Abstract
The most common mutational signature in urothelial carcinoma (UC), the most common type of urinary bladder cancer is assumed to be caused by the misdirected activity of APOBEC3 (A3) cytidine deaminases, especially A3A or A3B, which are known to normally restrict the propagation of exogenous viruses and endogenous retroelements such as LINE-1 (L1). The involvement of A3 proteins in urothelial carcinogenesis is unexpected because, to date, UC is thought to be caused by chemical carcinogens rather than viral activity. Therefore, we explored the relationship between A3 expression and L1 activity, which is generally upregulated in UC. We found that UC cell lines highly express A3B and in some cases A3G, but not A3A, and exhibit corresponding cytidine deamination activity in vitro. While we observed evidence suggesting that L1 expression has a weak positive effect on A3B and A3G expression and A3B promoter activity, neither efficient siRNA-mediated knockdown nor overexpression of functional L1 elements affected catalytic activity of A3 proteins consistently. However, L1 knockdown diminished proliferation of a UC cell line exhibiting robust endogenous L1 expression, but had little impact on a cell line with low L1 expression levels. Our results indicate that UC cells express A3B at levels exceeding A3A levels by far, making A3B the prime candidate for causing genomic mutations. Our data provide evidence that L1 activation constitutes only a minor and negligible factor involved in induction or upregulation of endogenous A3 expression in UC.
Highlights
The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3, A3) protein family of Zn2+-dependent DNA cytidine deaminases constitutes a defensive network of proteins restricting exogenous viruses (Chiu and Greene, 2008; Harris and Dudley, 2015) and endogenous transposable elements (Schumann, 2007; Schumann et al, 2010; Refsland and Harris, 2013; Salter et al, 2016)
As a first step to investigate if the expression of specific members of the A3 protein family of cytidine deaminases is a response to the upregulation of endogenous L1 expression, we profiled mRNA levels of all seven members of the A3 protein family (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) in 19 urothelial cancer cell lines (UCCs) and five independent primary cultures of normal urothelial cells (UPs) by RT-qPCR
The contribution of A3s is especially plausible in cancers elicited by viruses, such as cervical cancer (Henderson et al, 2014), but the high frequency of an APOBEC3related mutational signature in urothelial carcinoma (UC) (Alexandrov et al, 2013; Lawrence et al, 2013; Roberts et al, 2013) remains unexplained
Summary
The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3, A3) protein family of Zn2+-dependent DNA cytidine deaminases constitutes a defensive network of proteins restricting exogenous viruses (Chiu and Greene, 2008; Harris and Dudley, 2015) and endogenous transposable elements (Schumann, 2007; Schumann et al, 2010; Refsland and Harris, 2013; Salter et al, 2016). Many tumors of diverse entities display a characteristic mutational signature with strand-coordinated clusters of C→T transitions, which are frequently located in the proximity of chromosomal breakpoints This signature is often associated with increased A3A or A3B mRNA expression levels and is thought to be caused by misdirected A3 activity, partly in conjunction with viral infection (Burns et al, 2013; Roberts et al, 2013). The frequent occurrence of a characteristic mutational A3 signature (Lawrence et al, 2013) in urothelial carcinoma (UC) is puzzling, as these tumors are thought to be caused predominantly by chemical carcinogens rather than viral infections (Tolstov et al, 2014)
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