TGF‐β autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells

Abstract

Transforming growth factor beta (TGF) is a well-known inhibitor of myogenic differentiation as well as an autocrine product of rhabdomyosarcoma cells. We studied the role of the TGF-beta autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD. We previously reported that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and myogenic differentiation in these cells, which constitutively express muscle regulatory factors. We show that TPA inhibits the activation of secreted latent TGF-beta, thus decreasing the concentration of active TGF-beta to which the cells are exposed. This event is mediated by the TPA-induced alteration of the uPA/PAI serine-protease system. Complete removal of TGF-beta, mediated by the ectopic expression of a soluble type II TGF-beta receptor dominant negative cDNA, induces growth arrest, but does not trigger differentiation. In contrast, a reduction in the TGF-beta concentration, to a range of 0.14-0.20 x 10(-2) ng/ml (which is similar to that measured in TPA-treated cells), mimics TPA-induced differentiation. Taken together, these data demonstrate that cell growth and suppression of differentiation in rhabdomyosarcoma cells require overproduction of active TGF-beta; furthermore, they show that a 'critical' concentration of TGF-beta is necessary for myogenic differentiation to occur, whereas myogenesis is abolished below and above this concentration. By impairing the TGF-beta autocrine loop, TPA stabilizes the factor concentration within the range compatible for differentiation to occur. In contrast, in human primary muscle cells a much higher concentration of exogenous TGF-beta is required for the differentiation inhibitory effect and TPA inhibits differentiation in these cells probably through a TGF-beta independent mechanism. These data thus clarify the mechanism underlying the multiple roles of TGF-beta in the regulation of both the transformed and differentiated phenotype.

Myostatin inhibits rhabdomyosarcoma cell proliferation through an Rb-independent pathway

“…In agreement with the results described here, whereby RD cell proliferation is inhibited by myostatin, is a report showing that TGF-b can inhibit the proliferation of RD cells in a dose-dependent manner (Bouche et al, 2000). While the exact molecular mechanism for this inhibition in RD cells is not reported, other lines of evidence showing that TGF-b1 downregulates cyclin-E and cyclin-A mRNA and protein levels in epithelial cells without altering cyclin-D1 levels (Geng and Weinberg, 1993;Hu and Zuckerman, 2001) also support the findings described here.…”

“…Using an established cell line, RD which was derived from an RMS-E tumor (Houghton et al, 1981); the complete removal of TGF-bsignalling by the expression of a dominant-negative type II TGF-b receptor, induces growth arrest but does not trigger differentiation. In contrast, a reduction of TGFb to the range of 0.14-0.20 Â 10 À2 ng/ml was shown to induce myogenic differentiation (Bouche et al, 2000). This finding supports the cell cycle arrest and differentiation of RD cells by 12-O-tetradecanoylphorbol-13-acetate (TPA), which is suggested to occur through the TPA-induced loss of urokinase plasminogen activator (uPA) activity resulting in a reduction in the conversion of latent TGF-b to active TGF-b (Aguanno et al, 1990;Bouche et al, 1993Bouche et al, , 2000.…”

Hedgehog-independent overexpression of transforming growth factor-β1 in rhabdomyosarcoma of Patched1 mutant mice

“…This inhibition of differentiation by TGFB1 persists even in nonmuscle cell lines engineered to ectopically express members of the bHLH family (54)(55)(56). Recent studies have described that overproduction of TGFB1 and an autocrine TGFB1 loop is responsible for the growth of RMS (57) and that TGFB1 exhibits a growth-inducing effect in human airway smooth muscle cells through a mechanism that requires TGFBP3 expression (58). Interestingly, Tgfb1 and Igfbp3 were concomitantly overexpressed in Ptch1-asssociated RMS of our study.…”

Late relapse of myelodysplasia after allogeneic transplantation concomitant with new presentation of invasive liposarcoma as a secondary neoplasm

“…10,11 Perhaps (and similar to our case), other soft tissue sarcomas use the same mechanism, which at least in some tumors appears to be through a TGF-b autocrine loop, an idea supported in our case by the presence of both TGF-b and the TGF receptor on the sarcoma cell. 12,13 With these considerations, it can be speculated that the biologic benefit from donor immune engraftment (graft vs malignancy effect) may not be eradication of underlying malignancy, but rather successful suppression, similar to the recognized suppression of autoreactive immune effector cells in all individuals. Thus, when the donor:host immune homeostasis is destabilized, as in this case with the high tumor burden of the liposarcoma and its potential immune suppressive cytokines, relapse is experienced with an evolved host myelodysplastic clone selected for its aggressive phenotype.…”