Identification of selective inhibitors for diffuse-type gastric cancer cells by screening of annotated compounds in preclinical models

Background Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and poor patient prognosis. We have previously established an E-cadherin/p53 double conditional knockout (DCKO) mouse line as the first genetically engineered one, which morphologically and molecularly recapitulates human DGC. In this study, we explored low-molecular-weight drugs selectively eliminating mouse and human DGC cells. Methods We derived mouse gastric cancer (GC) cell lines from DGC of the DCKO mice demonstrating enhanced tumourigenic activity in immunodeficient mice and acquired tolerance to cytotoxic anti-cancer agents. Results We performed a synthetic lethal screening of 1535 annotated chemical compounds, and identified 27 candidates selectively killing the GC cell lines. The most potent drug mestranol, an oestrogen derivative, and other oestrogen receptor modulators specifically attenuated cell viability of the GC cell lines by inducing apoptosis preceded by DNA damage. Moreover, mestranol could significantly suppress tumour growth of the GC cells subcutaneously transplanted into nude mice, consistent with longer survival time in the female DCKO mice than in the male. Expectedly, human E-cadherin-mutant and -low gastric cancer cells showed higher susceptibility to oestrogen drugs in contrast to E-cadherin-intact ones in vitro and in vivo. Conclusions These findings may lead to the development of novel therapeutic strategies targeting DGC.


INTRODUCTION
Gastric cancer (GC) is estimated as the third leading cause of cancer-related death in the world. 1 GC is histologically classified into two major subtypes, intestinal-type and diffuse-type. Diffusetype gastric cancer (DGC) in particular demonstrates infiltrative growth, and occasionally metastases to lymph nodes, resulting in worse prognosis. 2 Although several clinical trials of chemotherapeutic drugs for advanced GC have been launched, overall survival rates have not been dramatically improved, approximately 20% in 5 years. [3][4][5] Germline mutations of CDH1 are frequently identified in hereditary DGC, while TP53, CDH1 and RHOA mutations in sporadic DGC, but molecular mechanisms underlying diffuse-type gastric carcinogenesis have not been completely clarified. 6,7 We have recently established a mouse model of DGC, in which E-cadherin (Cdh1) and p53 (Trp53) are inactivated specifically in gastric mucosae. 8 The penetrance is 100% for gastric neoplasm, contributing to the unfavourable mortality of 50% within a year. Poorly-differentiated and signet-ring cell adenocarcinoma cells are mainly distributed from mucosal to serosal layers in these mice. High frequency of lymph node dissemination and tumourigenicity in nude mice indicates the enhanced malignancy. Gene expression profiles of mouse DGC resemble those of human DGC, and mesenchymal markers and epithelial-mesenchymal transition (EMT)-regulators are overexpressed in mouse DGC as previously noted in human DGC. Taken together, the E-cadherin/p53 double conditional knockout (DCKO) mouse line is the first genetically engineered one which morphologically and molecularly recapitulates human DGC. 8 An in vitro system is required to further extend the mouse model-based research, and we therefore derived GC cell lines harbouring biological and molecular traits closely similar to those in vivo from DGC of the DCKO mice. The powerful platform of the cell lines and model mice could facilitate the drug development and preclinical testing for DGC treatment. In this study, considering the poor understanding of targets in E-cadherin-deficient DGC and the easy availability of agents in clinical practice, we performed synthetic lethal screening of a library of wellcharacterised compounds by using these cell lines, and evaluated Tumourigenicity in immunocompromised mice Cells were suspended in 100 μl Matrigel (BD Biosceiences) and subcutaneously injected into the male KSN nude mice. Frequency of tumourigenic cells and P-value was calculated by using extreme limiting dilution analysis. 10 The volume of the growing tumours was monitored once a week, and calculated by the formula; volume = length × width 2 × 0.5. Chemical agents were treated after tumour became palpable (about 500 mm 3 ). Tumour transplantation and drug administration were performed following the condition and schedule described in Supplementary Table 3.
Drug screening Cells were seeded in 100 μl of media including 1 × 10 3 cells into 96-well plates. A compound library composed of 1535 wellcharacterised and off-patent compounds was generously provided from Chemical Biology Screening Center of Tokyo Medical and Dental University (http://www.tmd.ac.jp/mri/SBS/cbsc/). At 12 h, 100 mM of diluted compound solution was transferred from the 96-well stock plates into the 96-well assay plates, resulting in 100 and 10 μM final concentration for all compounds. Forty eight hours after compound treatment, 10 μl of WST-8 Reagent (Dojindo) per well was added, and the absorbance was measured on a microplate reader (Bio-Rad Laboratories) at 450 nm with background subtraction at 630 nm at 4 h.
Cell cycle analysis Cells were plated in 6-cm dishes and grown overnight. Forty eight hours after drug treatment, the cells were harvested, washed with phosphate-buffered saline (PBS) and fixed with 70% ethanol overnight at 20°C. After rinsed with PBS containing 3% bovine serum albumin (BSA), the cells were resuspended in PBS with 50 μg/ml PI solution (Sigma-Aldrich, St. Louis, MO) and 10 μg/ml RNase A (Sigma-Aldrich) for 30 min on ice. The stained cells were counted by a FACSCalibur flow cytometer (BD Biosciences).
Apoptosis analysis Cells were seeded in 6-cm dishes and incubated overnight. Twenty four hours after each drug was administered, the cells were collected, rinsed with binding buffer (10 mM HEPES, pH 7.4; 140 mM NaCl; 2.5 mM CaCl 2 ), and incubated in 100 μl of binding buffer containing 5 μl of Annexin V-FITC Reagent (MBL International Corporation, Woburn, MA) for 30 min on the ice. The labelled cells were sorted by a FACSCalibur flow cytometer (BD Biosciences). For caspase inhibition assays, cells were seeded at a density of 2 × 10 4 cells per well in 12-well plates, and incubated overnight. Forty eight hours after treatment of 20 μM z-VAD-FMK (MedChem Express, Monmouth Junction, NJ) and 100 μM mestranol, cell viability was calculated by using MTT as described above.

Statistical analysis
Unsupervised clustering analysis based on Ward's method and principal component analysis for 505 genes with interquartile range (IQR) values greater than 4.0 were performed by using the R statistical software (version 3.0.3). Statistical analysis was also conducted by using the R statistical software. In cell viability assays, each P-value was calculated with the GraphPad Prism 7 software (San Diego, CA). P-value less than 0.05 was considered statistically significant.

Establishment of mouse E-cadherin/p53-deficient DGC cell lines
We first took into culture three transplanted tumours (#682, 792 and 773) of DGC of the DCKO mice as schematically illustrated in Fig. 1a, and isolated two cell lines rapidly expanding from each tumour (Supplementary Figure 1A). After cultured for a few passages, these six cell lines showed morphological heterogeneity, flat and round; the #682 and #773 tumours induced two flatdominant (MDGC1 and 2) and round-dominant cell lines (MDGC5 and 6), respectively, whereas the #792 did one flat-dominant (MDGC3) and one round-dominant (MDGC4) as presented in Fig. 1b. We also confirmed complete recombination of the Cdh1 and Trp53 loci by performing genomic PCR in all of the six MDGC cell lines (Supplementary Figure 1B).
We next investigated biological and molecular characteristics of these two types of cell lines. Cell proliferation assays (Fig. 1c) and wound-healing assays (Fig. 1d,e) displayed that the rounddominant cell lines exhibited greater mitogenic and motile properties than the flat-dominant ones. The round-dominant cell lines were more refractory than the flat-dominant to two cytotoxic drugs commonly used for treatment of patients with advanced GC, 5-fluorouracil and paclitaxel (Fig. 1f). Similarly to the gene expression signatures of primary DGC of the DCKO mice, the expression levels of mesenchymal markers (Vim and Cdh2) and EMT-regulators (Twist1 and Zeb2) were higher in the rounddominant cell lines than those in the flat-dominant cell lines (Fig. 1g). These findings suggested that mouse E-cadherin/p53deficient DGC might consist of two subtypes of cancer cells with morphologically, biologically and molecularly distinct features.
Evaluation of mouse E-cadherin/p53-deficienct DGC cell lines We succeeded in obtaining three subclones (MDGC4SC1, 6 and 7) composed of only flat cancer cells by limiting dilution, but it was difficult to maintain ones retaining the round phenotype for long periods. We then optimised culture conditions including sera, media and substrates, and newly established three cancer cell lines (MDGC7, 8 and 9) with the round shape from primary GC and lymph node metastasis of DGC of the DCKO mice (Fig. 2a), which harboured complete recombination of the Cdh1 and Trp53 loci as expected (Supplementary Figure 1B). We also possess cell lines derived from stomach mucosae of fetal p53-null mice. 9 We now compared the mouse flat and round Cdh1 −/− ;Trp53 −/− GC cell lines with the mouse Trp53 −/− gastric epithelial (GE) cell lines as control (Fig. 2b). We first cultured these three types of cell lines with serum-free media in ultra-low attachment culture dishes, an in vitro measurement of tumourigenic activity. The round GC cell lines showed a 30-fold increase in sphere-forming capacities relative to the flat GC and GE (Fig. 2c, Supplementary  Figure 2A). We next directly assessed the tumourigenic abilities by subcutaneously injecting them into nude mice. Tumours were generated with only 100 cells of the round GC, which were 100fold and 1000-fold less than were required for tumour seeding by the flat GC and GE cells, respectively (Fig. 2d). Tumours of the round GC cell lines grew much more rapidly than those of the flat GC (Fig. 2e). In transplanted tumours derived from the round GC cells, poorly differentiated cancer cells and signet ring carcinoma cells were abundantly distributed, and gland-like structures formed by moderately differentiated cancer cells were also scattered (Supplementary Figure 2B). In contrast, the flat GC cells generated tumours mainly composed of the gland-like structures, indicating that the round GC cells could maintain CSC-like properties and be at the higher level of tumour hierarchy than the flat GC cells. A strong resistance to conventional chemotherapeutic agents is also an important aspect of malignancy. Indeed, 5-fluorouracil and paclitaxel decreased the cell number of the round GC cell lines less than that of the flat GC and GE (Fig. 2f). As predicted from these in vitro results, when intraperitoneally administered into nude mice bearing palpable tumours of the MDGC7 cell line, 5-fluorouracil (50 mg/kg/week) failed to reduce tumour sizes (Supplementary Figure 2C). Unsupervised clustering analysis (Fig. 2g) and principle component analysis (Fig. 2h) of gene expression profiles could clearly distinguish these three cell types from each other, although the round and flat GC cells shared the similar genetic backgrounds. Among 505 differentially expressed genes, 142 genes were up-regulated in the round GC cell lines compared with the GE and flat GC included Twist1, 11 consistent with the above-mentioned data (Fig. 1g). Taken together, we established bona fide cancer cell lines of DGC of the DCKO mice.
First and second screenings of a library of known chemical compounds We speculated that drugs selectively killing the GC cell lines might be a silver bullet toward E-cadherin-deficient DGC. On the basis of this reasoning, we designed a proof-of-concept assay to identify such drugs (Fig. 3a), namely, we screened 1535 well-characterised compounds provided from Chemical Biology Screening Center of Tokyo Medical and Dental University by using the GE (GIF9), flat GC (MDGC4SC1) and round GC (MDGC7) cell lines. Before starting the ATP-based cell viability assays on 96-well plates, we examined the range where the cell number was proportionally correlated with the absorbance (Supplementary Figure 3A), and determined that seeding 1000 cells per well is appropriate for this assay. A screening window coefficient, Z'-factor, is requested to qualify a screening assay. 12 Under exposure of high concentration (5 mM) of 5-fluorouracil as positive control, Z'-factors of the cell lines were calculated as approximately 0.5, assuring the reliability of this screening system (Supplementary Figure 3B). We first screened 1535 test compounds including conventional chemotherapeutic drugs on this platform (Fig. 3b), and highlighted the difference between the GIF9 and MDGC7 cell viability (ΔCV) in a histogram (Supplementary Table 6). We identified 195 chemical compounds outside −1 standard deviation (SD) of the mean of ΔCV as candidates with selective toxicity toward the round GC cell lines. We next reevaluated the 195 hit compounds in duplicate, and created a histogram of ΔCV and a scatter plot (Fig. 3c). In the same way, 27 candidates outside −1 SD of the mean of ΔCV were enumerated in the table of Fig. 3d, which intriguingly contained some classes of clinically available drugs; mefenamic acid and diclofenac (red) are classified as non-steroidal anti-inflammatory drugs (NSAIDs), prophylactic use of which contributes to lower relative risks of gastrointestinal cancer; 13 oxybutynin and orphenadine (orange) are muscarinic antagonists widely prescribed for patients with benign prostatic hyperplasia, and Zhao and colleagues have recently documented that physiological and pharmacological inhibition of muscarinic acetylcholine receptors suppresses gastric tumourigenesis; 14 mestranol and equilin have oestrogen-like structure and function, consistent with the potential role of oestrogen in explaining the male predominance of stomach cancer; 15 betamethasone, desoxymetasone and desoxycorticosterone belong to the glucocorticoid class, and are generally used for palliative care of terminal cancer patients. These similarity could assist the accuracy of our scheme.
Third and fourth screenings of a library of known chemical compounds For further studies, after excluding antibiotics and antiseptics, we selected ten drugs and two alternates, thioridazine (a dopamine D2 receptor antagonist, ΔCV = −0.121) and catechin (an isomer of epicatechin, not included in the compound library), from the 27 candidate substrates listed in Fig. 3d in addition to four drugs, quercetin, salinomycin, flutamide and bicalutamide. Since thioridazine, 16 quercetin 17 and salinomycin 18 have been detected as drugs targeting cancer stem cells (CSCs) by screening of annotated compound libraries, we hypothesised that they could also selectively kill the round GC cell lines harbouring CSC-like properties (Fig. 2). Garnett and collaborators have screened a panel of several hundred cancer cell lines with 130 drugs under clinical and preclinical investigation, 19 and their public data suggest that somatic mutations of CDH1 gene are associated with cellular responses to an androgen receptor antagonist bicalutamide (P = 6.87 × 10 −3 ), categorised in the same class as flutamide (ΔCV = −0.271). Thus, we examined their effects across a range of doses, and confirmed that all of them provided evidence of selective toxicity toward the round GC cell lines (Fig. 4a, Supplementary Figure 4A). These results were not only supportive for the reliability of this screening assay, but also consistent with the previous reports of the drugs targeting CSCs [16][17][18] and Ecadherin-mutant cancer cells. 19 We compared drug sensitivity among three types of cell lines, the GE, flat and round GC, and found that the 12 candidate drugs were subdivided into two groups, ones selectively killing both the flat and round GC cell lines (e.g., mefenamic acid, rosuvastatin, dipyridamole and NADIDE) and ones doing only the round GC (Supplementary Figure 4B). Among the candidates, mestranol, NADIDE and betamethasone in particular had broad therapeutic windows between the GE and GC cell lines. We then performed flow cytometric analysis with propidium iodide (PI) staining with two types of cell lines to determine cell cycle profiles and apoptotic events. Treatment with mestranol, NADIDE and betamethasone increased the sub-G1 population only in the GC cell lines, consistent with the results of dose-response curves (Fig. 4b).
Effects of oestrogen on mouse E-cadherin-deficient GC We thus focused on mestranol, the most potent of the 1535 test compounds through the four steps of the screenings, and one of oestrogen derivatives frequently used for hormone replacement therapy. Observing that the 27 candidate substrates extracted by the second screening contained two oestrogen drugs (Fig. 3d), we retrospectively reanalysed the results of the first screening (Supplementary Table 6), and elucidated that the 195 hit compounds outside −1 SD of the mean of ΔCV included six compounds with oestrogen-like function such as 17β-oestradiol and tamoxifen, while the remainder did twelve, implying the class effect of oestrogen on cell viability of the GC cell lines (P = 0.0193, Fisher's exact test). This hypothesis was encouraged by the correlation between E-cadherin mutation and sensitivity of androgen receptor antagonists in this (Supplementary Figure 4A) and the previous comprehensive drug screenings. 19 We tried to explore the molecular mechanism and to identify oestrogen drugs with the most selective toxicity toward the GC cell lines (Fig. 5a, Supplementary Figure 5A). Exposure to not only 17α-ethinyl oestradiol (a metabolite of mestranol) and 17βoestradiol but also tamoxifen and raloxifene (recently reclassified as selective oestrogen receptor modulators), abrogated cell viability of the round GC cell lines, similarly to the results of mestranol. Two homologues of oestrogen receptors (ERs), ERα and ERβ, have different distribution and function among normal tissues as well as neoplasms including stomach cancer. 15 Propylpyrazole triol (ERα agonist) showed the similar cytotoxicity         against the GE and GC cell lines, whereas diarylpropionitrile (ERβ agonist) specifically killed the GC, suggesting that ERβ could mainly mediate cell death of the GC cell lines. These data were supported by the higher expression levels of ERβ in the GC than those in the GE (Fig. 5b), and consistent with preventive effects of ERβ on digestive system carcinogenesis. 15 Compared with normal gastric mucosae of Atp4b-Cre − ;Cdh1 loxP/loxP ;Trp53 loxP/loxP mice, ERβ was overexpressed in DGC of the DCKO mice at the RNA level (Supplementary Figure 5B). Oestrogen-induced cell death occurs through an increase in proapoptotic genes 20 or DNA doublestrand breaks. 21 Mestranol, 17β-oestradiol and tamoxifen triggered cell apoptosis in the round GC cell lines by using flow cytometric analysis with Annexin V-fluorescein isothiocyanate (FITC) and PI costaining (Fig. 5c). These events were accompanied with a significant increase of phosphorylation of H2A.X, not cleavage of caspase 3 (Fig. 5d), and an irreversible caspase inhibitor z-VAD-FMK could not rescue the cytotoxicity of the GC cells (Supplementary Figure 5C). Taken together, oestrogen drugs could induce DNA damage specifically in the GC cell lines via ERβ, and mestranol was the best candidate among them in the end. We then orally administered mestranol (0.5 mg/kg/day) into nude mice with palpable inoculated tumours of the MDGC7 cells, and observed tumour-growth inhibition (Fig. 5e). The effects of mestranol on sphere-forming efficiency were compatible to those on cell proliferation in the round GC cells (Fig. 4a and Supplementary Figure 5D), indicating that mestranol could exhibit toxic activities, but not inhibit stem cell-like properties. Moreover, the median survival time of the female DCKO mice was significantly longer than that of the male (P = 0.001, the log-rank test), implying the tumour-suppressive roles of oestrogen in DGC of the DCKO mice (Fig. 5f).

Effects of oestrogen on human E-cadherin-deficient GC cells
We examined whether oestrogen drugs could exert therapeutic function for human E-cadherin-deficient GC. We initially assessed the distribution and expression of E-cadherin in several human gastric cancer (HGC) cell lines at the protein level (Fig. 6a, Supplementary Figure 6A) as well as the mutation status at the mRNA and DNA levels (Supplementary Figures S6B and C), and divided them into three groups; E-cadherin-intact (MKN74 and MKN7), E-cadherin-mutant (MKN45 and KATOIII) and E-cadherinlow (AGS and HSC58). Treatment with four drugs with oestrogenlike activity including mestranol selectively impaired cell viability of the E-cadherin-mutant and -low HGC cell lines (Fig. 6c). Mestranol triggered cell apoptosis, not cell cycle arrest, in the Ecadherin-deficient HGC cell lines similarly to 17β-oestradiol and tamoxifen, which was preceded by DNA damage (Fig. 6d-f, Supplementary Figures S6D-S6F). Mestranol administration suppressed transplanted tumour growth of the E-cadherin-mutant and E-cadherin-low cell lines, but not that of the E-cadherin-intact ones (Fig. 6g, Supplementary Figure 6G). Thus, cellular responses of E-cadherin-deficient GC cells to oestrogen could be highly conserved across two different species.
To investigate the relationship between gender, histological subtype, E-cadherin/p53 status and prognosis in clinical samples of GC, we used public data provided from the Cancer Genome Atlas Research Network (TCGA), which included 176 tumours (76 DGC and 100 IGC) with gene mutation, copy number alteration, DNA methylation, mRNA expression and pathological data. Surprisingly, the female DGC patients had better prognosis in overall survival than the male (P = 0.006, the log-rank test), although there was no difference between the female and male IGC patients (P = 0.725) as shown in Supplementary Figure 7A. Next, we divided the 176 clinical specimens of TCGA data set into the E-cadherin-deficient and -intact groups with and without somatic mutation, biallelic loss, promoter hypermethylation (βvalue ≥ 0.5) or low expression (RSEM < 2000) of CDH1 gene, respectively. In 76 DGC samples, 10 of 29 E-cadherin-deficient tumours harboured somatic mutation or biallelic loss of TP53 gene, whereas 19 of 47 E-cadherin-intact tumours did, suggesting that E-cadherin/p53-deficiemt GC accounted for approximately 15% of DGC, and therefore was not rare (P = 0.636, Fisher's exact test). Although not in the E-cadherin-intact group (P = 0.362, the log-rank test), overall survival rate of the female patients was higher than that of the male in the E-cadherin-deficient group (P = 0.048), which was consistent with the prognosis of the DCKO mice ( Fig. 5f and Supplementary Figure 7B).
We performed Single Sample Gene Set Enrichment Analysis (ssGSEA) for tumour samples with high and low expression levels of CDH1, termed as CDH1-high and -low, with two gene sets associated with oestrogen signal transduction, ESTROGEN_RE-SPONSE_EARLY and ESTOGEN_RESPONSE_LATE. The ssGSEA scores of the CDH1-low group for both of the gene sets were significantly lower than those of the CDH1-high group (Supplementary Figure 7C), implying that only E-cadherin-deficient GC in which the oestrogen signal pathway was inactivated could survive.

DISCUSSION
The United States National Cancer Institute 60 human tumour cell lines anti-cancer drug screen (NCI60) emerged in the late 1980s as a powerful drug discovery tool. 22,23 However, cellular reactions in vitro are not able to reflect those in vivo due to two failings; cell lines have genetically, epigenetically and biologically changed under culture conditions; cell lines no longer maintain the tumour original properties present in the primary cancer. In an effort to address these shortcomings, patient-derived xenografts transplanted into immunodeficient rodents have recently been used for preclinical modelling. These models are appropriate for validating the in vivo effects of candidate drugs, but not for screening chemical libraries due to difficulty of primary culture. Since tumour genotype and epigenotype variation in tumour and between patients (intratumour and interpatient heterogeneity) is non-negligible, large-scale screenings are required for discovery of potent agents targeting the common molecular mechanism. We then solved these problems by a mouse model-based study, that is, establishing a genetically engineered mouse model recapitulating human cancer, deriving cell lines from the mouse tumour, performing a synthetic lethal screening by using the cell lines, and validating tumour suppressor activity of candidate drugs against human cancer. Early passage cells from cancer of a genetically engineered mouse model harbour the biological and molecular traits of the original tumours, and a few of them are sufficient for screening assays due to similar genetic background. In addition, by using the mice with carcinoma in situ, anti-cancer effects of the Fig. 6 Effects of oestrogen drugs on human E-cadherin-deficient gastric cancer cells. a Representative fluorescence microscopy images of the HGC cells stained with antibodies against E-cadherin (red). Nuclei were counterstained with DAPI (blue). b Representative phase-contrast images. c Dose-response curves of oestrogen drugs against the HGC cell lines. Bars show standard deviations. P-value was calculated from the ANOVA table. NS not significant. d Flow cytometric analysis with PI staining. The left and right panels show the representative histograms and percentage graphs of cells in each cell cycle, respectively. e Flow cytometric analysis with Annexin V-FITC and PI costaining. f Immunoblots of phosphorylated H2A.X after treatment with vehicle (V), mestranol (M) and 17β-oestradiol (E) in the E-cadherin-mutant (MKN45) and -low (AGS) cells. g Tumour-growth curves of the E-cadherin-mutant (MKN45) and -low (AGS) cells in nude mice under treatment with mestranol (0.5 mg/ kg/day, orally administered). Bars show standard errors. P-value was calculated by Welch's t-test Screening of known drugs by using DGC mouse model S Shimada et al.
candidate drugs can be precisely evaluated on the platform mimicking tumour microenvironment including cancer-associated fibroblasts and immune cells.
Currently, most molecularly targeted drugs are inhibitors of driver oncogenes, because it appears more straightforward to repress a hyperactivated oncogene than to restore the function of inactivated tumour suppressor genes. 24 Among several promising strategies of drug development for cancer with mutations in tumour suppressor genes, we here applied a comprehensive approach for E-cadherin-deficient DGC, that is, a screening with a collection of well-established annotated compounds. This process of finding new uses outside the scope of the original medical indication for existing drugs is known as repositioning, 25 and offers two significant advantages over conventional de novo drug discovery and development; firstly, from the molecular function of compounds selectively killing cancer cells, the addicted signal pathway could be predicted; secondly, safer and shorter routes to the clinic are possible because in vitro and in vivo screenings, lead optimisation and chemical toxicology have already been completed. Excellent examples of this concept "repositioning" are thioridazine and salinomycin. Thioridazine, a Food and Drug Administration (FDA)-approved antipsychotic dopamine receptor antagonist, was reprofiled as an anti-CSC drug from libraries of known compounds, and demonstrated that dopamine D2 receptor antagonism could account for the loss of stemness. 16 Since Gupta et al. initially did, several other groups have noted that salinomycin attenuates CSC-like properties in various types of cancer, and identified that this potassium ionophore overcomes ABC transporter-mediated multidrug resistance and inhibits oxidative phosphorylation in mitochondria on which CSCs mainly rely more than on glycolysis. Our screening assay with 1535 wellcharacterised compounds also revealed that differentially expressed ERβ could be a potential target for E-cadherindeficient DGC, and provided convincing evidence of clinical use of oestrogen drugs including mestranol for prevention and treatment of this subtype of GC.
Our mouse model-based study could hint favourable prognosis of female patients with DGC. It sounds odd that oestrogen protects against the development of DGC, which is believed to be encountered in young female patients, although there is a strong and enigmatic male dominance in the incidence of GC with a male-to-female ratio of approximately 2:1. 15 The cumulative risk of hereditary DGC by age 60 was estimated to be higher for females than for males, but not significantly. 26 Patients with DGC younger than 40 years showed a nearly equal male-to-female ratio (1.3:1), whereas patients older than 40 showed a male preponderance (2.3:1) in a series of 66 GC probands for germline CDH1 mutations. 27 In a large-scale cohort study, the diffuse-type is more common in males than in females for nearly all age groups. 28 Females had a significantly lower risk of dying compared to males in patients whose tumours were poorly differentiated. 29 There is also mounting evidence for the clinical potency of exogenous oestrogen on GC. A seemingly protective effect of hormone replace therapy, such as mestranol treatment, on GC risk has been reported in several human studies from different populations. 15 In animal models, administration of oestrogen to N-methyl-N'-nitro-nitrosoguanidine (MNNG)-treated rat 30 and Helicobacter pylori-infected INS-GAS mice 31 decreased their incidence of GC. Thus, an intrinsic tumour-suppressor role of oestrogen in Ecadherin-deficient DGC is assisted by these epidemiological and experimental findings.
Since tamoxifen administration in mice causes extensive parietal cell damage by active acid secretion through H,K-ATPase, 32 it is possible that estrogen analogues are also toxic to the GC cells, which were originated from parietal cells of the DCKO (Atp4b-Cre + ;Cdh1 loxP/loxP ;Trp53 loxP/loxP ) mice. In immunohistochemical analysis, cleaved caspase 3 was not detected in parietal cells of the DCKO mice orally treated with mestranol (0.5 mg/kg/day) for a week, suggesting few adverse effects of mestranol on parietal cells (Supplementary Figure 8A). Because RT-PCR analysis demonstrated that the expression levels of Atp4a and Atp4b encoding H, K-ATPase subunits were extremely lower in the GE and GC cell lines than those in normal gastric mucosae containing parietal cells (Supplementary Figure 8B), it did not seem that mestranol specifically eliminated the GC cells by inducing acid secretion through H,K-ATPase. The two evidences disproved the hypothesis above.
We here screened the compound library composed of offpatent drugs, and highlighted a single potent drug, mestranol, targeting E-cadherin-deficient DGC. It is also of interest to investigate how the other 26 candidates listed in Fig. 3d could exhibit the specific toxicity to this subtype of GC, and to perform screening with other libraries containing novel molecularly targeted agents by using this platform.