Role of APOBEC2 in Rhabdomyosarcoma and its Involvement in the Pannexin-1 Mediated Inhibition of Malignant Properties

Abstract

Rhabdomyosarcoma (RMS), the most common soft tissue cancer in Canadian children, is an aggressive childhood malignancy with a poor prognosis. To improve the outcome of RMS patients, an enhanced understanding of the molecular mechanisms involved in RMS pathogenesis and regulating its malignant properties is imperative. RMS tumor cells are thought to arise from muscle precursors that fail to completely differentiate into skeletal muscle. Our laboratory recently identified pannexin 1 (PANX1) as a novel regulator of myogenesis. PANX1 levels are downregulated in patient-derived cell lines and primary tumour specimens as compared to differentiated skeletal muscle myoblasts and tissue, respectively. Further work demonstrated that PANX1 overexpression inhibits RMS malignant properties through a mechanism that is independent of its canonical channel function. As this was the first time that a channel-independent function had been attributed to pannexins, RNA-sequencing was performed in RMS cells expressing PANX1 compared to their control. It was found that APOBEC2 was significantly downregulated in PANX1-expressing cells. APOBEC2 is solely found in differentiated skeletal and cardiac striated muscle and acts as a negative regulator of differentiation. We hypothesized that RMS malignant properties will decrease when APOBEC2 is downregulated and that APOBEC2 is involved in the signaling pathway by which PANX1 alleviates RMS malignancy. Here, we found that APOBEC2 is downregulated in RMS patient-derived cell lines compared to that of differentiating human skeletal muscle myoblasts. Although we found that APOBEC2 does not regulate RMS cell viability or proliferation, its overexpression promoted tumour spheroid growth. PANX1 overexpression prevents the upregulation of APOBEC2 in proliferating Rh28, Rh30, and RD but not in the other three cell lines. Our data suggests that the RMS cell lines in which PANX1 regulates APOBEC2 levels are those in which PANX1 overexpression triggers cell fusion. Notably, we found that the overexpression of APOBEC2 reversed the PANX1-mediated induction of RMS cell fusion. Together these results suggest that APOBEC2 regulates tumor growth in vitro in which an increase in APOBEC2 expression promotes RMS malignancy. At the same time, we identify a further role for APOBEC2 in cell fusion shedding additional light onto the mechanism by which PANX1 functions to alleviate RMS malignant properties.