Targeted inhibition of Ninjurin2 promotes chemosensitivity in chemoresistant gastric cancer by suppressing cancer-initiating cells

Abstract

Background: The combination of epirubicin, cisplatin, and 5-fluorouracil (ECF) is widely used for gastric cancer treatment. However, cancer cells can acquire chemoresistance over multiple treatment cycles, leading to recurrence. This study aimed to investigate a novel biomarker for predicting ECF resistance and its biological roles in gastric cancer.

Methods: ECF-resistant (ECF-R) gastric cancer cell lines were established through stepwise ECF treatment. Transcriptome analysis was performed to identify resistance-related genes, which were validated in tumor organoids and in vivo models. Additionally, gastric cancer patient tumor tissues were analyzed for clinical relevance.

Results: Transcriptome analysis revealed that NINJURIN2 and CD44 were highly expressed in ECF-R cells but rarely expressed in normal gastric tissues. NINJURIN2 inhibition significantly increased chemosensitivity to ECF in vitro and in vivo. Liquid chromatography-tandem mass spectrometry identified periostin as a binding partner of NINJURIN2, mediating chemoresistance. Furthermore, VAV2 phosphorylation was markedly upregulated in ECF-R cells but was inhibited by NINJURIN2 knockdown. Clinical analysis showed that high NINJURIN2 expression correlated with poor survival outcomes in gastric cancer patients.

Conclusion: Our findings suggest that NINJURIN2 can be used as a novel biomarker for chemoresistant gastric cancer patients and that inhibiting NINJURIN2 along with standard chemotherapy could prevent chemoresistance-associated relapse in gastric cancer.

Keywords: Cancer initiating cells; Chemoresistance; Gastric cancer cells; Ninjurin2; Organoid.

Fig. 1

NINJ2 expression in ECF-R gastric cancer cells. (A) Representative IC₅₀ values for parent (WT) and ECF-resistant (ECF-R) MKN-74 and SNU-484 cells. The IC₅₀ values are given in an equation using a four-parameter logistic curve. (B) Parent and ECF-R MKN-74 cells were individually transplanted into nude mice and left until the tumor reached a volume of 100 mm³. The tumor-bearing mice were given ECF, and the tumor volume was measured. *p < 0.05 versus parent at each time point. (C) (Left) Venn diagram showing top genes with 15-fold changes in the RNA-sequencing of WT and ECF-R genes related to “Integral component of plasma membrane (GO:0005887)” and “Cell adhesion (GO:0007155).” (Right) Heatmap showing the gene expression level associated with resistance and the two gene ontology categories. (D) RGV analysis for the human NINJ2 isoform based on RNA-sequencing of WT and ECF-R (hg19 base) cells. (E) mRNA levels of NINJ2 was analyzed by quantitative real-time PCR in parent and ECF-R cells from the MKN-28/74, MKN-74, MKN-45, SNU-484, SNU-520, and SNU-688 lines. (F) Protein levels of NINJ2 in parent and ECF-R MKN-74 and SNU-484 cells were determined by Western blot analysis. ECF, epirubicin, cisplatin, and 5-fluorouracil; ECF-R, ECF-resistance. Data are presented as mean ± SD

Fig. 2

NINJ2 induces CD44 expression and cancer-initiating cells. (A) (Left) FACS plot showing the surface expression of NINJ2 on parent and ECF-R MKN-74 cancer cells. The I and III populations are NINJ2-expressing cells among parental and ECF-R cells, respectively. The II population is NINJ2⁻ cells among ECF-R cells. (Right) Quantification of NINJ2⁺ cells. (B) (Left) FACS plot showing surface expression of CD44 on parent and ECF-R MKN-74 cancer cells. (Right) Quantification of CD44^high cells. (C) (Left) FACS plot showing CD44 expression in gated NINJ2⁻ and NINJ2⁺ populations of ECF-R MKN-74 cancer cells. (Right) Quantification of the NINJ2⁺CD44^hi population. (D) NINJ2 and CD44 correlation analysis in human gastric adenocarcinoma (n = 426) from the The Cancer Genome Atlas (TCGA) and oncoSG dataset. (E) NINJ2 isoform-1 (NP_057617.3) and isoform-3 (NP_001281275.1) overexpression efficiency was analyzed by quantitative real-time PCR. (F) CD44 mRNA level in NINJ2-overexpressing MKN-74 cells was measured by quantitative real-time PCR. (G) (Left) Percentage of CD44^high cells in stable mock-, NINJ2 isoform 1-, and NINJ2 isoform 3-overexpressing MKN-74 cells measured by FACS analysis. (Right) Quantification of the CD44^high cells. O/E, overexpressing. Data are presented as mean ± SD

Fig. 3

NINJ2 in ECF-R regulates chemo-sensitivity and cell cycle arrest. (A) ECF-R MKN-74 cells were transduced with shRNA lentiviral particles targeting human NINJ2 (clone 1 and clone 2) or the pLKO.1-puro empty control. Viability analysis using crystal violet staining (Left) and the CCK-8 assay (Right) 3 weeks after ECF treatment in mock and NINJ2 K/D ECF-R MKN-74 cells. (B) Representative ECF IC₅₀ values for MKN-74 cells overexpressing NINJ2 isoform-1 and isoform-3. (C) (Left) Cell cycle status of mock-, NINJ2 isoform-1-, and isoform-3-O/E MKN-74 cells. (Right) Quantification of the left FACS plots. *p < 0.05 versus mock (S-phase reduction), #p < 0.05 versus mock (G0/G1-phase increase). (D) Western blot analysis of NINJ2, p27^KIP1, CDK2, CDK4, CDK6, CDC25a, cyclin D1, cyclin E1, Rb (p-S780), Rb (p-S795), total Rb, and GAPDH in scramble siRNA and NINJ2 K/D MKN-74 cells. K/D, knockdown. Data are presented as mean ± SD

Fig. 4

NINJ2 activates VAV2 signaling by interacting with periostin. (A) NINJ2/periostin interaction from stable NINJ2-HaloTag MKN-74 cancer cells was confirmed by co-immunoprecipitation. (B) Kinetics for NINJ2/periostin interaction was estimated by SPR analysis. The extracellular domain of NINJ2 immobilized through amine coupling and periostin at the five concentration (231, 116, 58, 29 and 14 nM) were flowed. (C) periostin mRNA levels in parent or ECF-R MKN-74 cancer cells. (D) Signaling analysis from NINJ2-overexpressing MKN-74 cells analyzed through site-specific and phospho-specific antibodies. (E) Western blot analysis of VE-cadherin, VAV2, and JunD in mock or NINJ2-overexpressing MKN-74 cells. (F) ECF-R MKN-74 cells were treated with NINJ2 siRNA or control siRNA to analyze the expression of p-VAV2/VAV2 by western blotting. (G) ECF-R MKN-74 cells with NINJ2 or scramble siRNA knockdown were evaluated for cell viability using CCK-8 assay after ECF (IC₅₀) treatment. (H) Expression of CD44 mRNA level in NINJ2 or scramble siRNA-treated ECF-R MKN-74 cells. Data are presented as mean ± SD

Fig. 5

Role of NINJ2 in chemoresistant relapse of gastric cancer. (A) Parent or ECF-R MKN-74 cells were subcutaneously injected into nude mice and tumor growth was observed for 30 days. Scale bar, 5 mm. (B) Tumor volume, (C) and tumor weight. (D) Representative images of IHC for NINJ2, CD44, and Cyclin D1 in tumors from each group. Scale bar, 50 μm. (E-G) Parent MKN-74, NINJ2 or scrambled siRNA-treated ECF-R MKN-74 cells were subcutaneously injected into nude mice treated weekly with ECF. (E) Representative tumor images. Scale bar, 5 mm. (F) Tumor weight and (G) tumor volume. (H) NINJ2 mRNA level was measured with quantitative real-time PCR in tumors. Data are presented as mean ± SD

Fig. 6

NINJ2 is upregulated in ECF-R tumor organoids and shows clinical importance. (A) Representative images of parental and ECF-R human gastric tumor organoids. Scale bar, 40 μm. (B) ECF IC₅₀ values of parental and ECF-R tumor organoids. (C) NINJ2 and CD44 mRNA levels in parental and ECF-R human gastric tumor organoids (D) Kaplan–Meier curves for overall survival of gastric cancer patients in a public dataset (GSE15459). (E) Expression of NINJ2 in tumor tissues and their corresponding normal tissues. Scale bar, 50 μm. (F) Chemotherapy response variability of patients according to NINJ2 expression in gastric cancer tissues, stratified into low and high expression groups. Data are presented as mean ± SD