Dual-modified antisense oligonucleotides targeting oncogenic protocadherin to treat gastric cancer

Abstract

Background: The objective of this study was to develop an innovative treatment strategy utilizing antisense oligonucleotides (ASOs) that target the gene encoding protocadherin alpha 11 (PCDHA11) and to elucidate the role of PCDHA11 in gastric cancer cells.

Methods: We designed and screened 54 amido-bridged nucleic acid (AmNA)-modified ASOs, selecting them based on PCDHA11-knockdown efficacy, in vitro and in vivo activity, and off-target effects. We assessed the impact of AmNA-modified anti-PCDHA11 ASOs on cellular functions and signaling pathways, and investigated the effects of Pcdha11 deficiency in mice.

Results: AmNA-modified anti-PCDHA11 ASOs significantly reduced the proliferation of gastric cancer cells and other solid tumors, whereas overexpression of PCDHA11 enhanced cell proliferation. The selected ASOs inhibited cellular functions related to the metastatic potential of gastric cancer cells, including migration, invasiveness, spheroid formation, and cancer stemness. Our findings revealed that AmNA-modified anti-PCDHA11 ASOs disrupted the AKT/mTOR, Wnt/β-catenin, and JAK/STAT signaling pathways. In mouse models of peritoneal metastasis (gastric and pancreatic cancer), systemic metastasis, and established subcutaneous tumors, administration of AmNA-modified anti-PCDHA11 ASOs inhibited tumor growth. ASO treatment induced reversible, dose- and sequence-dependent liver damage. Pcdha11-deficient mice demonstrated normal reproductive, organ, and motor functions.

Conclusions: AmNA-modified anti-PCDHA11 ASOs offer a promising therapeutic strategy for the treatment of gastric cancer and other solid malignancies.

Fig. 1. Clinical relevance of PCDHA11 expression.

a Gastric cancer tissues expressed higher mean levels of PCDHA11 mRNA than corresponding normal adjacent tissues. *P < 0.05 between the two groups. b Prognosis and PCDHA11 expression in the institutional, TCGA, and Kaplan-Meier-plotter cohorts. c Immunohistochemical analysis of PCDHA11 expression in stage II or III gastric cancer. PCDHA11 expression at the primary cancer component was associated with greater frequency of disease recurrence. *P < 0.05 between the two groups. d qRT-PCR analysis of PCDHA11 mRNA in the indicated human gastric cancer cell lines and representative results of PCR array showing positive correlations between PCDHA11 and IL1RN, SNAI1, TGFB1, and ZEB1 mRNA.

Fig. 2. Effects of candidate ASOs, hPCDHA11-2382 and -3716, on knockdown of PCDHA11 mRNA expression, cellular functions of cancer cells and in vivo tumor growth.

a Structures of AmNA-modified anti-PCDHA11ASOs. b Concentration-dependent knockdown efficacy of PCDHA11 mRNA expression by hPCDHA11-2382 and -3716. c The proliferation rate of gastric cancer cells was attenuated by hPCDHA11-2382 and -3716. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs at Day 6. Invasiveness (d) and migration (e) of gastric cancer cell lines were attenuated when transfected with hPCDHA11-2382 and 3716. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs. f Intraperitoneal administration of hPCDHA11-3716 in mouse models of peritoneal metastasis of gastric and pancreatic cancers. In both models, 4 weeks administration of hPCDHA11-3716 inhibited the growth of peritoneal nodules. *P < 0.05 between ASO-NEG and hPCDHA11-3716. Error bars indicate the standard deviation.

Fig. 3. Design and influence of hPCDHA11-2255, -3319 and -3969 on PCDHA11 mRNA and protein expression, in vitro and in vivo cancer cell growth.

a The predicted loop structure of hPCDHA11-3969. b Concentration-dependent knockdown efficacy of PCDHA11 mRNA expression by hPCDHA11-2255, -3319 and -3969. c The proliferation rate of gastric cancer cells was attenuated by hPCDHA11-2255, -3319 and -3969. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs at Day 6. d Confirmation of knockdown of PCDHA11 protein expression by hPCDHA11-2255, -3319 and -3969. e Intraperitoneal administration of hPCDHA11-3319 and -3969 attenuated the growth of peritoneal metastasis of gastric cancer cells. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs. f Knockdown of PCDHA11 expression decreased the formation of systemic metastasis of gastric cancer cells injected into the tail vein. Microscopic findings are shown (400× magnification). g Treatment effects of AmNA-modified anti-PCDHA11 ASOs in mouse subcutaneous xenograft models of gastric cancer. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs at day 28. Error bars indicate the standard deviation.

Fig. 4. Specificity and influence of AmNA-modified anti-PCDHA11 ASOs on caspase activity, apoptosis, cancer stemness, and functions associated with metastatic potential.

a Proliferation assay of KATO-III cell transfected with empty and PCDHA11-overexpressing vectors. *P < 0.05 between the empty vector and overexpression vector at day 5. b Little inhibitory effects of AmNA-modified anti-PCDHA11 ASOs on proliferation of Pcdha1-knockout cells. c In HGC-27 parental cells, hPCDHA11-2255 and -3969 increased caspase activities compared to controls, whereas little changes in caspase activities by AmNA-modified anti-PCDHA11 ASOs were observed in Pcdha1-knockout cells. d Growth of parental and PCDHA11-knockout HGC-27 xenografts in mouse peritoneal metastasis models. *P < 0.05. e Annexin V staining revealed that the proportion of apoptotic cells detected was significantly increased by AmNA-modified anti-PCDHA11 ASOs. Effects of UV irradiation served as a positive control. *P < 0.05. f Comparison of spheroid formation by gastric cancer cells. g Proportions of ALDH-positive living gastric cancer cells (right upper panels) were decreased by hPCDHA11-3969. h Proportion of cancer cells adhering to human primary mesothelial cells was decreased by hPCDHA11-2255, -3319 and -3969. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs. Error bars indicate the standard deviation.

Fig. 5. Toxicities of hPCDHA11-2255, -3319 and -3969.

a Toxicity test treatment schedule. b Changes in body weight after administration of 0.6 mg ASOs. c Liver function after administration of 0.6 mg ASOs. d Serum amylase levels and renal function after administration of 0.6 mg ASOs. e Appearance; histological findings; and volumes of the liver, kidney, and brain after administration of 0.6 mg ASOs. Error bars indicate the standard deviation.

Fig. 6. Effect of AmNA-modified anti-PCDHA11 ASOs on signal transduction pathways.

a Volcano plot of expression levels of cadherin superfamily members in gastric cancer tissues with distant metastasis and those of candidate ligands. b Results of the western blot digital imaging analysis. c Hypothetical working model of AmNA-modified anti-PCDHA11 ASOs.

Fig. 7. Characterization of Pcdha11-deficient mice.

a Genotyping of Pcdha11^+/+, Pcdha11^+/−, and Pcdha11^−/− mice. b Gross appearance and major organs. Microscopic findings (×400 magnification). c Rotarod test of motor coordination and learning administered to Pcdha11^+/+, Pcdha11^+/−, and Pcdha11^−/− mice. Blood counts (d), liver functions (e), and renal functions (f) of Pcdha11^+/+, Pcdha11^+/−, and Pcdha11^−/− mice. Error bars indicate the standard deviation.