Epigenomic and Functional Characterization of Junctophilin 3 (JPH3) as a Novel Tumor Suppressor Being Frequently Inactivated by Promoter CpG Methylation in Digestive Cancers

Abstract

Junctophilin (JPH) proteins stabilize junctional membrane complexes between plasma membrane and endoplasmic reticulum, also implicated in some human diseases. JPH3 mutations are linked to Huntington's disease-like 2 syndrome. Through epigenomic study of a colon cancer cell line pair (HCT116 and DKO), we identified JPH3 as a methylated novel tumor suppressor gene (TSG) candidate at 16q24. We further studied its epigenetic alterations and functions in digestive tumorigenesis. JPH3 expression at the RNA level was found to be frequently silenced or reduced in colorectal and gastric cancers due to its promoter CpG methylation, which is associated with tumor progression and poor survival of digestive cancer patients. Ectopic expression of JPH3 inhibited tumor cell growth in vitro and in vivo. JPH3 expression upregulated the cytosolic Ca²⁺ levels, and unfolded protein response gene expression upon endoplasmic reticulum stress. JPH3 also induced calpain activation and subsequent mitochondrial membrane depolarization and cell apoptosis. Thus, JPH3 was identified as a novel TSG methylated in colorectal and gastric tumors which promotes mitochondrial-mediated apoptosis, also as a potential metastasis and survival biomarker for digestive cancers.

Keywords: junctophilin, tumor suppressor gene; metastasis, 16q24.; methylation.

Figure 1

JPH3 expression and methylation in colorectal and gastric cancer cell lines and primary tumors. (A) CpG methylome study identified JPH3 as a methylated target in HCT116. JPH3 gene structure, promoter and exon 1 (UCSC Genome Browser NCBI36/hg18) are shown on the top panel. e1: exon 1. Positive methylation signal peak (blue) in HCT116 was identified by MeDIP-chip. (B) Broad expression of JPH3 in a normal adult tissue panel by semi-quantitative RT-PCR (primers detecting both variant 1 and 4), with GAPDH as a control (upper). Representative immunohistochemical staining for JPH3 in normal colonic and gastric mucosa. Original magnification: 200x, 400x (lower). (C) Structure of the JPH3 promoter CpG island (CGI). CpG sites are shown as short vertical lines. MSP primer sites and BGS region analyzed are also indicated. (D) Downregulation and silencing of JPH3 in colorectal and gastric cancer cell lines as a result of its CGI methylation, with RT-PCR primers detecting both variant 1 and 4. (E) Pharmacological demethylation with 5-Aza and TSA restored JPH3 expression in methylated and silenced cell lines. Representative results are shown. (F) Representative BGS results of cell lines. Vertical lines indicate individual CpG sites. Cloned BGS-PCR products were sequenced and each colony is shown as an individual row, representing a single allele of the CGI. Filled circles represent methylated and open circles unmethylated CpG sites. U: unmethylated; M: methylated. A: 5-Aza; T: TSA; CRC, colorectal cancer; GsCa, gastric cancer.

Figure 2

JPH3 inhibited the growth and migration of cancer cells. (A) Representative colony formation assay by monolayer culture and quantitative analysis. (B) Representative results of soft agar assays and quantitative analysis. (C) JPH3 expression in stably transfected cells confirmed by RT-PCR and Western blot. (D) Effect of JPH3 expression on tumor growth in nude mice. Result of tumor growth in nude mice inoculated subcutaneously with MKN-28/vector or MKN-28/JPH3 cells. *: p<0.05. (E) Migration assay with a 24-transwell system and quantitative analysis. The pictures were taken 24 h after seeding. Original magnification: 100x. (F) Wound-healing assay. Photos were taken every 24 h. Original magnification: 100x. All values are the mean ± s.d. of three independent experiments. *: p<0.05.

Figure 3

Effects of ectopic JPH3 expression on the apoptosis and UPR of tumor cells with or without BFA treatment. (A) Quantitative analysis results of annexin V-FITC/PI staining before and after BFA treatment in HCT116 and MKN-28 cells. Values are the mean ± s.d. of three independent experiments. *: p<0.05. (B) Cleaved Caspase 3, Caspase 9 and PARP were significantly induced by JPH3 expression. (C) Expression of several key UPR markers examined by Western blot.

Figure 4

Effects of ectopic JPH3 expression induced digestive cancer cell apoptosis through mitochondria-mediated apoptosis pathway. (A) Mitochondrial membrane potential was significantly decreased by JPH3 expression. (B) JPH3 expression increased the release of Cyto C and Smac into the cytosol. (C) Western blot and (D) immunofluorescence results showed that JPH3 expression induced translocation of mitochondrial AIF into the nucleus significantly after BFA treatment.

Figure 5

Effects of ectopic JPH3 expression on the level of cytosolic Ca²⁺, Calpain activity, several important protein kinases and BCL2 family protein expression. (A-B) JPH3 expression significantly increased the level of cytosolic Ca²⁺ and (C) Calpain activity. (D) JPH3 expression significantly decreased the pro-survival Bcl2 family proteins including Bcl2, P-Bcl2 (Ser70), Mcl-1 and Bcl-xL, but had no apparent effect on the pro-apoptosis Bcl2 family proteins. (E) JPH3 expression significantly decreased p-Akt (S473) expression and had no apparent effects on the others.

Figure 6

JPH3 reduction by promoter methylation in colorectal and gastric primary tumors and its correlation with clinicopathological features. Representative analysis of JPH3 methylation in primary tumors (T) and normal tissues (N) by (A) MSP and (B) BGS. (C) Representative immunohistochemical staining for JPH3 in normal gastric and colonic mucosa, as well as their primary carcinoma tissues. Original magnification: 200x. (D) Overall survival analysis results in colorectal and gastric cancer patients. (E) Correlation of JPH3 methylation and expression in primary colorectal and gastric tumors. (F) Analysis of JPH3 expression and methylation in colorectal and gastric cancer patients from the TCGA database, as well as their correlation with clinicopathological parameters.

Figure 7

Diagram showing the mechanisms with which JPH3 expression induced cancer cell apoptosis by unfolded protein response involving perturbed calcium homeostasis.