My research program focuses on two areas of cell biology: the molecular mechanisms of mammalian cell differentiation and mitotic checkpoint control. Though seemingly unrelated, both areas as described below continue to provide fertile ground for the analysis of molecular mechanisms and have direct application to cancer biology. In the most general terms, we are exploring the role of the Id proteins in controlling the growth and differentiation of tumor cells and the vasculature that supports their growth and metastatic progression. In addition, we are examining the role of the mitotic checkpoint gene Mad2 in maintaining genome stability by ensuring proper chromosome segregation during mitosis.
Molecular Mechanisms of Differentiation and Metastasis
Tumor metastasis, or the spread of cancer cells to distant organs, is the leading cause of death among cancer patients. The process of metastasis involves a number of critical steps including invasion of the tumor cells into a blood vessel, survival and travel in the bloodstream, exiting the blood vessel at a distant site, and then regrowth once the cells arrive at the new organ site. Each step of this process is controlled by sets of proteins that are now beginning to be identified. My laboratory focuses on the expression of a family of proteins called Id proteins, which are essential for the last step of metastasis — namely the reinitiation of tumor growth once the cancer cell has taken up residence at a new site. Inhibiting the activity of these proteins has been shown in animal models to severely inhibit the dissemination of cancer cells and presents a novel therapeutic approach to the management of metastasis in patients.
The Id proteins were first identified as proteins that controlled the growth and specialization of cells in the early embryo. It was shown that high levels of Id expression were required for cells in the embryo to undergo the appropriate number of rounds of cell division before they began to develop into specialized cells of certain tissues (such as muscle and brain and blood vessels), at which time they stop dividing and perform the necessary function of those particular tissue types. Remarkably, when the Id proteins are re-expressed in cancer cells, they do a very similar thing — they allow the tumor cells to continue to divide, particularly when they arrive at distant organ sites during metastasis. In collaboration with Sloan Kettering Institute Director Joan Massague’s laboratory at SKI, we have shown that when Id proteins are depleted, breast tumor cells fail to reinitiate growth when they arrive in the lung, a major metastatic site in breast cancer progression.
The growth of blood vessels at metastatic sites is also under the control of Id proteins. When metastatic tumors begin to grow, they recruit rapidly growing blood vessels that re-express the Id proteins. Importantly, Id proteins are not expressed in normal “resting” blood vessels, making them attractive therapeutic targets. Our lab has shown over the past decade that the re-expression of the Id proteins is essential for the formation of a functioning blood-vessel network at sites of metastasis. The Id-expressing, blood-vessel-forming cells are recruited from the bone marrow to the site of metastasis. Inhibiting these proteins in the bone marrow in mouse models decreases the integrity of the vasculature at metastatic sites and thereby starves the growing cancer cells of necessary oxygen and nutrients.
Mechanism of action of AGX51, an ID degrader
In an effort to apply these findings to the development of a new class of drugs, we have identified a small molecule (AGX51) capable of binding the Id proteins which causes a change in their conformation which in turn leads to their ubiquitlyation and degradation. Importantly, the binding of AGX51 to Id proteins is in a highly conserved region of the protein that cannot tolerate mutations and retain activity so acquired resistance to AGX51 by mutation of the binding pocket is not observed. Amplification of the target Id proteins, another common resistance mechanism, also does not override the strong protein degradation response. Therefore the hope is as this class of compounds is reaches clinical application in cancer, the response to therapy will be durable, targeting both the tumor cell itself as well as the supporting vasculature.
Molecular Mechanisms of Maintenance of Chromosome Stability
Dividing cells must ensure that each of the two daughter cells receives an exact duplicate of all chromosomes to ensure fitness. The acquisition of aneuploidy, or abnormal chromosome numbers, has long been associated with tumor relapse, metastasis, and poor prognosis in a wide variety of human cancers. Recently it has been shown in a large number of mouse modeling studies, including those from our lab, that aneuploidy is not simply a passenger but rather can drive the generation of more aggressive disease and fuel recurrence after tumor regressions. In addition to the gains in tumor-promoting oncogenes or loss of tumor suppressors that result from numerical chromosome changes, it is now becoming clear that chromosome missegregation events can lead directly to DNA damage and changes in metabolism that can further fuel the rapid growth and fitness of tumor cells.
The mitotic checkpoint (MC) ensures proper segregation of chromosomes by delaying anaphase until all kinetochores are bound to microtubules. This inhibitory signal is composed of a complex containing Mad2, which inhibits anaphase progression. The proteins that execute the MC are, somewhat surprisingly, often overexpressed in cancer because their expression is repressed by gene products (namely p53 and Rb) that are often lost in cancer. Overexpression of components of the MC lead to a failure of chromosomes to segregate properly as a result of a failure to degrade the proteins (cohesins) which encircle the two copies of each replicated chromosome. The timing of cohesin loss is important to allow each chromosome to find its way to the proper daughter cell.
The MC complex can be disassembled by p31comet and TRIP13; however, TRIP13 knockdown has been shown to cause only a mild mitotic delay. Overexpression of MC genes is correlated with chromosomal instability (CIN) in cancer, but the initial effects of Mad2 overexpression are prolonged mitosis which ultimately result in the missegregation events . We have shown that TRIP13 overexpression significantly reduced, and TRIP13 reduction significantly exacerbated, the mitotic delay associated with Mad2 overexpression, but not that induced by microtubule depolymerization. The combination of Mad2 overexpression and TRIP13 loss reduced the ability of checkpoint complexes to disassemble and significantly inhibited the proliferation of cells in culture and tumor xenografts. These results identify an unexpected dependency on TRIP13 in cells overexpressing Mad2.By exploring the molecular mechanisms that control the proper expression of genes involved in the SAC and understanding how the SAC itself is extinguished once all chromosomes are properly attached to the spindle, we hope to uncover new vulnerabilities in the aggressive and often drug-resistant cancer cells that have acquired abnormal chromosome numbers.