Tumour-Suppressive Effects of Pannexin 1 and Its Mechanism of Action and Regulation in Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is a paediatric neoplasm of skeletal muscle in dire need of novel therapeutic strategies. We explored the role of pannexin 1 (PANX1), an emerging regulator of skeletal muscle differentiation, in the two major histological subtypes of RMS: embryonal RMS (eRMS) and alveolar RMS (aRMS). We found that PANX1 levels are down-regulated in eRMS and aRMS tissue specimens and patient-derived cell lines. While PANX1 transcripts were expressed in RMS, we showed that the majority lacked the putative 5’ untranslated region (5’UTR) and the first ATG start codon, which contributed to low PANX1 expression. Re-introduction of PANX1 in eRMS and aRMS patient-derived cell lines induced their partial differentiation and suppressed their malignant properties in vitro and in vivo. Notably, our data suggest that the PANX1-mediated tumour-inhibitory function in RMS does not depend on its canonical channel function. Furthermore, our genome-wide transcriptomic study found that PANX1 over-expression in RMS cells induced changes in expression of genes in various cellular processes and signaling pathways involved in tumour-suppression including connexin 43 (Cx43), which was previously shown to induce RMS cell differentiation. At the protein level, we showed that the PANX1 interactome in RMS cells involved plasma membrane and cytoskeleton associated proteins including the neuroblast differentiation-associated protein AHNAK, also known as desmoyokin. We demonstrated that PANX1-mediated tumour-suppression in RMS cells depended on its interaction with AHNAK. We further constructed the first searchable PANX1 transcriptome and interactome databases and made them available for the scientific community. We then searched for a means by which to increase PANX1 levels as a new therapeutic approach for RMS. We found quercetin, a clinical approved natural flavonoid, as a PANX1 up-regulator in RMS cells. This induction of PANX1 expression involved alternative transcription of a PANX1 mRNA variant containing a translationally active 5’UTR, which depended, in part, on binding of the transcription factor ETV4 (ETS Variant Transcription Factor 4) in the PANX1 promoter. Moreover, quercetin treatment induced RMS cell differentiation, inhibited tumour spheroid growth, and induced regression of established tumour spheroids. Collectively, we demonstrate down-regulation of PANX1 in RMS and the dramatic tumour-inhibitory effects of restoring PANX1 expression in this malignancy. We show that PANX1-induced tumour-suppression does not rely on its canonical channel function but rather by signaling transduction via interaction with AHNAK. We reveal that PANX1 expression can be regulated at the translational level by the 5’UTR of its mRNA via quercetin-induced alternative transcription. We further demonstrate quercetin as a potential therapeutic agent for RMS by showing its tumour-suppressive effects in RMS. These studies not only substantiate our current understanding of PANX1, but also establish PANX1 as a therapeutic target in RMS and provide potential therapeutic options for its treatment.