G1 Cell Cycle Dysregulation in Cancer
The focus of my laboratory is on G1 cell cycle control and its dysregulation in cancer cells. Specifically, we are investigating the function of the retinoblastoma protein, (pRb), D-type cyclins, cdk4 and cdk6 in programs of cell cycle exit. Together, along with p16INK4a, a negative regulator of cdk4 and cdk6, these proteins regulate a critical decision making point in progression to S phase and thus DNA replication. In almost all human tumors, one of these proteins is lost or overexpressed, thus favoring proliferation and tumor growth. Although these proteins regulate S phase in response to genome integrity (DNA damage), we and others have shown that they are also critically involved in cell fate determination and in the permanent cell cycle withdrawal associated with differentiation and senescence.
pRb in Bone Cell Differentiation and Senescence
Recently, work from our lab has identified important roles for each of these proteins that extends beyond a role in checkpoint control. We are presently studying the role of the retinoblastoma protein in bone cell differentiation and senescence, with the aim of identifying specific genes regulated in these processes. In addition, we are constructing mouse models of pRb loss in the bone, mimicking a common event in human osteosarcoma. This has allowed us to begin careful studies of the phenotypic consequences of pRb loss in the bone as well as provide a source of genetically defined primary cells for culture-based studies of osteoblast differentiation. These studies have revealed a key role fo pRb in the osteoprogenitor cell that restricts this cell to the bone lineage. Loss of pRb leads to an increase in the number of multipotent progenitors, with concomitant changes in cell migration, invasion, and resistance to terminal cell cycle withdrawal. These changes are reminiscent of those ascribed to the “tumor stem cell” suggesting that pRb may be a key suppressor of the generation of tumor cells from normal tissue progenitors in the bone.
Figure 1. Rb loss increases the osteoblast/adipocyte progenitor, as shown by increased adipogenesis in mouse calvarial cells, bottom.
Cyclin D1 in Development and Tumorigenesis
In a separate albeit conceptually related project, we have produced "knock-in" alleles of cyclin D1 to test for novel roles for this protein in development and tumorigenesis. The most interesting of these alleles is one that binds cdk4/6 but fails to activate the kinase. We have found that mice homozygous for this mutation have extensive mammary and retinal development, unlike their cyclin D1-null counterparts. An important goal of these experiments is to clearly define the role of cyclin D1 in mammary tumorigenesis. To that end, we crossed knockin 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. Interestingly, mice bearing defective cyclin D1 in the mammary gland remain resistant to tumor formation even after multiple pregnancies, and show progressive loss of lobular epithelial progenitor cells that we now identify as the initiating cells for ErbB2-dependent tumors. Further, glands lacking the oncogene show a significant shift in the types of epithelial cells produced, underscoring the role of cyclin D1 in progenitor cell expansion and fate choice in the mammary epithelium.
Figure 2. The drawing illustrates the ways by which cyclin D1 affects mammary gland development.
Cdk4 and Cdk6 in Tumorigenesis
Another major emphasis in the lab centers on the role of the cdk4 and cdk6 subunits themselves in tumorigenesis. In collaboration with Karl Münger, we have found that cdk6 plays an important but undefined role in oral cancer, apparently in collaboration with cdk4. These studies suggest the two kinases are not of equivalent function in at least some cell types, and our main goal is to understand the unique roles of cdk6 in development and tumors. To do this, we have undertaken a series of cell culture experiments using various active and dominant negative alleles of each kinase and are now extending these studies to include siRNA-mediated knockdown of the subunits. To supplement this, we have built mice bearing knockin alleles of hyperactive and “kinase dead” cdk6 (as well as null control). These animals are of great benefit as a source of primary cells for in vitro study, allow significant genetic experiments in crosses with existing animals prone to a variety of cancers, and will prove to be of lab-wide utility since cdk6 is likely to play roles in many of the processes studied in each of the above-mentioned areas. All of these studies have culminated in an overarching hypothesis that places the RB pathway at the center of cell fate decision-making steps in stem- and progenitor cells, and our major focus is now on understanding how this role of the RB pathway influences its role in cancer and development.
Figure 3. Cdks play a key role in bone development as illustrated by the effects of the K43M knock-in allele on bone mineral density (BMD).
pRb in Senescence
Finally, each of these areas of focus on development and tumorigenesis is complemented by our ongoing studies of the mechanisms of cellular senescence. pRb is a key player in this process and we have begun to dissect its biochemical role in detail. Our recent published and unpublished work has identified several new downstream elements such as cdk5, rac1 and ERM proteins, that are key to pRb’s instigation of the tumor-suppressive senescent phenotype. We are avidly pursuing the regulation of these and their role in tumor suppression. These studies, combined with our genetic approaches described above, promise to deliver considerable new insight into the molecular basis of cancer.