Abstract
Rhabdomyosarcoma (RMS) is a deadly cancer of skeletal muscle origin. Pannexin 1 (PANX1) is down-regulated in RMS and increasing its levels drastically inhibits RMS progression. PANX1 upregulation thus represents a prospective new treatment strategy for this malignancy. However, the mechanisms regulating PANX1 expression, in RMS and other contexts, remain largely unknown. Here we show that both RMS and normal skeletal muscle express a comparable amount of PANX1 mRNAs, but surprisingly the canonical 5′ untranslated region (5′ UTR) or 5′ leader of the transcript is completely lost in RMS. We uncover that quercetin, a natural plant flavonoid, increases PANX1 protein levels in RMS by inducing re-expression of a 5′ leader-containing PANX1 transcript variant that is efficiently translated. This particular PANX1 mRNA variant is also present in differentiated human skeletal muscle myoblasts (HSMM) that highly express PANX1. Mechanistically, abolishing ETV4 transcription factor binding sites in the PANX1 promoter significantly reduced the luciferase reporter activities and PANX1 5′ UTR levels, and both quercetin treatment in RMS cells and induction of differentiation in HSMM enriched the binding of ETV4 to its consensus element in the PANX1 promoter. Notably, quercetin treatment promoted RMS differentiation in a PANX1-dependent manner. Further showing its therapeutic potential, quercetin treatment prevented RMS in vitro tumor formation while inducing complete regression of established spheroids. Collectively, our results demonstrate the tumor-suppressive effects of quercetin in RMS and present a hitherto undescribed mechanism of PANX1 regulation via ETV4-mediated transcription of a translationally functional 5′ leader-containing PANX1 mRNA.
Highlights
Pannexin 1 (PANX1; known as Panx1 in rodents) is a transmembrane glycoprotein forming single membrane channels that are considered major conduits for ATP release [1,2,3,4,5]
PANX1 5’ UTR and protein levels are positively correlated in RMS and human skeletal muscle myoblasts (HSMM) Unexpectedly, the RNA-sequencing (RNA-seq) result from Rh30 cells revealed an abundance of endogenous PANX1 transcripts which mapped to all five exons with the exception of the 5′ untranslated region (5′ UTR) region (Fig. 1A)
RNA-seq data on adult skeletal muscle tissue specimens deposited in the Gene Expression Omnibus (GEO) database showed a clear mapping of PANX1 transcripts onto its 5′ UTR region (Fig. 1A)
Summary
Pannexin 1 (PANX1; known as Panx in rodents) is a transmembrane glycoprotein forming single membrane channels that are considered major conduits for ATP release [1,2,3,4,5]. We have previously shown that PANX1 levels are highly upregulated during human skeletal muscle myoblast (HSMM) differentiation [6], as well as during murine skeletal muscle development and regeneration [7]. PANX1 over-expression in undifferentiated HSMM promoted their differentiation while PANX1 channel blockade inhibited this process [6]. PANX1 levels are down-regulated in RMS tissue specimens and patientderived cell lines. Ectopic expression of PANX1 in RMS cells dramatically suppressed their malignant properties in vitro and in vivo [12]. Our study of the PANX1 transcriptome and interactome in RMS further implicated PANX1 in the regulation of genes involved in various key cellular processes, such as migration and apoptosis, and uncovered its tumor-inhibitory interaction with the neuroblast differentiation-associated protein AHNAK, respectively [13]
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