Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

Abstract

The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation.

The Satellite Cell Niche Regulates the Balance between Myoblast Differentiation and Self-Renewal via p53

“…Nutlin-3, a small molecule compound that blocks the interaction between p53 and the E3 ligase MDM2, is often used to increase the levels of p53 protein in the cell ( Vassilev, 2005 ). Nutlin-3 treatment of C2C12 myoblasts leads to a generalized increase in p53 protein levels and both proliferation and differentiation inhibition ( Walsh et al., 2015 ), thus supporting our hypothesis that a sustained increase in p53 levels in primary myoblasts might lead to apoptosis, senescence, or quiescence, as well as differentiation inhibition. To test this hypothesis we treated primary dispersed myoblasts with increasing concentrations of Nutlin-3 and verified that indeed Nutlin-3 inhibits proliferation ( Figure S5 A).…”

“…Thus, it could be speculated that the upregulation of p53 that we observe in myofiber-associated myoblasts drives differentiation. However, it has been recently shown that a sustained increase in p53 levels, such as that due to genotoxic stress, or treatment with the MDM2 inhibitor Nutlin-3, impairs myogenic differentiation, possibly via direct inhibition of myogenin expression ( Walsh et al., 2015 , Yang et al., 2015 ). Moreover, p53 mediates hypoxia-induced inhibition of myoblast differentiation ( Wang et al., 2015 ).…”

Aberrant Caspase Activation in Laminin-α2-Deficient Human Myogenic Cells is Mediated by p53 and Sirtuin Activity

“…Our finding that sirtinol had a negative effect on MDC1A myogenic cells, as measured by increased caspase activation, is consistent with studies showing that sirtinol can induce apoptosis in multiple cell types [ 56–58 ]. Our results are also consistent with previous work showing that sirtinol-induced apoptosis is p53-dependent [ 56 ] and that SIRT1 inhibition can increase KU70 acetylation [ 38, 59 ]. However, it remains to be determined if the decreased caspase activation caused by SIRT1 overexpression is dependent on p53.…”

“…Differentiated cultures of healthy control cells also showed increased caspase activity upon treatment with Nutlin-3a, but both the ∼2X increase and final caspase levels were less in the differentiated control cultures than in MDC1A cultures. Much as seen previously in cultures of mouse C 2 C 12 myogenic cells [ 38 ], we also found that nutlin-3a inhibited myotube formation when it was added to proliferating MDC1A or control myoblasts at the time of switching to differentiation medium (not shown). Because nutlin-3a prevents HDM2 from binding to and inhibiting p53, these findings suggest that MDC1A cells, as well as healthy control cells, have functional HDM2 that at least partially restrains p53-mediated caspase activation.…”

Roles of pRB in the Regulation of Nucleosome and Chromatin Structures

“…Therefore, inhibiting pRB inactivation is a relevant strategy to suppress cancer progression. Some efficacious compounds and small molecules have been investigated, such as CDK4/6 inhibitors to suppress pRB phosphorylation [127] and Nutlin-3, a small molecule inhibitor of Mdm2, to regulate Mdm2-mediated regulation of pRB expression [128–130]. The CDK4/6 inhibitor palbociclib is currently in phase II development, and ribociclib and abemaciclib are in phase I development.…”