Transmembrane and Tetratricopeptide Repeat Containing 4 Is a Novel Diagnostic Marker for Prostate Cancer with High Specificity and Sensitivity

Abstract

The histopathologic diagnosis of prostate cancer (PCa) from biopsies is a current challenge if double or triple staining is needed. Therefore, there is an urgent need for development of a new reliable biomarker to diagnose PCa patients. We aimed to explore and compare the expression of TMTC4 in PCa cells and tissue specimens and evaluate its sensitivity and specificity. The expression of TMTC4 in PCa and normal prostate epithelial cells was determined by real-time PCR and Western blot analyses. Immunohistochemical (IHC) staining of TMTC4 was performed on tissues collected from PCa and benign prostatic hyperplasia (BPH). Our results show a high expression of TMTC4 on mRNA and protein levels in PCa versus BPH1 and normal cells (p < 0.05). IHC results show strong cytoplasmic expressions in PCa cases (p < 0.001) as compared to BPH cases. The overall accuracy as measured by the AUC was 1.0 (p < 0.001). The sensitivity and specificity of the protein were 100% and 96.6%, respectively. Taken together, we report a high TMTC4 expression in PCa cells and tissues and its ability to differentiate between PCa and BPH with high sensitivity and specificity. This finding can be carried over to clinical practice after its confirmation by further studies.

Keywords: TMTC4; biomarker; immunohistochemical staining; prostatic cancer; sensitivity and specificity.

Figure 1

TMTC4 study design and its expression in prostate cancer cells. (A) The flowchart of the experimental design of the current study. (B) Real-time PCR was performed to determine the expression of tmtc3 and tmtc4 in PCa and normal cells using gapdh as a housekeeping gene. Fold change was calculated regarding PEC and RWPE1 cells. (C) Sixty µg protein lysate was resolved on SDS-PAGE using TMTC4 antibody. GAPDH antibody was used as a housekeeping protein. Data significance was considered at p-value < 0.05 regarding normal and BPH cells, respectively. Experiments repeated at least twice.

Figure 2

Immunohistochemistry of TMTC4 expression in BPH and prostate cancer patients: (A,B) TMTC4 staining in BPH showing negative expression in prostatic glands (green arrows); (C,D) mild cytoplasmic expression of TMTC4 in BPH (white arrows); and (E,F) strong cytoplasmic expression of TMTC4 in PCa tissues at Gleason score 3 (small glands but still separated from each other by stroma as shown by blue arrows). Magnification: 40× (A,C,E); and 400× (B,D,F).

Figure 3

Immunohistochemistry of TMTC4 expression in PCa patients at high Gleason score and perineural invaded tissues: (A,B) strong cytoplasmic protein expression of TMTC4 in PCa at Gleason score 4 (fused glands with no intervening stroma as depicted by white arrows); (C,D) strong cytoplasmic staining of TMTC4 in Gleason pattern 5 shows single cells without glandular lumen formation as depicted by arrowhead; and (E) strong expression of TMTC4 in perineural invasion of malignant prostatic glands (the yellow arrow indicates the nerve and the arrow heads pointing to malignant glands surrounding the nerve). Magnification: 40× (A,C); and 400× (B,D,E).

Figure 4

Immunohistoscore of TMTC4 expression in PCa tissues and its sensitivity and specificity: (A) immunohistoscore (H-score) of TMTC4 in PCa (n = 43) compared to BPH (n = 29) tissues; (B–D) H-sore of the protein based on Gleason score, age, and perineural invasion, respectively; and (E) ROC curve analysis for H- score based on TMTC4 protein expression in PCa and BPH tissue specimens. Significance of data was considered at p < 0.05.

Figure 5

Upregulation of TMTC4 transcript in tissues of patients with PCa using TCGA available data. Available data were retrieved from TCGA, as indicated in the methods. (A) Expression of TMTC4 in tumor (n = 497) compared to normal (52) tissue specimens. (B,C) Differential expression of TMTC4 transcripts according to Gleason Score (GS) and molecular features of PCa. * depicts significance at p < 0.01.