USP13 genetics and expression in a family with thyroid cancer

Abstract

Purpose: Papillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma and its incidence has greatly increased in the last 30 years. Ubiquitin-specific protease 13 (USP13) is a class of deubiquitinating enzymes (DUBs) and plays an important role in cellular functions such as cell cycle regulation, DNA damage repair, and different cell signaling pathways. Studies regarding the role of USP13 in cancer development and progression are divergent and there are no previous data regarding the role of USP13 gene in PTCs. In this study, we investigated the genetic cause of PTC diagnosed in multiple members of a Brazilian family.

Methods: Whole exome sequencing (WES) was performed to identify the genetic cause of PTC. Cycloheximide chase assay and clonogenic assay were performed to study USP13 stability and function in vitro.

Results: WES analysis identified a heterozygous missense variant c.1483G > A (p.V495M) in the USP13 gene that fully segregates with the disease. In silico modeling suggests that this variant may cause protein structural perturbations. USP13 overexpression increased the potential of a single cell to form colonies. The USP13 c.1483G > A variant enhanced the effects seen in USP13 overexpression and preserved protein stability for longer hours compared to the non-mutated USP13 protein.

Conclusion: Our study suggests that USP13 overexpression may play a role in tumorigenesis of PTCs; and that the USP13 p.V495M (c.1483G > A) variant enhances USP13 estability.

Keywords: Oncogene; Papillary thyroid carcinoma; Pathogenic variant; Ubiquitin-specific protease 13.

Fig 1. USP13 variant analysis.

(a) Genealogical tree and USP13 c.1483G>A; p.V495M variant analysis. Open symbols represent individual that are wild type for USP13. Filled symbols indicate individuals that are carriers of the USP13 c.1483G>A variant. Black filled symbols indicate individuals diagnosed with papillary thyroid carcinoma, gray symbols indicate individuals diagnosed with other thyroid problems (Hashimoto Thyroiditis or goiter). Truncated symbols indicate deceased individuals. (b) Electropherogram of part of USP13 sequence including the position c.1483 of each member represented in the genealogical tree. Diagnosis of papillary thyroid carcinoma or other thyroid complications are indicated above the electropherogram.

Fig 2. In silico prediction of the USP13 structure.

(a) Protein sequence alignment evidencing the USP13 p.V495M variant within the USP13 domain and amino acid residue conservation among different species. (b) I-TASSER predicted ab-initio model of USP13 c.1483G>A; p.V495M variant. Secondary structure of USP domain with a focus on the USP13 c.1483G>A; p.V495M variant.

Fig 3. USP13 siRNA in MDA-T32 cells.

(a) USP13 and PTEN expression analysis of MDA-T32 cells transfected with USP13 siRNA performed by western blot. (b) Band densitometry quantification of USP13 and (c) PTEN normalized by the non-target siRNA. *p>0.0001. (d) Representative images of clonogenic assay to access the capacity of MDA-T32 cells transfected with USP13 siRNA for 48 hours to establish colonies. Cells were stained with Crystal Violet after 7 days of transfection. (e) Absorbance plot of the colony staining. *p>0.0001. (f) Cell viability accessed by MTT assay in MDA-T32 cells transfected with USP13 siRNA for 24, 48 and 72 h. *p=0.013.

Fig 4. USP13 c.1483G>A; p.V495M functional assessment.

(a) Electropherogram of part of USP13 sequence including the position c.1483 in the pCMV6 USP13 WT and mutant vector. (b) Cycloheximide chase assay test performed in MDA-T32 cells transfected with pCMV6 USP13 WT and mutant vector. After 24 hours of transfection, cells were treated with cycloheximide for additional 3, 8 and 24 hours. Representative blots of USP13 expression analyzed by western blot. (c) Band densitometry quantification of USP13 expression for all the different times comparing USP13 WT and mutant. *p=0.01 (d) Representative images of clonogenic assay to access the capacity of MDA-T32 cells transfected with USP13 WT or USP13c.1483G>A for 48 hours to establish colonies. Cells were stained with Crystal Violet after 7 days of transfection. (e) Absorbance plot of the colony staining. *p>0.001. WT: wild type.