Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer

Abstract

Background: Patients with hereditary diffuse gastric cancer (HDGC), a cancer predisposition syndrome associated with germline mutations of the CDH1 (E-cadherin) gene, have few effective treatment options. Despite marked differences in natural history, histopathology, and genetic profile to patients afflicted by sporadic gastric cancer, patients with HDGC receive, in large, identical systemic regimens. The lack of a robust preclinical in vitro system suitable for effective drug screening has been one of the obstacles to date which has hampered therapeutic advances in this rare disease.

Methods: In order to identify therapeutic leads selective for the HDGC subtype of gastric cancer, we compared gene expression profiles and drug phenotype derived from an oncology library of 1912 compounds between gastric cancer cells established from a patient with metastatic HDGC harboring a c.1380delA CDH1 germline variant and sporadic gastric cancer cells.

Results: Unsupervised hierarchical cluster analysis shows select gene expression alterations in c.1380delA CDH1 SB.mhdgc-1 cells compared to a panel of sporadic gastric cancer cell lines with enrichment of ERK1-ERK2 (extracellular signal regulated kinase) and IP3 (inositol trisphosphate)/DAG (diacylglycerol) signaling as the top networks in c.1380delA SB.mhdgc-1 cells. Intracellular phosphatidylinositol intermediaries were increased upon direct measure in c.1380delA CDH1 SB.mhdgc-1 cells. Differential high-throughput drug screening of c.1380delA CDH1 SB.mhdgc-1 versus sporadic gastric cancer cells identified several compound classes with enriched activity in c.1380 CDH1 SB.mhdgc-1 cells including mTOR (Mammalian Target Of Rapamycin), MEK (Mitogen-Activated Protein Kinase), c-Src kinase, FAK (Focal Adhesion Kinase), PKC (Protein Kinase C), or TOPO2 (Topoisomerase II) inhibitors. Upon additional drug response testing, dual PI3K (Phosphatidylinositol 3-Kinase)/mTOR and topoisomerase 2A inhibitors displayed up to >100-fold increased activity in hereditary c.1380delA CDH1 gastric cancer cells inducing apoptosis most effectively in cells with deficient CDH1 function.

Conclusion: Integrated pharmacological and transcriptomic profiling of hereditary diffuse gastric cancer cells with a loss-of-function c.1380delA CDH1 mutation implies various pharmacological vulnerabilities selective to CDH1-deficient familial gastric cancer cells and suggests novel treatment leads for future preclinical and clinical treatment studies of familial gastric cancer.

Keywords: Differential gene expression profiling; Hereditary diffuse gastric cancer (HDGC); High throughput drug screening; Therapeutic leads; c.1380delA CDH1 gastric cancer cells.

Fig. 1

Pedigree of family with hereditary diffuse gastric cancer harboring c.1380delA germline mutation of the CDH1 gene. Squares indicate males; circles indicate females. Symbols with a shaded portion indicate individuals whom have been diagnosed with cancer. Type of cancer and age at diagnosis are indicated, symbols with slashes indicate deceased family members

Fig. 2

Characteristics of patient-derived c.del1380CDH1 SB.mhdgc-1 cancer cells. a Light microscopy image of c.del1380 CDH1 SB.mhdgc-1 cells (10× magnification). b SB.mhdgc-1 cells lack E-cadherin expression but express CEA. Immunoflurescence of SB.mhdgc-1 cells stained with anti-E-cadherin (left) and anti-CEA (right; both green), nuclei stained with DAPI (blue). Gastric cancer lines N87 and AGS (for E-cadherin), BxPC3 and HeLa cells (for CEA) shown as positive and negative controls. c Karyotype and SKY images of SB.mhdgc-1 cells show features consistent with human cancer cells including aneuploidy (such as in chromosomes 1 and 2) and chromosomal translocations (in chromosomes 3:5 and 4:8). d Light microscopy of c.del1380CDH1 SB.mhdgc-1 cancer cells grown under ultra-low attachment conditions in FBS-free media. Top (14 days of culture), three dimensional multicellular spheroid (MCS) clusters with compact, amorphous center. Bottom (33 days of culture), c.del1380CDH1 SB.mhdgc-1 spheroids have rounded up, became more compact, and formed basal membranes (arrows; 20× magnification). e Patient-derived SB.mhdgc-1 cells harbor c.1380del CDH1 germline mutation. Deep sequencing of Hg19 CDH1 locus (chr16: 68,771,195-68,869,444, NM_004360) in c.del1380CDH1 SB.mhdgc-1 spheroids (top) and SB.mhdgc-1 cells 2D monolayer cells (bottom); 40 base sequences of 14 genomic DNA fragments around c.1380 are shown. Artifacts of alignment as identified by BWA are shown in blue

Fig. 3

c.1380delA CDH1 SB.mhdgc-1 harbors select transcriptomic alterations compared to sporadic gastric cancer cells. a Unsupervised hierarchical cluster analysis and associated heat map of baseline transcriptomic profiles. Columns represent individual probes while rows represent individual cell lines. The color of each probe reflects log₂ ratio of normalized expression values for each cell line compared to the median from all cell lines (see scale, top 200 upregulated and 100 downregulated (FC > 2; p < 0.05) in c.1380delA CDH1 SB.mhdgc-1 shown). b Most relevant network selective for SB.mhdgc-1 cells by GeneSpring GX analyze networks (AN) algorithm using shortest paths algorithm with main parameters (1) relative enrichment and (2) relative saturation of networks with canonical pathways. Networks are prioritized based on the number of fragments of canonical pathways in the network. c c.1380delA CDH1 SB.mhdgc-1 gastric cancer cells harbor increased phosphoinositide-derived messengers. Immunofluoresence of SB.mhdgc-1 and sporadic SB.msgc-1 gastric cancer cells measuring anti-phosphatidylinositol 4,5-bisphosphate (top) and anti-phosphatidylinositol 3,4,5-trisphosphate levels (bottom). Mean of staining intensity normalized to DAPI of 100 cells of SB.mhdgc-1 and SB.msgc-1 shown on the right. d Reduced cell adhesion including extracellular matrix substrate adhesion of c.1380delA CDH1 SB.mhdgc-1 versus SB.msgc-1 cells. Time course of ratios of adherent versus non-adhered cells (student’s t test; two images were acquired of each triplicate and the mean taken). e Increased p-ERK: total ERK and p-AKT: total AKT protein expression ratios in c.del1380A CDH1 SB.mhdgc-1 cells compared to sporadic gastric cancer cell lines. Immunoblots with antibodies indicated on the right

Fig. 4

Comparative quantitative high-throughput screening (qHTS) using compounds from the MIPE Oncology 4.0 library identifies compounds with preferential activity against c.del1380A CDH1 SB.mhdgc-1 cells compared to SB.msgc-1 gastric cancer cells. a Bubble diagram of drug phenotypes by compound class of SB.mhdgc-1 versus SB.msgc-1 cells depicting class activities (number of compounds per drug class) measured by area under the curve (AUC) (drug class activities ≥2 standard deviations from the mean of ΔAUC(% AUC(c.1380delA SB.mhdgc-1/SB.msgc-1) being considered significantly more active in the respective cell line). b Drug classes with activity in both c.del1380A CDH1 SB.mhdgc-1 and SB.msgc-1 gastric cancer cells by maximum response (MAXR) <30%. Enrichment (number of active compounds in a target class relative to total number of compounds for that target class versus enrichment for each target, compared to background; Fishers exact test, 2-tailed)

Fig. 5

c.1380 CDH1 SB.mhdgc-1 gastric cancer cells show vulnerabilities to toposisomerase II and PI3K/mTOR inhibition. a Drug response curves of a panel of gastric cancer cell lines treated with a range of concentrations of mitoxantrone, etoposide (both TOPO2A inhibitors), or PI-103 (dual class IA phosphatidylinositol 3 kinase/mTOR inhibitor) for 72 h. X-axis indicates log [concentration] tested, y-axis indicates cell viability percentage normalized to vehicle-control samples. Mean cell viability values are plotted with standard error of the mean (SEM) from at least 2 independent experiments done in triplicate. b Rate of apoptosis induced by 24-h treatment of 1 µM etoposide, mitoxantrone, or PI-103 normalized to DMSO-treated samples in sporadic gastric cancer SNU-16 and hereditary c.1380delA SB.mhdgc-1 cells. Flow cytometry profiles of FITC-labeled anti-BrdU staining of 3′-hydroxyl (OH) termini of double- and single-stranded DNA, relative BrdU fractions normalized to DMSO-treated control shown on the right