Pannexin 1 inhibits rhabdomyosarcoma progression through a mechanism independent of its canonical channel function

Abstract

Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma of childhood thought to arise from impaired differentiation of skeletal muscle progenitors. We have recently identified Pannexin 1 (PANX1) channels as a novel regulator of skeletal myogenesis. In the present study, we determined that PANX1 transcript and protein levels are down-regulated in embryonal (eRMS) and alveolar RMS (aRMS) patient-derived cell lines and primary tumor specimens as compared to differentiated skeletal muscle myoblasts and tissue, respectively. While not sufficient to overcome the inability of RMS to reach terminal differentiation, ectopic expression of PANX1 in eRMS (Rh18) and aRMS (Rh30) cells significantly decreased their proliferative and migratory potential. Furthermore, ectopic PANX1 abolished 3D spheroid formation in eRMS and aRMS cells and induced regression of established spheroids through induction of apoptosis. Notably, PANX1 expression also significantly reduced the growth of human eRMS and aRMS tumor xenografts in vivo. Interestingly, PANX1 does not form active channels when expressed in eRMS (Rh18) and aRMS (Rh30) cells and the addition of PANX1 channel inhibitors did not alter or reverse the PANX1-mediated reduction of cell proliferation and migration. Moreover, expression of channel-defective PANX1 mutants not only disrupted eRMS and aRMS 3D spheroids, but also inhibited in vivo RMS tumor growth. Altogether our findings suggest that PANX1 alleviates RMS malignant properties in vitro and in vivo through a process that is independent of its canonical channel function.

Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its tumor inhibitory interaction with AHNAK

“…The significantly regulated genes were classified by gene ontology (GO) according to BP using DAVID [28] (Fig. 1D), which revealed numerous highly enriched (Fisher's Exact P value < 0.05) GO_BP terms in accordance with our previous observations [10], including regulation of apoptotic processes ( Fig. 1E) and negative regulation of migration ( Fig.…”

“…Based on this, we explored whether PANX1 levels are altered in RMS and if so, whether restoration of its expression could reduce RMS malignant properties. We found that PANX1 expression is downregulated in patientderived RMS cell lines and tumor specimens when compared to differentiated skeletal muscle cells and tissue [10]. Increasing PANX1 levels abrogated the proliferative and migratory potential of eRMS and aRMS cells, inhibited 3D tumor spheroid growth and induced regression of established spheroids through the induction of apoptosis [10].…”

“…We found that PANX1 expression is downregulated in patientderived RMS cell lines and tumor specimens when compared to differentiated skeletal muscle cells and tissue [10]. Increasing PANX1 levels abrogated the proliferative and migratory potential of eRMS and aRMS cells, inhibited 3D tumor spheroid growth and induced regression of established spheroids through the induction of apoptosis [10]. Moreover, while control tumors grew rapidly in mice, PANX1 overexpression significantly reduced eRMS and aRMS growth [10].…”

“…We have previously shown that PANX1 overexpression in RMS cells reduced their proliferation, migration, and inhibited tumor growth via induction of apoptosis [10]. As our first step toward understanding the PANX1 downstream signaling in RMS, Rh30 (aRMS) cells expressing ectopic PANX1 or control GFP (Fig.…”

“…Increasing PANX1 levels abrogated the proliferative and migratory potential of eRMS and aRMS cells, inhibited 3D tumor spheroid growth and induced regression of established spheroids through the induction of apoptosis [10]. Moreover, while control tumors grew rapidly in mice, PANX1 overexpression significantly reduced eRMS and aRMS growth [10]. As ectopic PANX1 effectively alleviates RMS tumor growth, deciphering its downstream signaling pathways would bring insight into the molecular mechanism by which PANX1 reduces RMS malignant properties while offering an opportunity to identify potential new therapeutic targets.…”