylori, and on gastric tissues of infected patients and mice. LATS2 and YAP1 were silenced using small interfering RNAs. A transcriptional enhanced associated domain (TEAD) reporter assay was used. Cell proliferation and invasion were evaluated. Results LATS2 and YAP1 appear co-overexpressed in the infected mucosa, especially in gastritis and intestinal metaplasia. H pylori via CagA stimulates LATS2 and YAP1 in a coordinated biphasic pattern, characterized by an early transient YAP1 nuclear accumulation and stimulated YAP1/TEAD transcription, followed by nuclear LATS2 up-regulation leading to YAP1 phosphorylation and targeting for degradation. LATS2 and YAP1 reciprocally positively regulate each other’s expression. Loss-of-function experiments showed that LATS2 restricts H pylori–induced EMT marker expression, invasion, and intestinal metaplasia, supporting a role of LATS2 in maintaining the epithelial phenotype of gastric cells and constraining H pylori–induced preneoplastic changes. Conclusions H pylori infection engages a number of signaling cascades that alienate mucosa homeostasis, including the Hippo LATS2/YAP1/TEAD pathway. In the host–pathogen conflict, which generates an inflammatory environment and perturbations of the epithelial turnover and differentiation, Hippo signaling appears as a protective pathway, limiting the loss of gastric epithelial cell identity that precedes gastric carcinoma development., Graphical abstract

Helicobacter pylori infection, the main risk factor for gastric cancer (GC), leads to an epithelial&ndash, mesenchymal transition (EMT) of gastric epithelium contributing to gastric cancer stem cell (CSC) emergence. The Hippo pathway effectors yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) control cancer initiation and progression in many cancers including GC. Here, we investigated the role of TAZ in the early steps of H. pylori-mediated gastric carcinogenesis. TAZ implication in EMT, invasion, and CSC-related tumorigenic properties were evaluated in three gastric epithelial cell lines infected by H. pylori. We showed that H. pylori infection increased TAZ nuclear expression and transcriptional enhancer TEA domain (TEAD) transcription factors transcriptional activity. Nuclear TAZ and zinc finger E-box-binding homeobox 1 (ZEB1) were co-overexpressed in cells harboring a mesenchymal phenotype in vitro, and in areas of regenerative hyperplasia in gastric mucosa of H. pylori-infected patients and experimentally infected mice, as well as at the invasive front of gastric carcinoma. TAZ silencing reduced ZEB1 expression and EMT phenotype, and strongly inhibited invasion and tumorsphere formation induced by H. pylori. In conclusion, TAZ activation in response to H. pylori infection contributes to H. pylori-induced EMT, invasion, and CSC-like tumorigenic properties. TAZ overexpression in H. pylori-induced pre-neoplastic lesions and in GC could therefore constitute a biomarker of early transformation in gastric carcinogenesis.

Cancer stem cells (CSCs) present chemo-resistance mechanisms contributing to tumour maintenance and recurrence, making their targeting of utmost importance in gastric cancer (GC) therapy. The Hippo pathway has been implicated in gastric CSC properties and was shown to be regulated by leukaemia inhibitory factor receptor (LIFR) and its ligand LIF in breast cancer. This study aimed to determine LIF&rsquo, s effect on CSC properties in GC cell lines and patient-derived xenograft (PDX) cells, which remains unexplored. LIF&rsquo, s treatment effect on CSC markers expression and tumoursphere formation was evaluated. The Hippo kinase inhibitor XMU-MP-1 and/or the JAK1 inhibitor Ruxolitinib were used to determine Hippo and canonical JAK/STAT pathway involvement in gastric CSCs&rsquo, response to LIF. Results indicate that LIF decreased tumorigenic and chemo-resistant CSCs, in both GC cell lines and PDX cells. In addition, LIF increased activation of LATS1/2 Hippo kinases, thereby decreasing downstream YAP/TAZ nuclear accumulation and TEAD transcriptional activity. LIF&rsquo, s anti-CSC effect was reversed by XMU-MP-1 but not by Ruxolitinib treatment, highlighting the opposite effects of these two pathways downstream LIFR. In conclusion, LIF displays anti-CSC properties in GC, through Hippo kinases activation, and could in fine constitute a new CSCs-targeting strategy to help decrease relapse cases and bad prognosis in GC.

Highlights • Secretion of progastrin is a signature event of early malignant transformation in the colon. • In the healthy epithelium, progastrin is produced by a subset of enteroendocrine cells expressing both Bmi1 and Prox1. • LGR5-high intestinal stem cells are a primary source of progastrin production in early mouse and human intestinal adenomas., 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.

Gastric cancer is the third leading cause of cancer mortality worldwide. Cancer stem cells (CSC) are at the origin of tumor initiation, chemoresistance, and the formation of metastases. However, there is a lack of mouse models enabling the study of the metastatic process in gastric adenocarcinoma (GC). The aims of this study were to develop original mouse models of patient-derived primary GC orthotopic xenografts (PDOX) allowing the development of distant metastases as preclinical models to study the anti-metastatic efficiency of drugs such as the phosphatidylinositol 3-kinase (PI3K) inhibitor Buparlisib (BKM120). Luciferase-encoding cells generated from primary GC were injected into the stomach wall of immunocompromised mice, gastric tumor and metastases development were followed by bioluminescence imaging. The anti-CSC properties of BKM120 were evaluated on the GC cells&rsquo, phenotype (CD44 expression) and tumorigenic properties in vitro and in vivo on BKM120-treated mice. After eight weeks, PDOX mice formed tumors in the stomach as well as distant metastases, that were enriched in CSC, in the liver, the lung, and the peritoneal cavity. BKM120 treatment significantly inhibited the CSC properties in vitro and reduced the number of distant metastases in mice. These new preclinical models offer the opportunity to study the anti-metastatic efficiency of new CSC-based therapeutic strategies.

International audience; Colorectal cancer lethality usually results from post-treatment relapse in the majority of stage II-IV patients, due to the enhanced resistance of Cancer Stem Cells (CSCs). Here, we show that the nuclear receptor Pregnane X Receptor (PXR, NR1I2), behaves as a key driver of CSC-mediated tumor recurrence. First, PXR is specifically expressed in CSCs, where it drives the expression of genes involved in self-renewal and chemoresistance. Clinically, high levels of PXR correlate with poor recurrence-free survival in a cohort of >200 stage II/III colorectal cancer patients treated with chemotherapy, for whom finding biomarkers of treatment outcome is an urgent clinical need. shRNA silencing of PXR increased the chemo-sensitivity of human colon CSCs, reduced their self-renewal and tumor-initiating potential, and drastically delayed tumor recurrence in mice following chemotherapy. This study uncovers PXR as a key factor for CSC self-renewal and chemoresistance and targeting PXR thus represents a promising strategy to minimize colorectal cancer relapse by selectively sensitizing CSCs to chemotherapy.

Resume Le processus metastatique est defini comme « une dissemination de cellules neoplasiques dans un site secondaire non contigu et distant, au sein duquel ces cellules proliferent pour former une masse extravasculaire de cellules incompletement differenciees ». La majorite des deces imputables aux cancers solides est la consequence de l’apparition et du developpement de metastases disseminees qui sont responsables a terme de la dysfonction d’organes vitaux. Dans cette revue un certains de nombre de points, soit recemment decouverts soit sujets a controverse, sont abordes. En effet il sera discute de la transition epithelio-mesenchymateuse (EMT), des cellules tumorales circulantes, de la dormance tumorale, de la colonisation dans un organe distant et enfin des cellules souches cancereuses.

MicroRNAs are key factors in the regulation of gene expression and their deregulation has been directly linked to various pathologies such as cancer. The use of small molecules to tackle the overexpression of oncogenic miRNAs has proved its efficacy and holds the promise for therapeutic applications. Here we describe the screening of a 640-compound library and the identification of polyamine derivatives interfering with in vitro Dicer-mediated processing of the oncogenic miR-372 precursor (pre-miR-372). The most active inhibitor is a spermine-amidine conjugate that binds to the pre-miR-372 with a KD of 0.15 µM, and inhibits its in vitro processing with a IC50 of 1.06 µM. The inhibition of miR-372 biogenesis was confirmed in gastric cancer cells overexpressing miR-372 and a specific inhibition of proliferation through de-repression of the tumor suppressor LATS2 protein, a miR-372 target, was observed. This compound modifies the expression of a small set of miRNAs and its selective biological activity has been confirmed in patient-derived ex vivo cultures of gastric carcinoma. Polyamine derivatives are promising starting materials for future studies about the inhibition of oncogenic miRNAs and, to the best of our knowledge, this is the first report about the application of functionalized polyamines as miRNAs interfering agents.

Signal transducer and activator of transcription 3 (STAT3) signaling is a major driver of colorectal cancer (CRC) growth, however therapeutics, which can effectively target this pathway, have so far remained elusive. Here, we performed an extensive screen for STAT3 inhibitors among a library of 1167 FDA-approved agents, identifying Ponatinib as a lead candidate. We found that Ponatinib inhibits STAT3 activity driven by EGF/EGFR, IL-6/IL-6R and IL-11/IL-11R, three major ligand/receptor systems involved in CRC development and progression. Ponatinib was able to inhibit CRC migration and tumor growth in vivo. In addition, Ponatinib displayed a greater ability to inhibit STAT3 activity and mediated superior anti-proliferative efficacy compared to five FDA approved SRC and Janus Kinase (JAK) inhibitors. Finally, long-term exposure of CRC cells to Ponatinib, Dasatinib and Bosutinib resulted in acquired resistance to Dasatinib and Bosutinib occurring within six weeks. However, acquired resistance to Ponatinib was observed after long-term exposure of &gt, 4 months. Overall, our results identify a novel anti-STAT3 property of Ponatinib and thus, Ponatinib offers a potential therapeutic strategy for CRC.

International audience; Gastric cancer is the third leading cause of cancer-related deaths worldwide and has still a poor prognosis. Therefore, new therapeutic strategies are needed: among them, targeting cancer stem cells (CSCs) could offer new opportunities. The aim of our study was to evaluate the anti-tumoural effect of metformin on gastric cancer in vitro and in vivo and especially, to determine whether this molecule could target the gastric CSCs. Metformin effects were evaluated on the proliferation and tumourigenic properties of the gastric CSCs from patient-derived primary tumour xenografts (PDXs) and cancer cell lines (MKN45, AGS and MKN74) in vitro in conventional 2 dimensional (2D) and in 3 dimensional (3D) culture systems, in which only CSCs are able to form tumourspheres and in mouse xenograft models in vivo. Metformin induced a cell cycle arrest, which decreased cell proliferation in the 2D cultures. In a 3D culture system, metformin decreased the number of tumourspheres, revealing its capacity to target the CSCs. This effect was confirmed by the study of the expression of CSC markers (CD44 and Sox2) and differentiation markers (Kruppel-like factor 4 and MUC5AC), which were decreased or increased in response to metformin, respectively. Finally, in vivo treatment of PDXs with metformin led to a tumour growth delay and decreased the self-renewal ability of the CSCs. These results suggest that the use of metformin could represent an efficient strategy to inhibit tumour growth by targeting gastric CSCs.

Subpopulations of cancer stem–like cells (CSC) are thought to drive tumor progression and posttreatment recurrence in multiple solid tumors. However, the mechanisms that maintain stable proportions of self-renewing CSC within heterogeneous tumors under homeostatic conditions remain poorly understood. Progastrin is a secreted peptide that exhibits tumor-forming potential in colorectal cancer, where it regulates pathways known to modulate colon CSC behaviors. In this study, we investigated the role of progastrin in regulating CSC phenotype in advanced colorectal cancer. Progastrin expression and secretion were highly enriched in colon CSC isolated from human colorectal cancer cell lines and colon tumor biopsies. Progastrin expression promoted CSC self-renewal and survival, whereas its depletion by RNA interference–mediated or antibody-mediated strategies altered the homeostatic proportions of CSC cells within heterogeneous colorectal cancer tumors. Progastrin downregulation also decreased the frequency of ALDHhigh cells, impairing their tumor-initiating potential, and inhibited the high glycolytic activity of ALDHhigh CSC to limit their self-renewal capability. Taken together, our results show how colorectal CSC maintain their tumor-initiating and self-renewal capabilities by secreting progastrin, thereby contributing to the tumor microenvironment to support malignancy. Cancer Res; 76(12); 3618–28. ©2016 AACR.

Gastric carcinoma is the third leading cause of cancer-related death worldwide. This cancer, most of the time metastatic, is essentially treated by surgery associated with conventional chemotherapy, and has a poor prognosis. The existence of cancer stem cells (CSC) expressing CD44 and a high aldehyde dehydrogenase (ALDH) activity has recently been demonstrated in gastric carcinoma and has opened new perspectives to develop targeted therapy. In this study, we evaluated the effects of all-trans-retinoic acid (ATRA) on CSCs in human gastric carcinoma. ATRA effects were evaluated on the proliferation and tumorigenic properties of gastric carcinoma cells from patient-derived tumors and cell lines in conventional 2D cultures, in 3D culture systems (tumorsphere assay) and in mouse xenograft models. ATRA inhibited both tumorspheres initiation and growth in vitro, which was associated with a cell-cycle arrest through the upregulation of cyclin-dependent kinase (CDK) inhibitors and the downregulation of cell-cycle progression activators. More importantly, ATRA downregulated the expression of the CSC markers CD44 and ALDH as well as stemness genes such as Klf4 and Sox2 and induced differentiation of tumorspheres. Finally, 2 weeks of daily ATRA treatment were sufficient to inhibit gastric tumor progression in vivo, which was associated with a decrease in CD44, ALDH1, Ki67 and PCNA expression in the remaining tumor cells. Administration of ATRA appears to be a potent strategy to efficiently inhibit tumor growth and more importantly to target gastric CSCs in both intestinal and diffuse types of gastric carcinoma.