DNA methylation downregulated ZDHHC1 suppresses tumor growth by altering cellular metabolism and inducing oxidative/ER stress-mediated apoptosis and pyroptosis

Abstract

Cancer progression is an intricate biological process profiled by not only unscheduled proliferation, but also altered metabolism mechanisms. In this article, we introduced a novel tumor suppressor gene (TSG), Zinc Finger DHHC-Type Containing 1 (ZDHHC1, also known as ZNF377), frequently silenced due to epigenetic modification among various cancers, which exerts significant anti-tumor effects through metabolic regulation. Methods: Quantitative reversed-transcription PCR (qRT-PCR), reverse transcription PCR (RT-PCR) and Western blot were employed to demonstrate transcriptional and protein levels of targeted regulators. Methylation of ZDHHC1 promoter was detected by bisulfite genomic sequencing (BGS) and methylation specific PCR (MSP). Proteomics were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) and gas chromatography-mass spectrometry (GC-MS) were utilized for metabolomics analysis. Cellular functions were examined via corresponding approaches. Nude mice were used for xenograft tumor models. Indirect immunofluorescence staining was utilized to obtain precise location and expression of target proteins. Oxidative and ER stress indicators were detected using specific kits. Results: We found that ZDHHC1 expression was frequently silenced in multiple tumor cells and specimens due to methylation. Restoration of ZDHHC1 expression can curb cancer cell progression via stimulating apoptosis and cell cycle arrest, repressing metastasis, and reversing EMT transition and cell stemness. ZDHHC1's salient anti-tumor abilities were recognized in vivo as well. Metabolomic and proteomic analyses predicted inhibitory role of ZDHHC1 in glucose metabolism pathways in a CYGB-dependent manner, and in pentose phosphate pathway (PPP), which was validated by examining altered key factors. Moreover, we unraveled that ZDHHC1 dedicates to the increment of oxidative stress and endoplasmic reticulum (ER) stress to promote pyroptosis for anticancer purposes. Conclusion: Our study for the first time indicates ZDHHC1 is a potential tumor-suppressor frequently silenced due to promoter methylation, capable of negatively regulating metabolisms of tumor cells while stimulating oxidative stress and ER stress to expedite cell death through induction of pyroptosis and apoptosis, which can be exploited for development of new cancer prevention and therapies.

Keywords: CYGB; ER stress; Oxidative stress; ZDHHC1; zinc finger protein.

Figure 1

The expression of ZDHHC1 in multiple normal tissues and tumor cells. (A). RT-PCR assays demonstrated a wide range expression of ZDHHC1 among nearly all normal adult tissues and fetal tissues. (B). RT-PCR assays showed downregulated or silenced ZDHHC1 in a number of cancer cell lines. (C). The methylation state of the ZDHHC1 promoter was detected by methylation-specific PCR (MSP).

Figure 2

Promoter CpG Methylation Mediates ZDHHC1 Downregulation in multiple Cancers. (A). Pharmacologic and genetic demethylation reactivated ZDHHC1 expression in carcinoma cell lines. Aza (A), Trichostatin A (T). (B-C). Methylation alleles of ZDHHC1 demonstrated by BGS in multiple tumor cells, every row of circles indicated an individual promoter allele clone sequenced. Filled circles represented methylated CpG sites, blank ones referred to unmethylated sites. Aza (A), Trichostatin A (T). (D-F). The methylation status of ZDHHC1 in multiple normal and cancer tissues measured by MSP.

Figure 3

ZDHHC1 inhibited growth, migration and invasion of MCF7, and HONE1 cells. Analysis of nude mice xenografts generated by ZDHHC1- overexpressing cell. (A). Ectopic expression of ZDHHC1 was confirmed by RT-PCR and Western blot. (B). Measurement of cell proliferation for vector- and ZDHHC1-transfected carcinoma cells through CCK-8 assay. (C). Histogram statistics of CFA. MCF7 and HONE1 cells transfected with ZDHHC1 or empty vector were used for colony formation assay to measure proliferation rates. (D). Histogram for flow cytometry analysis of cell cycle progression. (E). Histogram for flow cytometry analysis of cellular apoptosis. Vector and ZDHHC1 transfected MCF7 and HONE1 cells were double stained with Annexin-V-FITC and PI for apoptosis measurement. (F). Histogram of quantified migration cells/field, Transwell assays measuring cell migration ability, using two cells transfected with Vector or ZDHHC1 24 h after seeding. (G). Histogram of quantified invasion cells/field, Transwell assays measuring cell invasion ability, using MCF7 and HONE1 cells transfected with Vector or ZDHHC1. Three independent experiments were carried out. **: p < 0.01; ***: p < 0.001. (H-J). Tumor growth was significantly stalled in nude mice xenograft model injected with MCF7 cells (n=6 /group). (H) Representative images of xenografts before and after resection in Vector and ZDHHC1 groups. (I) Line graphs of tumor volume at various time points after injection. (J) Histogram of tumor weight by the time of resection. (K). (left) Representative immunofluorescent staining of ZDHHC1 in MCF7 xenografts. Red for ZDHHC1, blue for nucleus DNA counterstained with DAPI. (right) H&E staining, and immunohistochemistry staining of Ki67 and PCNA in MCF7 xenografts overexpressing ZDHHC1 or vector. (L) TUNEL assays of MCF7 xenografts overexpressing ZDHHC1 or vector. Green for fluorescein-dUTP, and blue for DNA counterstained with DAPI. *p < 0.05, ** p < 0.01, ***p < 0.001. (M) Western blot results of critical apoptosis indicators in the xenograft tissues (Cleaved caspase-7, caspase-3 and PARP). GAPDH was taken as internal control.

Figure 4

Ectopic ZDHHC1 inhibited EMT and disrupted stemness biomarkers in carcinoma cells. (A). Morphology changes of MCF7 and HONE1 cells observed under phase contrast microscopy. (B). Immunofluorescent staining of epithelial markers (E-cadherin, Occludin) and mesenchymal markers (N-cadherin, Vimentin) in MCF7 cells transfected with ZDHHC1 or Vector. (C). Western blot assays of epithelial markers (E-cadherin, Occludin) and mesenchymal markers (N-cadherin, Vimentin) in MCF7 and HONE1 cells transfected with ZDHHC1 or Vector. β-actin was taken as internal control. (D). RT-PCR assays of cancer stemness markers (NANOG, SOX2, OCT4, CD44, ABCG2 and BMI1) in MCF7 and HONE1 cells, with ZDHHC1 or empty vector transfected. β-actin was taken as control. (E-F). Spheroid formation assay showed that ZDHHC1 decreased the size of spheres formed by MCF7 and HONE1 cells. ***p < 0.001.

Figure 5

Effects of ectopic ZDHHC1 expression on proliferation, apoptosis and glucose metabolism in a CYGB-dependent way. Effects of ectopic ZDHHC1 on ER stress and oxidative stress and thereafter stimulated pyroptosis and apoptosis. (A). The efficiency of CYGB knockdown was confirmed by qPCR. (B-G). Effects of ZDHHC1 overexpression and knock-down of CYGB on proliferation, apoptosis and glucose metabolism in carcinoma cell lines. ** p < 0.01, ***p < 0.001. (H). Intracellular superoxide levels were detected with dihydroethidium (DHE). (I). Intracellular ROS was detected by means of oxidation-sensitive fluorescent probe (DCF). (J). NADPH oxidase activity was assessed. (K). Total antioxidant capacity in the cell was assessed by (T-AOC Assay Kit). (L-M). ER stress pyroptosis and apoptosis related markers were detected by quantitative PCR and Western blot. The above experiments were all conducted with MCF7 and HONE1 cells transfected with ZDHHC1 or Vector, respectively. (N). Morphology of MCF7 cell undergoing pyroptosis was observed under electronic microscopy. (O). Immunohistochemistry staining of pMLKL in MCF7 xenografts overexpressing ZDHHC1 or vector.

Figure 6

ZDHHC1 promoted NLRP3 expression is dependent of its enzymatic activity. (A-B) ER stress and pyroptosis markers alteration after drug treatment for separate intentions. GRP78, CHOP, NLRP3 and IL-1β levels were captured by qPCR and Western blot. (C). GSDMD and GSDME were detected by quantitative PCR. (D). Multiple cysteine residues within NLRP3 are likely to be palmitoylated using palmitoylation site prediction tool CSS-PALM4.0 (http://csspalm.biocuckoo.org/). (E). Indirective immunofluorescence assays showed that when we introduced a point mutation (C164A) in the DHHC motif of ZDHHC1, NLRP3 expression was no longer increased in HONE1 cells.