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  • Breast cancer is the most common neoplasm in women with

    2019-07-17

    Breast cancer is the most common neoplasm in women, with approximately 231,840 new cases of invasive breast cancer, 60,290 additional cases of in situ breast cancer and 40,290 deaths occurring in 2015 in the United States. Meanwhile, about 2350 men will be diagnosed with breast cancer, and 440 men will die from the disease. Metastasis is the most devastating and life-threatening problem in women with breast cancer and therefore has become an important focus for research (Werbeck et al., 2014). Feline mammary carcinoma (FMC) has been proposed as a model for human breast cancer based on age of incidence, risk factors, histopathology, prognostic aspects, metastatic pattern and response to therapy (Hahn et al., 1994). The annual incidence of feline mammary neoplasia was estimated at 13 to 25 per 100,000 female cats (Hassan et al., 2017), and the feline mammary tumors are the most frequent neoplasm causing death in cats (Jemal et al., 2003), which occur predominantly in middle-aged to older female cats with a mean age of 10 to 12years (Zappulli et al., 2005). Like human breast cancer, FMCs usually present as in situ or invasive carcinomas (Soares et al., 2016), typically classified according to criteria of the World Health Organization (WHO). Changes in the expression of genes at the mRNA and protein levels have been described in both human breast cancer and FMCs (Zappulli et al., 2005). Since FMC has a high rate of malignancy, it will be useful to investigate the pathogenesis of metastasis and expression of tumor-related genes. In the current study, we described the isolation of feline BCA2 (fBCA2), analyzed the autoubiquitination ability of fBCA2 and compared it with that of human BCA2 (hBCA2). Full-length fBCA2 cDNA was isolated from CrFK hsp70 inhibitor in culture and cloned into a mammalian or prokaryotic expression vector. We then showed evidence that fBCA2 has E3 ubiquitin ligase activity, and its RING domain was found to be essential for its autoubiquitination. Our findings showed that fBCA2 may be a potential biomarker for FMC, which will help to contribute to an animal model for human breast cancer.
    Materials and methods
    Results
    Discussion RING E3 ligases play key roles in protein degradation and receptor-mediated endocytosis. BCA2 is a RING finger protein possessing ubiquitination ligase activity (Burger et al., 2005), which is over-expressed in invasive breast cancers and is a potential target for therapeutic interventions (Burger et al., 1996, Burger et al., 1998, Burger et al., 2005). FMC has been proposed as a model for human breast cancer based on age of incidence, risk factors, histopathology, prognostic aspects, metastatic pattern and response to therapy (Hahn et al., 1994). In the present study, an E3 ubquitin ligase fBCA2 was first identified and characterized from feline CrFK cells. The protein sequence was conserved in various species (human, mouse, bird, frog and fish), suggesting important functions for this autoubiquitination activity. fBCA2 encodes a RING domain as well, which is essential for ubiquitin transfer activity and thought to mediate protein interactions (Lorick et al., 1999, Karbowski et al., 2007). The autoubiquitination activity of hBCA2 requires the presence of an E2 as demonstrated with UbcH5s (Amemiya et al., 2008). We demonstrated here that UbcH5b could coordinate with both human and feline BCA2. The autoubiquitination activity of BCA2 was also found to depend upon the RING domain, as BCA2 with a mutated RING domain lacking key cysteines could not interact with UbcH5b, but it could still be modified by ubiquitin in 293T cells; meanwhile, it could not be modified by ubiquitin in vitro, which suggests that UbcH5b is not the only E2 that can transfer ubiquitin to BCA2 in vivo. In this study we have also identified the BZF domain in fBCA2, which is responsible for binding ubiquitin non-covalently in hBCA2 (Amemiya et al., 2008). We found that the mutation of lysines contained within the BZF domain could still be modified with ubiquitin both in vivo and in vitro, which is not exactly consistent with the results from a previous report (Amemiya et al., 2008). We hypothesized that the lysines in the BZF domain may not be the only two amino acid residues that can interact with ubiquitin.