TWIST2: A new candidate tumor suppressor in prostate cancer

Abstract

Background: Prostate cancer (PCa) is a leading cause of cancer morbidity and mortality in men worldwide; however, PCa incidence and mortality rates vary widely across geographic regions and ethnic groups. The current study was designed to elucidate the pivotal factors involved in PCa occurrence and development.

Methods: We performed RNA sequencing on the prostate tumor and adjacent normal tissues from Chinese PCa patients. Genes identified via genome-wide expression profile analysis were validated by quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. Hypermethylation of CpG islands was assessed by nested methylation-specific PCR. Whole genome microarray analysis was performed using an Affymetrix GeneChip.

Results: We identified nine possible abnormally expressed genes (P < .05) and then revealed TWIST2 as having strikingly lower expression in tumors than in control tissues (P < .01). Low messenger RNA expression levels of TWIST2 were associated with hypermethylation of CpG islands in its promoter region. In accordance with these findings, PCa tumor tissues showed markedly decreased TWIST2 protein expression compared to that in both normal and prostatic intraepithelial neoplasia tissues by immunohistochemical staining. Ectopic expression of TWIST2 in LNCap cells not only inhibited cell proliferation and colony formation in vitro and tumor growth in vivo but also induced transcriptional repression of a cell proliferation-related gene cohort, including androgen receptor signaling mediators, cyclins, homeobox genes, forkhead box genes, and SOX2.

Conclusions: Our results suggest that TWIST2 could function as a tumor suppressor involved in the pathogenesis of PCa by influencing the expression of target genes and that hypermethylation of the TWIST2 promoter in prostate tumors may be an underlying mechanism for TWIST2 transcriptional silencing.

Keywords: RNA sequencing; TWIST2; hypermethylation; prostate cancer; tumor suppressor.

Figure 1

Heatmap of 42 selected genes and confirmation of selected genes by reverse‐transcription polymerase chain reaction (RT‐PCR). A, Fold‐changes in expression between the tumor and adjacent normal samples in the 10 pairs of samples. B, RT‐qPCR confirmed five genes with downregulated expression and four with upregulated expression in 18 pairs of tumor and normal tissues. The Wilcoxon W test was used to determine significance [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Reduced TWIST2 expression in human prostate tumors. A, Reads per kilo bases per million mapped reads levels of TWIST2 in 494 prostate cancer (PCa) pathological tissues from The Cancer Genome Atlas and 106 human normal postmortem prostate tissues from GTEx. The Wilcoxon W test was used to determine significance. Immunostaining of the TWIST2 protein in human PCa tissue microarrays. B, Adjacent normal tissue. C, Prostatic intraepithelial neoplasia (PIN) tissue. D, Tumor tissue. E, Statistical plots of the immunoreactive intensity of TWIST2 staining in 67 pairs of PCa samples and adjacent normal tissues. The Fisher exact χ ² test was used to analyze the staining scores of adjacent normal tissues, PIN tissues, and tumor tissues. The data are shown as mean ± SD (***P < .001) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

The TWIST2 gene promoter is hypermethylated in PCa specimens. Methylation patterns of CpG islands in TWIST2 in tumor and matched adjacent normal tissues. The CpG positions +100 ~ +183 are indicated relative to the transcription start codon. Each circle in the figure represents a single CpG site. For each DNA sample, the percentage of demethylation at a single CpG site reflects the methylation degree from the sequencing results. The results show methylated TWIST2 in tumors (T1‐T18) and demethylated TWIST2 in matched adjacent normal tissues (N1‐N18). Pearson correlation was used to analyze the degree of methylation at each CpG site with TWIST2 messenger RNA levels (*P < .05, **P < .01). PCa, prostate cancer

Figure 4

TWIST2 inhibits LNCap cell proliferation and colony formation in vitro, and growth of LNCap tumors in vivo. A, Western blot analysis of FLAG, TWIST2, and GAPDH. B, Cell counting kit‐8 assays of the cell proliferation of LNCap, LNCap‐vector, and LNCap‐TWIST2 cells. C, Number of colonies formed by the three cell lines. D‐F, Colony formation assay of LNCap, LNCap‐vector, and LNCap‐TWIST2 cells. G, I, Ten male BALB/c nude mice aged 6 to 8 weeks were randomly divided into two groups. Vector‐ or TWIST2‐expressing LNCap cells (1 × 10⁶) were subcutaneously injected into the BALB/c nude mice. H, J, Solid tumors isolated from each mouse subcutaneously injected with vector‐ or TWIST2‐expressing LNCap cells. K, Growth of tumors derived from nude mice 14 days after injection. Student t test (two‐sided) was used to determine significance. The data are shown as the mean ± SD (*P < .05) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Multiple gene transcription changes occur in response to overexpression of TWIST2 in LNCap cells. A, Cluster of genes upregulated/downregulated in LNCap‐TWIST2, LNCap‐vector, and LNCap cells. Two LNCap‐TWIST2 cell lines have almost the same expression pattern, while those in the LNCap‐vector and LNCap wild‐type cell lines are similar. B, Scatter plot displays two‐fold upregulated/downregulated (green and red dots, respectively) genes in the LNCap‐TWIST2 cell line with respect to the LNCap‐vector cell line. The plot filters genes with a fold change value between 0.5 and 2 (black dots). C, Selected genes disregulated by overexpression of TWIST2 in LNCap‐TWIST2 vs LNCap‐vector cells. The RSEM levels of TWIST2 and selected genes from the TCGA Prad provisional database and Z‐scores from the TCGA Prad mkscc database in 61 PCa pathological tissues. Spearman correlations were analyzed of each selected gene with TWIST2 in the two databases (*P < .05, **P < .01, ***P < .001). PCa, prostate cancer [Color figure can be viewed at wileyonlinelibrary.com]