Multiple Biochemical Roles of Cyclin D1 in Development and Cancer

We have produced "knock-in" alleles of the pRb regulator cyclin D1 to test for novel roles for this protein in development and tumorigenesis. The most interesting of these alleles, KE, is one that binds cdk4/6 but fails to activate the kinase. We have found that mice homozygous for this mutation have normal mammary and retinal development, unlike their cyclin D1-null counterparts. These animals do have aberrant cerebellar development, express low levels of serum IGF-1, display decreased body size and neurological defects, however, providing us with novel insight into tissues that depend on cdk4 function for normal development. An important goal of these experiments is to clearly define the role of cyclin D1 in mammary tumorigenesis. To that end, we have begun crosses of knock-in animals with those prone to breast cancer (e.g. MMTV-neu mice) to ask the important question of whether kinase activation is needed for tumorigenesis in the breast. The development of breast cancers in knock-in mice will stand in contrast to the resistance of such pathology in cyclin D1-null mice and will indicate that cdk4 activation may not be a suitable target for intervention in this disease. Conversely, a lack of tumor development in mutant mice that undergo normal development will underscore a key role for cyclin D1-kinase activity in tumorigenesis, but not in mammary development. Extending this approach, we are producing conceptually similar knock-in mice bearing specific point mutations in the cdk6 gene, a cyclin D1-dependent kinase that can also regulate pRb but likely has additional roles in specific tumor types. Combined, these experiments will allow a comprehensive understanding of the pRb-dependent and independent roles of cyclin D1, cdk4 and cdk6 in tumorigenesis.

The Role of the Retinoblastoma Protein in Senescence

The focus on development and tumorigenesis described above is complemented by our ongoing studies of the mechanisms of cellular senescence. Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-ß-galactosidase (SA-ß-gal). Moreover, recent work from the Campisi lab has shown that senescent fibroblasts in a tumor microenvironment can actually augment tumor growth, underscoring the need to understand this process in molecular detail in order to fully understand the impact of senescence-inducing therapies on tumorigenesis. Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated downregulation of Rac1 activity and phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-ß-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth. These studies, combined with our genetic approaches described above, promise to deliver considerable new insight into the molecular basis of cancer.