It has long been recognized that skeletal muscle differentiation is intimately coupled to cell cycle arrest. The Rb protein acts in two ways during this process: It preserves cell cycle arrest and it fosters high expression of muscle-specific genes induced by MyoD and other myogenic bHLH transcription factors. Without Rb, muscle development is compromised in vivo and in cell culture models.
Beyond muscle development, we know that the coordination between cell cycle arrest and muscle differentiation could be relevant to rhabdomyosarcoma, a common type of childhood cancer. This type of cancer is composed of skeletal myoblast-like cells in which the Rb protein function is usually compromised by deregulated Cyclin D/Cdk activity. Further, myogenic transcription factors are present in rhabdomyosarcoma cells but they fail to robustly induce the full complement of muscle differentiation genes and they cannot foster cell cycle arrest. Because normal muscle differentiation is coupled to irreversible cell cycle arrest, we feel that understanding why these processes fail may eventually point toward new treatment strategies.
To identify the key factors that control the initial steps in the transition from a proliferating myoblast to a post-mitotic, differentiating myocyte, we are employing a number of genetic and pharmacological strategies based on candidate genes and unbiased screens. We are using complementary cell culture and mouse models as well as human tissue samples and accompanying genomics data.
The cellular response to loss of Rb – a benefit of “senescence”?
Germ-line loss of one Rb allele leads to retinoblastoma – a highly malignant ocular tumor – in nearly all children lacking the gene; a smaller subset (10 percent to 15 percent) also develop a pineoblastoma, a highly malignant brain tumor in the pineal gland. Given the central role that Rb plays in cell cycle regulation, it’s rather remarkable that rather few cancers arise – raising the questions as to what are the secondary tumor suppressor effects that are engaged?
The story of mouse Rb is somewhat different than human Rb. First, mice that are born with only one normal Rb allele do not develop retinoblastoma (or pineoblastoma). It is now thought that this is due to functional redundancy or compensation for Rb loss by two other Rb-related proteins: p107 and p130.
As an alternative to studying Rb loss, we have engineered a mouse model in which Cyclin D1, a key negative regulator of the Rb protein (and also an important oncogene), is ectopically expressed in the retina and the pineal gland. We did this in order to model the tri-lateral retinoblastoma that often occurs in children. Remarkably, the forced expression of this potent oncogene is unable to drive tumor formation in either tissue. However, when the p53 tumor suppressor is also inactivated, high-grade pineoblastomas develop and cause severe morbidity within 2-3 months.
We have used this mouse model to begin to understand why tumors fail to develop unless p53 is inactivated. We are focusing on oncogene-induced senescence – which we now know is caused by Cyclin D1 expression in the pineal gland and can be modeled in vitro in cultured cells. We are using complementary mouse and cell culture models to understand both how p53 activity is engaged (surprisingly, Arf plays no role) and also what p53 does to drive cells to this senescent state. We are also interested in molecular mechanisms to maintain senescence, and we are developing a new mouse model in which the cellular response to Cyclin D1 can be assessed in other tissues.