Inactivating germline mutations in the CDH1 gene (encoding the E-cadherin protein) are the genetic hallmark of hereditary diffuse gastric cancer (HDGC), and somatic CDH1 mutations are an early event in the development of sporadic diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In this study, histone deacetylase (HDAC) inhibitors were tested for their ability to preferentially inhibit the growth of human cell lines (MCF10A and NCI-N87) and murine organoids lacking CDH1 expression. CDH1^−/− breast and gastric cells were more sensitive to the pan-HDAC inhibitors entinostat, pracinostat, mocetinostat and vorinostat than wild-type cells, with an elevated growth inhibition that was, in part, attributable to increased apoptosis. CDH1-null cells were also sensitive to more class-specific HDAC inhibitors, but compared to the pan-inhibitors, these effects were less robust to genetic background. Increased sensitivity to entinostat was also observed in gastric organoids with both Cdh1 and Tp53 deletions. However, the deletion of Tp53 largely abrogated the sensitivity of the Cdh1-null organoids to pracinostat and mocetinostat. Finally, entinostat enhanced Cdh1 expression in heterozygous Cdh1^+/− murine organoids. In conclusion, entinostat is a promising drug for the chemoprevention and/or treatment of HDGC and may also be beneficial for the treatment of sporadic CDH1-deficient cancers.

Individuals born with intrauterine growth retardation (IUGR) are more prone to cardio-metabolic diseases as adults, and environmental changes during the perinatal period have been identified as potentially crucial factors. We have studied in a preclinical model early-onset molecular alterations present before the development of a clinical phenotype. We used a preclinical mouse model of induced IUGR, in which we modulated the nutrition of the pups during the suckling period, to modify their susceptibility to cardio-metabolic diseases in adulthood. Mice born with IUGR that were overfed (IUGR-O) during lactation rapidly developed obesity, hepatic steatosis and insulin resistance, by three months of age, whereas those subjected to nutrition restriction during lactation (IUGR-R) remained permanently thin and highly sensitive to insulin. Mice born with IUGR and fed normally during lactation (IUGR-N) presented an intermediate phenotype and developed insulin resistance by 12 months of age. Molecular alterations to the insulin signaling pathway with an early onset were observed in the livers of adult IUGR-N mice, nine months before the appearance of insulin resistance. The implication of epigenetic changes was revealed by ChIP sequencing, with both posttranslational H3K4me3 histone modifications and microRNAs involved. These two changes lead to the coherent regulation of insulin signaling, with a decrease in Akt gene transcription associated with an increase in the translation of its inhibitor, Pten. Moreover, we found that the levels of the implicated miRNA19a-3p also decreased in the blood of young adult IUGR mice nine months before the appearance of insulin resistance, suggesting a possible role for this miRNA as an early circulating biomarker of metabolic fate of potential use for precision medicine.

Germline inactivating variants of CDH1 are causative of hereditary diffuse gastric cancer (HDGC), a cancer syndrome characterized by an increased risk of both diffuse gastric cancer and lobular breast cancer. Because loss of function mutations are difficult to target therapeutically, we have taken a synthetic lethal approach to identify targetable vulnerabilities in CDH1-null cells. We have previously observed that CDH1-null MCF10A cells exhibit a reduced rate of endocytosis relative to wildtype MCF10A cells. To determine whether this deficiency is associated with wider vulnerabilities in vesicle trafficking, we screened isogenic MCF10A cell lines with known inhibitors of autophagy, endocytosis, and sphingolipid metabolism. Relative to wildtype MCF10A cells, CDH1^−/− MCF10A cells showed significantly greater sensitivity to several drugs targeting these processes, including the autophagy inhibitor chloroquine, the endocytosis inhibitors chlorpromazine and PP1, and the sphingosine kinase 1 inhibitor PF-543. Synthetic lethality was confirmed in both gastric and mammary organoid models of CDH1 loss, derived from CD44-Cre/Cdh1^fl/fl/tdTomato mice. Collectively, these results suggest that both sphingolipid metabolism and vesicle trafficking represent previously unrecognised druggable vulnerabilities in CDH1-null cells and may lead to the development of new therapies for HDGC.

The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for the cancer syndrome hereditary diffuse gastric cancer (HDGC). CDH1-deficient gastric cancers exhibit high AKT serine/threonine kinase 3 (AKT3) expression, but specific drugs against this AKT isoform are not available. We therefore used two publicly available datasets to identify AKT3-associated genes which could be used to indirectly target AKT3. Reactome analysis identified an enrichment of extracellular matrix remodelling genes in AKT3-high gastric cancers. Of the 51 genes that were significantly correlated with AKT3 (but not AKT1), discoidin domain receptor tyrosine kinase 2 (DDR2) showed the strongest positive association. Treatment of isogenic human cells and mouse gastric and mammary organoids with dasatinib, a small molecule inhibitor of multiple kinases including SRC, BCR-ABL and DDR2, preferentially slowed the growth and induced apoptosis of E-cadherin-deficient cells. Dasatinib treatment also preferentially slowed the growth of gastric and mammary organoids harbouring both Cdh1 and Tp53 mutations. In organoid models, dasatinib treatment was associated with decreased phosphorylation of total AKT, with a stronger effect seen in Cdh1-deficient organoids. Treatment with combinations of dasatinib and an inhibitor of AKT, MK2206, enhanced the effect of dasatinib in breast MCF10A cells. In conclusion, targeting the DDR2-SRC-AKT3 axis with dasatinib represents a promising approach for the chemoprevention and chemotherapy of gastric and breast cancers lacking E-cadherin.

Nutrition during the early postnatal period can program the growth trajectory and adult size. Nutritionally regulated hormones are strongly suspected to be involved in this physiological regulation. Linear growth during the postnatal period is regulated by the neuroendocrine somatotropic axis, whose development is first controlled by GHRH neurons of the hypothalamus. Leptin that is secreted by adipocytes in proportion to fat mass is one of the most widely studied nutritional factors, with a programming effect in the hypothalamus. However, it remains unclear whether leptin stimulates the development of GHRH neurons directly. Using a Ghrh-eGFP mouse model, we show here that leptin can directly stimulate the axonal growth of GHRH neurons in vitro in arcuate explant cultures. Moreover, GHRH neurons in arcuate explants harvested from underfed pups were insensitive to the induction of axonal growth by leptin, whereas AgRP neurons in these explants were responsive to leptin treatment. This insensitivity was associated with altered activating capacities of the three JAK2, AKT and ERK signaling pathways. These results suggest that leptin may be a direct effector of linear growth programming by nutrition, and that the GHRH neuronal subpopulation may display a specific response to leptin in cases of underfeeding.