RAGE signaling deficiency in rhabdomyosarcoma cells causes upregulation of PAX7 and uncontrolled proliferation

Abstract

BSTRACTEmbryonal rhabdomyosarcomas (ERMSs) show elevated levels of PAX7, a transcription factor that marks quiescent adult muscle stem (satellite) cells and is important for proliferation and survival of activated satellite cells and whose timely repression is required for myogenic differentiation. However, the mechanism of PAX7 accumulation in ERMSs and whether high PAX7 causes uncontrolled proliferation in ERMS remains to be elucidated. The receptor for advanced glycation end-products (RAGE, encoded by AGER) transduces a myogenic and anti-proliferative signal in myoblasts, and stable transfection of the ERMS cell line TE671, which does not express RAGE, with AGER results in reduced proliferation and formation of tumor masses in vivo, and enhanced apoptosis and myogenic differentiation. Herein, we show that RAGE expression is low or absent in human ERMSs. We also show that in ERMS cells (1) PAX7 accumulates owing to absent or low RAGE signaling; (2) elevated PAX7 levels reduce RAGE expression and levels of MyoD and myogenin, muscle-specific transcription factors required for myoblast proliferation arrest and differentiation, respectively; (3) PAX7 supports myoblast proliferation by reducing the levels of MyoD, primarily by promoting its degradation; and (4), when ectopically expressed in ERMS cells, that RAGE upregulates myogenin which upregulates MyoD and downregulates PAX7, with consequent inhibition of proliferation and stimulation of differentiation. Thus, failure to express RAGE and, hence, MyoD and myogenin above a critical level in ERMS cells might result in deregulated PAX7 expression leading to uncontrolled proliferation and, potentially, to rhabdomyosarcomagenesis.

Defective RAGE activity in embryonal rhabdomyosarcoma cells results in high PAX7 levels that sustain migration and invasiveness

“…RAGE has emerged as a promyogenic cell surface receptor playing a key role in promoting cell cycle exit and differentiation in muscle precursor and ERMS cells (8)(9)(10)(11)14). Here we show that ectopic expression of RAGE in TE671 ERMS cells translates into reduced ability to metastasize lungs following i.p.…”

“…Immunofluorescence analyses confirmed that myoblasts transfected with PAX7 were characterized by undetectable levels of NCAM, whereas high fluorescence for NCAM was found in cells overexpressing myogenin ( Figure 5C). Because myogenin reduces PAX7 levels and vice versa (14), in order to assess whether myogenin or PAX7 is responsible for the observed changes in NCAM levels we knocked down either Myog, Pax7 or both in WT primary myoblasts in DM. We found that NCAM mRNA and protein levels correlated negatively with PAX7 and showed no correlation with myogenin levels in the experimental conditions tested ( Figure 5D and E), suggesting that a repressor activity of PAX7 rather than a stimulatory activity of myogenin is involved in the regulation of NCAM expression in muscle precursor cells.…”

“…The ERMS variant is believed to originate from proliferating/differentiating muscle precursor cells and typically expresses high PAX7 levels which have been proposed to contribute to tumorigenesis in the absence of chromosomal translocations (4). Indeed, PAX7 expression is inversely related to the expression of the terminal differentiation marker, myogenin, in fetal/adult myoblasts and ERMS tumors (18,24,25), and upregulation of PAX7 in muscle precursor cells is causally linked to their high proliferation (14) and prevents their terminal differentiation (18,26).…”

“…Cell culture TE671 (ATCC #CRL-8805), RD (ATCC #CCL-136) and TE671/RAGE cells obtained by stable transfection of TE671 cells with full-length human RAGE (11) were cultured in growth medium (GM) or differentiation medium (DM) as decribed (14). After receipt, TE671 and RD cells were expanded and aliquots were frozen in liquid nitrogen; each aliquot was used for no more than 10 passages.…”

“…Additionally, myogenin reduces PAX7 levels by a proteasome-mediated mechanism (13). We recently found that ERMS cell lines express no or low RAGE which results in high PAX7 levels that in turn cause reduction of RAGE, MyoD and myogenin levels ultimately leading to unrestricted proliferation and absence of myogenic differentiation (14). In this study, we analyzed the possibility that loss of RAGE expression/activity in muscle precursor cells and ERMS cells and the consequent upregulation of PAX7 expression might contribute to the acquisition of a metastatic behavior.…”