A novel method to detect hPG80 (human circulating progastrin) in the blood.

hPG80 (human circulating progastrin) is produced and released by cancer cells. We recently reported that hPG80 is detected in the blood of patients with cancers from different origins, suggesting its potential utility for cancer detection. To accurately measure hPG80 in the blood of patients, we developed the DxPG80 test, a sandwich Enzyme-Linked Immunosorbent Assay (ELISA). This test quantifies hPG80 in EDTA plasma samples. The analytical performances of the DxPG80 test were evaluated using standard procedures and guidelines specific to ELISA technology. We showed high specificity for hPG80 with no cross-reactivity with human glycine-extended gastrin (hG17-Gly), human carboxy-amidated gastrin (hG17-NH2) or the CTFP (C-Terminus Flanking Peptide) and no interference with various endogenous or exogenous compounds. The test is linear between 0 and 50 pM hPG80 (native or recombinant). We demonstrated a trueness of measurement, an accuracy and a variability of hPG80 quantification with the DxPG80 test below the 20% relative errors as recommended in the guidelines. The limit of detection of hPG80 and the limit of quantification were calculated as 1 pM and 3.3 pM respectively. In conclusion, these results show the strong analytical performance of the DxPG80 test to measure hPG80 in blood samples.

The oncogenic and druggable hPG80 (Progastrin) is overexpressed in multiple cancers and detected in the blood of patients.

BACKGROUND: In colorectal cancer, hPG80 (progastrin) is released from tumor cells, promotes cancer stem cells (CSC) self-renewal and is detected in the blood of patients. Because the gene GAST that encodes hPG80 is a target gene of oncogenic pathways that are activated in many tumor types, we hypothesized that hPG80 could be expressed by tumors from various origins other than colorectal cancers, be a drug target and be detectable in the blood of these patients. METHODS: hPG80 expression was monitored by fluorescent immunohistochemistry and mRNA expression in tumors from various origins. Cancer cell lines were used in sphere forming assay to analyze CSC self-renewal. Blood samples were obtained from 1546 patients with 11 different cancer origins and from two retrospective kinetic studies in patients with peritoneal carcinomatosis or hepatocellular carcinomas. These patients were regularly sampled during treatments and assayed for hPG80. FINDINGS: We showed that hPG80 was present in the 11 tumor types tested. In cell lines originating from these tumor types, hPG80 neutralization decreased significantly CSC self-renewal by 28 to 54%. hPG80 was detected in the blood of patients at significantly higher concentration than in healthy blood donors (median hPG80: 4.88 pM versus 1.05 pM; p < 0.0001) and shown to be correlated to GAST mRNA levels in the matched tumor (i.e., lung cancers, Spearman r = 0.8; p = 0.0023). Furthermore, we showed a strong association between longitudinal hPG80 concentration changes and anti-cancer treatment efficacy in two independent retrospective studies. In the peritoneal carcinomatosis cohort, median hPG80 from inclusion to the post-operative period decreased from 5.36 to 3.00 pM (p < 0.0001, n = 62) and in the hepatocellular carcinoma cohort, median hPG80 from inclusion to remission decreased from 11.54 pM to 1.99 pM (p < 0.0001, n = 63). INTERPRETATION: Because oncogenic hPG80 is expressed in tumor cells from different origins and because circulating hPG80 in the blood is related to the burden/activity of the tumor, it is a promising cancer target for therapy and for disease monitoring. FUNDINGS: ECS-Progastrin.

Targeting the Wnt Pathway and Cancer Stem Cells with Anti-progastrin Humanized Antibodies as a Potential Treatment for K-RAS-Mutated Colorectal Cancer.

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 ß-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.

Essential requirement for ß-arrestin2 in mouse intestinal tumors with elevated Wnt signaling.

ß-Arrestins (Arrb) participate in the regulation of multiple signaling pathways, including Wnt/ß-catenin, the major actor in human colorectal cancer initiation. To better understand the roles of Arrb in intestinal tumorigenesis, a reverse genetic approach (Arrb(-/-)) and in vivo siRNA treatment were used in Apc(Δ14/+) mice. Mice with Arrb2 depletion (knockout and siRNA) developed only 33% of the tumors detected in their Arrb2-WT littermates, whereas Arrb1 depletion remained without significant effect. These remaining tumors grow normally and are essentially Arrb2-independent. Unsupervised hierarchical clustering analysis showed that they clustered with 25% of Apc(Δ14/+);Arrb2(+/+) tumors. Genes overexpressed in this subset reflect a high interaction with the immune system, whereas those overexpressed in Arrb2-dependent tumors are predominantly involved in Wnt signaling, cell adhesion, migration, and extracellular matrix remodeling. The involvement of Arrb2 in intestinal tumor development via the regulation of the Wnt pathway is supported by ex vivo and in vitro experiments using either tumors from Apc(Δ14/+) mice or murine Apc(Min/+) cells. Indeed, Arrb2 siRNAs decreased the expression of Wnt target genes in cells isolated from 12 of 18 tumors from Apc(Δ14/+) mice. In Apc(Min/+) cells, Arrb2 siRNAs completely reversed the increased Wnt activity and colony formation in soft agar induced by Apc siRNA treatment, whereas they did not affect these parameters in basal conditions or in cells expressing constitutively active ß-catenin. We demonstrate that Arrb2 is essential for the initiation and growth of intestinal tumors displaying elevated Wnt pathway activity and identify a previously unsuspected molecular heterogeneity among tumors induced by truncating Apc mutations.