Recent large-scale molecular subtyping efforts have opened novel individualized treatment avenues for gastric cancer [
23‐
25]. For example, patients with metabolic subtype of gastric cancer derived from large gene expression profiling of multiple cohorts of clinical gastric cancer specimens show improved clinic outcome upon treatment with 5-FU [
23]. While cancer cells of the metabolic subtype were measured more sensitive to 5-FU, gastric cancer cells of the mesenchymal subtype responded to a greater degree to PI3K/mTOR inhibition [
23]. Similarly, the recently released data from The Cancer Genome Atlas (TCGA) initiative suggests genomically defined leads, for example, of increased sensitivity to receptor tyrosine kinase or cell cycle modulating agents in the chromosomal unstable (CIN) identified gastric cancers, or increased sensitivity to PI3K kinase, JAK2, and immune checkpoint blockade in the EBV positive subtype [
25]. In contrast, leads for drug sensitivities unique to familial gastric cancer due to germline CDH1 mutations are to date largely derived from a synthetic lethality and drug screen in an isogenic CDH1 knockout breast fibroblast model (MCF10A CDH1(−/−)) [
12], CDH1-negative systems overexpressing wild type versus mutant forms of CDH1 [
26,
27], or correlative tissue studies of the early unique T1a lesions of prophylactic gastrectomy specimens [
8,
10]. That hereditary diffuse gastric cancer (HDGC) due to germline CDH1 variants, and sporadic gastric cancers with somatic CDH1 perturbations are unique subtypes of gastric cancer, likely to harbor different drug sensitivity profiles and hence offer opportunities for genotype-directed personalized treatment approaches, is supported by a number of observations; for example, in a detailed CDH1 profiling effort of 174 sporadic and 72 familial gastric cancer specimens Corso et al. reported significant differences in clinical outcomes depending on presence and type of CDH1 alternation with the worst survival rates observed across all examined gastric cancers in cases with structural CDH1 defects and in particular in familial tumors [
11]. Or, eleven percent of the 205 primary gastric adenocarcinoma, examined as part of the TCGA effort, harbored somatic mutations in the CDH1 gene, which occurred nearly exclusively in the genomically stable (GS) gastric cancer subtype (37% of GS cases) which, by far, comprised of the largest number of cancers with diffuse histology [
25].
Similar to the MCF10A CDH1(−/−) mutant cells, c.1380delA CDH1 SB.mhdgc-1 gastric cancer cells showed altered gene expression of genes involved in cellular component organization, cytoskeletal organization, and cell adhesion (Additional file
4: Figure S2), including microtubule nucleation involving genes like TUBB2 [
28]. We found c.1380delA CDH1 SB.mhdgc-1 cells sensitive to taxanes targeting TUBB1 as well as agents targeting mitosis like the aurora kinase inhibitors, a finding also made by Telford and colleagues in the MCF10A CDH1 (−/−) system [
12,
29]. Elevated phosphoinositide signaling, both by direct detection of PI(4,5)P2 and PI(3,4,5)P3s messenger intermediates as well as by network analysis of differential gene expression profiling may be a consequence of increased GPCR signaling, which was the most enriched functional cluster (enrichment score = 10.01) in the synthetic lethality screen and confirmed by increased sensitivity to JAK2 inhibition, an immediate downstream effector kinase of GPCR signaling complexes, in the isogenic MCF10A CDH1 (−/−) system studied by Telford et al. [
12]. Overall, drug sensitivities with overlap to Telford and colleagues’ findings in the CDH1(−/−) isogenic mutant MCF10A model include inhibitors of the PI3K/mTOR axis, including PI-103 followed up in our validation studies, mTOR, aurora kinase inhibitors as well as inhibitors of c-Src kinase. Our findings are also in line with the characteristics, both on a molecular and drug sensitivity level, of a large gene expression profiling effort across 258 tumors which identified three subclasses of gastric cancer [
23]. In this large study of well-validated patient specimens, gene expression analysis identified three subgroups, mesenchymal, proliferative and metabolic, based on transcriptomic differences. The mesenchymal subtype most frequently harbored diffuse gastric cancers (up to 92.5%), showed cell adhesion and cell motility as well as focal adhesion and ECM receptor gene expression aberrations and displayed sensitivity to inhibitors of the PI3K/AKT pathway. These tumors were frequently hyper-methylated and of high grade and appear to be most similar to the genomically stable (GS) subtype by TCGA which consists of 75% Lauren classification type diffuse tumors, which has the highest rate of CDH1 mutations, as well as frequent variants involving genes of cytoskeletal, cell polarity, and cellular component organization, both findings in line with the GO process analysis and drug phenotype in the c.1380delA CDH1 SB.mhdgc-1 cells [
23,
25]. On the other, there were also significant differences between the pharmacological profile of MCF10A CDH1 (−/−) mutant cells reported by Telford and coworkers and the drug phenotype of c.1380delA CDH1 SB.mhdgc-1 in our study. Among these, for example, sensitivity to MEK or EGFR inhibition was not seen in E-cadherin deficient MCF10A cells and sensitivity to the c-Src kinase inhibitor saracatinib was only shown at one of the three concentrations tested to be significant between the CDH1 MCF10A isogenic cell line pair with overall generally a more modest effect on cell viability [
12]. Also, there was discordancy between the sensitivities to HDAC inhibitors and anti-apoptosis inhibitors to BCL2 and XIAP between the two systems. We attribute these differences to the more evolved stage of the patient-derived c.1380delA CDH1 SB.mhdgc-1 cells compared to the MCF10A system. In the sentinel studies of Humar and colleagues, who examined signal transduction aberrations of the T1a stage with phospho-immunohistochemistry in a family with a c.1008G>T CDH1 germline mutation, detailed pathology analysis showed that deficiency in E-cadherin is sufficient to initiate diffuse gastric cancer in the absence of hyperproliferation and that early intramucosal signet-ring cell carcinoma (SRRC) is initially slow proliferating in the upper neck of the gastric glands with loss of expression of junctional molecules including actin, p120, or Lin-7 homologue A of the cell polarity complex [
9,
10]. Expansion and progression beyond the early HDGC base is associated with c-Src kinase activation, including activation of downstream effectors FAK and signal transducer and activator of transcription 3 (STAT3), and described as one of the sentinel events of progression beyond the gastric mucosa and transformation to poorly differentiated cells and the development of an EMT phenotype [
10]. It is thus perceivable that the MCF10A system, which is not tumorigenic per se and has no oncogenic addiction, represents the very early intramucosal T1a stage in the slow proliferating phase with no, or limited, response to inhibitors c-Src inhibitors but capturing the perturbations associated with defective CDH1 adhesion and cell polarity signaling, whereas the transformed c.1380delA CDH1 SB.mhdgc-1 cells derived from the ascites of the HDGC patient with diffuse gastric cancer are dependent on c-Src, MAPK kinase, or other late occurring signal transduction signaling aberrations [
12,
28]. It is known that elevated c-Src or MAPK signaling pathways are involved in the activation of EMT transcription factors [
30‐
32]. Thus, while previous results including pharmacological leads from the isogenic E-cadherin deficient MCF10A model, or from a study in a CDH1 null drosophila model, might be mostly applicable to the early, intramucosal T1a stage, we propose that leads derived from patient-derived c.1380delA CDH1 SB.mhdgc-1 cells capture later tumor stages shifting from a drug profile with primarily chemopreventative merit to a therapeutic one. The anecdotal use of anti-EGFR therapy with cetuximab in HDGC patients with advanced gastric cancers applied by some medical oncologists in the field appears to be in line with the detected sensitivities to EGFR, PI3K and MEK inhibition as well as elevation of MAPK kinase signaling in c.1380delA CDH1 SB.mhdgc-1 cells. Similar to KRAS wild type colon cancer, in the absence of constitutively active RAS reduction of upstream input to the MAPK and PI3 kinase signal transduction pathways, EGFR inhibition might have merit as a molecular therapy option which might be improved by the use of select downstream mTOR inhibition considering alternative non-erb receptor ligand activation of the PI3K pathway via increased phosphoinositol-mediated signaling, c-Src or protein kinase C signaling via ERK-mediated release of TSC1/2 and mTORC1 inhibition.
Together, combined comparative gene expression profiling and qHTS in patient-derived hereditary c.1380delA CDH1 SB.mhdgc-1 cells may open new avenues to improved individualized treatment options for familial gastric cancer.