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Purpose: Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct β-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses.Experimental Design: Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed in vitro and in vivo, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling.Results: We show that anti-progastrin antibodies decrease self-renewal of CSCs both in vitro and in vivo, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity in vitro is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors.Conclusions: Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS–mutated colorectal patients, for which there is a crucial unmet medical need. Clin Cancer Res; 23(17); 5267–80. ©2017 AACR.

This table presents the Top 10 most enriched KEGG pathways identified from the panel of miRNAs regulated by claudin-2, along with their enrichment score and p-value.

Contain all the supplementary methods linked to this paper

This table contains the combined list of genes (Column I) involved in self-renewal processes that were used to analyze potential enrichments for miRNA targets. This list was established using Gene Ontology keywords ('Self-renewal', columns A-B; 'Stem cell', columns C-D) or extracted from prior publications.

This table contains the de-identified clinical data on all patients from the Tissue microarray used in Figure 1 and S1.

This table contains the sequence of all primers used for qPCR, of siren and shRNAs against claudin-2, as well as details about miRNA mimics and inhibitors used in this work.

This table contains identified self-renewal target genes for the three candidate miRNA experimentally tested in our study, along with their prediction score. Only those targets with prediction score >0.85 are included.

This file contains all supplemental Figures, as well as legends for both Supplemental Figures and Supplemental Tables, as well as Supplemental methods and associated references

Contain all the supplemetary data linked to this paper

Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance in vitro. While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil–based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal in vitro and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDHHigh CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDHHigh phenotype. Next-generation sequencing in ALDHHigh cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer.Significance: Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. Cancer Res; 78(11); 2925–38. ©2018 AACR.

Annexin II expression is not enriched in cancer stem cell populations.

Description of additional methods and procedures used in the study.

Progastrin inhibition decreases colonosphere incidence, CSC frequency and ALDHhigh cell proportion in vitro.

ALDHhigh CRC cells display CSC characteristics and elevated progastrin expression.

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.

Legend for Supplemental Figures S1-S6.

Promotion of a glycolytic profile by progastrin is essential for the self-renewal ability of ALDHhigh cells.

Progastrin regulates the self-renewal of ALDHhigh colorectal CSCs.

shRNA-mediated down regulation of progastrin in vitro and in vivo.

Background: In spite of wide application of sorafenib for advanced hepatocellular carcinoma (HCC) treatment, and systemic chemotherapy cocktails (5-fluorouracil, irinotecan, and oxaliplatin) for metastatic colorectal cancer, the prognostic for both cancers remains poor. In recent years, highly tumorigenic sub-populations of cancer cells named Cancer Stem Cells (CSCs) have been claimed as responsible of some tumor recurrences. Indeed, CSCs are resistant to chemotherapy, and they have the ability to regenerate all the tumor bulk with its heterogeneous cell type populations. For this reason, new drugs with original mechanism of action which target CSC properties would likely improve cancer treatment Material and methods: Antitumor activity of GNS561 was tested on a panel of cancer cell lines and primary tumors. GNS561 impact on CSCs subpopulation in patient derived cells from colorectal hepatic metastatic tumors was assessed by flow cytometry (ALDH activity). In HCC, the effect of this drug was evaluated by sphere formation assay as readout to estimate CSC survival. Tolerance and plasma and liver pharmacokinetic were evaluated after single and repeated dosing in mice and rats. In vivo GNS561 activity was tested in orthotopic mouse model Results: GNS561 demonstrated multiple cellular effects such as inhibition of autophagy, induction of apoptosis and cell cycle modulation. It showed antitumor activity against several human cancer cell lines. Furthermore, GNS561 was effective against a panel of HCC tumors even from patients harboring sorafenib resistance. GNS561 showed nonetheless an original dose-response cytotoxic activity against the whole tumor populations but also against a subpopulation displaying high ALDH activity in three CRC patient-derived cell lines established from fresh liver metastasis biopsies. Consequently, on the same model this compound induced a striking decrease of sphere formation. In HCC cell lines GNS561 was active on both whole populations (mean EC50 2µM) and subpopulations displaying CSC features (Epcam high). In addition, in the opposite of sorafenib, GNS561 decreases the HCC cell capacity to form spheroids. In mouse, GNS561 was found well tolerated and highly selectively trapped in the liver (exposure ratio liver/plasma about 170 animals). In HCC PDX mouse model, tumor growth was significantly reduced by GNS561 with a dose-response manner, this tumor regression was associated with AFP level decreases by 72% with GNS561 (p=0.002) and 54% with sorafenib (p=0.046) compared to control Conclusions: GNS561 is a liver selective drug which offers great promise for HCC and liver metastatic tumors treatment. By simultaneously targeting the cancer stem cell subpopulation and tumor bulk, both cell heterogeneity, plasticity and recurrences could be overcome at least in colorectal cancer and HCC. GNS 561 is now aimed to further reach clinical development in patients in 2017 Note: This abstract was not presented at the meeting. Citation Format: Firas Bassissi, Elena Patricia Gifu, Sonia Brun, Jerome Courcambeck, Antoine Beret, Jean Marc Pascussi, Julie Pannequin, Eric Raymond, Philippe Halfon, Philippe Merle, Claude Caron de Fromentel. GNS561 is a new quinoline derivative with high efficacy on cancer stem cells from colorectal liver metastasis and hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1914. doi:10.1158/1538-7445.AM2017-1914