Carolinas Research Day 2021

Transmembrane and Tetratricopeptide Repeat Containing isoforms as novel biomarkers for diagnosis of prostate cancer Alexander S. Hernandez, OMS-I, Dahir Hassan, OMS-I, Zakaria Y. Abd Elmageed, MS, PhD Edward Via College of Osteopathic Medicine (VCOM), Department of Biomedical Sciences, Monroe, Louisiana

Abstract # BIOM-7

Abstract

Results

Conclusions

Introduction: Prostate cancer (PCa) is the most commonly diagnosed cancer in elderly men. Although prostate specific antigen (PSA) is a routine marker for PCa, its sensitivity is compromised and may mislead treatment decisions. We aimed to determine the clinical utility of novel TMTC isoforms at different stages of PCa. Methods: The expression of TMTC3 and TMTC4 in PCa cells in addition to normal cells was determined by Real-Time PCR and Western blot analyses. Immunohistochemical (IHC) staining of TMTC4 was performed on paraffin- embedded sections of 72 cases diagnosed with benign prostatic hyperplasia (BPH) and PCa in 29 and 43 cases, respectively. . Results: Protein analysis showed that TMTC3 and 4 are differentially expressed (p<0.01) in PCa cells versus normal or BPH1 cells. IHC studies revealed that TMTC4 are overexpressed (P<0.001) in tumors versus hyperplastic PCa tissues. The overall accuracy measured by the AUC was 1 (p<0.001). Conclusion: TMTC 3 and 4 are overexpressed in PCa cells and tissues compared to normal controls and can be used as diagnostic markers. This study warrants further investigations to understand their underlying mechanisms in cancer progression.

• Expression of TMTC4 in PCa Tissue Specimens

• Specificity and sensitivity of TMTC4 in differentiation of PCa from benign tissues.

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Figure 4. TMTC4 distinguished PCa from BPH with AUC value of 1

Conclusions

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References • TMTC4 is differentially expressed in PCa cells and human tissue specimens. • TCGA available data of TMTC4 suggests that the protein is significantly expressed (p<0.0001) in PCa patients (n=497) versus normal (n=52), GS 6 to 9 (p<0.001)) and ERG, ETV1/4 7 SPOP (p<0.01). • ROC curve was depicted for TMTC4 expression in PCa and BPH. The overall accuracy measured by the AUC was 1 (P<0.001). • TMTC4 protein can be utilized as a biomarker and may act as a potential therapeutic target for treating PCa patients. 1. Siegel RL, Miller KD, Jemal A. Cancer statistics. CA Cancer J Clin 2020. 70: 7-30. 2. Ali HEA, Lung PY, Sholl AB, Gad SA, Bustamante JJ, Ali HI, Rhim JS, Deep G, Zhang J, Abd Elmageed ZY. Dysregulated gene expression predicts tumor aggressiveness in African-American prostate cancer patients. Sci Rep , 2018. 8(1): p. 16335. 3. Abd Elmageed ZY, Moroz K, Srivastav S, Crawford B, Moparty K, Thomas R, Abdel-Mageed AB. The importance of Estrogen-ER-beta axis in health disparity of Prostate Cancer. Carcinogenesis 2013. 34 (9): 2017-23.

Methods and gene expression

• A panel of PCa cell lines were utilized to examine the expression of TMTC4 in these cells in addition to normal prostate cells. • Reconstitution of TMTC4 in low-expressing PCa cells was performed by transfection of TMTC4-based vector into PCa cells in addition to empty vector as a control as previously described (2). • For evaluating its protein expression, immunohistochemical (IHC) studies were performed on human PCa and BPH tissue specimens (3).

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Figure 2. Expression of TMTC4 in PCa tissues. Tissue slides were immunostained with TMTC4 antibody. Tissue slides procured from benign prostatic hyperplasia (A, B), and PCa at Gleason score 6 (C, D), and GS 8 (E, F) were incubated with TMTC4 antibody, counterstained and photographed. Magnification was at 40X (A, C, E) and 400X (B, D, F), respectively. Quantification of TMTC4 and its correlation with Gleason score, age and perineural invasion was performed (G).

• Gene expression of TMTC4 in PCa Tissue Specimens

• Expression of TMTC3 & 4 in PCa Cell Lines

FUNDING This work was supported in part by Edward Via College of Osteopathic Medicine (Abd Elmageed, PI). Email: zelmageed@ulm.vcom.edu

Figure 3. Expression of TMTC4 transcript in tissues of PCa patients. Expression data were retrieved from TCGA (A), classified according to Gleason Score “GS” (B), and molecular features of PCa (C). *depicts significance at p<0.01

Figure 1. TMTC4 expression in PCa and normal cells. A: qPCR was performed for TMTC3 & 4 in PCa and normal cells. B: Sixty µg protein lysate was resolved on SDS-PAGE using TMTC4 antibody. GAPDH was used as a housekeeping protein.

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