We study on how cell growth and division are regulated during normal development and in disease. We also study what happens to cells that, defying the cell cycle quality control mechanisms, undergo a faulty cell division and hence become aneuploid.
Preventing Aneuploidy from Occurring: Understanding the Mechanisms that Ensure Accurate Cell Division
Regulation of Exit from Mitosis by the Mitotic Exit Network. Exit from mitosis is the final cell cycle transition when cells disassemble their mitotic spindles, reform the nuclear envelope and undergo cytokinesis. In budding yeast, this transition is triggered by the protein phosphatase Cdc14, whose activity in turn is controlled by a Ras-like GTPase signaling pathway known as the Mitotic Exit Network (MEN) in yeast and the Hippo pathway in higher eukaryotes. We study the mechanistic basis of MEN signaling and the signals that control MEN activity. Key among the MEN regulatory signals are spindle position and anaphase onset. Our studies have led to the formulation of a hypothesis that explains how spindle position and anaphase onset control MEN activity and hence exit from mitosis.
Correctly positioning the mitotic spindle within a cell is essential for accurate chromosome segregation. Thus, mechanisms must exist that sense spindle position and convey this information to the cell cycle machinery. We found that the localization of MEN components and MEN regulators is at the heart of spindle position sensing: The MEN constituents localize to the cytoplasmic face of spindle pole bodies (SPBs), the budding yeast equivalent of centrosomes (Figure 1). The MEN activator Lte1 localizes to the bud, the MEN inhibitor Kin4 localizes to the mother cell. Thus, the cell is divided into a MEN inhibitory zone in the mother cell, where Kin4 resides, and a MEN activating zone in the bud, where Lte1 resides (Figure 1). The MEN component carrying SPB functions as the sensor. Only when the MEN bearing SPB escapes the MEN inhibitor Kin4 in the mother cell and moves into the bud during anaphase where the MEN activator Lte1 resides can exit from mitosis occur. Thus, by targeting activators and inhibitors of signal transduction pathways to specific cellular locations spatial information is sensed and translated into a chemical signal. Determining how Kin4 and Lte1 are targeted to the mother cell and bud, respectively and how these proteins control MEN activity has been a recent focus of our lab. For example, we found that Lte1’s function is to inhibit Kin4 that leaks into the bud.
We also study the mechanisms that ensure that the MEN is only active during anaphase. We have identified a regulatory network, known as the FEAR network, that restricts MEN activity to anaphase. The FEAR network transiently activates Cdc14 during early anaphase. Cdc14 in turn dephosphorylates multiple MEN components foremost the GTPase effector kinase, thereby priming MEN activity. Dephosphorylation by Cdc14 allows the MEN to become fully active once its GTPase is delivered into the bud by the SPB. These findings have significant implications for GTPase signaling pathway regulation. They indicate that GTPase signaling pathways are not only regulated by the nucleotide binding state of their GTPases but that signal integration occurs at multiple steps in the signaling cascade. In the case of the MEN, a spindle position signal is sensed by the GTPases and anaphase onset by the effector kinase.
Importance of cellular shape and size for cell division. Every cell type has its characteristic shape and size. The importance of maintaining the correct cellular dimensions is not understood. We study how cell shape and size affects cell proliferation in budding yeast and mammals. We found that in yeast, increasing cell size without increasing genome copy number has a dramatic effect on cell proliferation and genome instability (Figure 2). This finding indicates that maintenance of a cell-type specific DNA to cytoplasm ratio is critical for cell function. Currently, we are investigating which aspects of cell proliferation and genome stability are affected by altering DNA to cytoplasm ratio. Remarkably, decreasing the DNA to cytoplasm ratio has the hallmarks of replicative aging. This observation raises the interesting possibility that increasing cell size, as occurs during replicative aging in yeast, is a major cause of aging at least in yeast.
Cell shape and polarity are also important for accurate cell division. Our studies in mammalian cells show that disruption of epithelial architecture and hence loss of cell shape and polarity decreases chromosome segregation fidelity (Figure 3). These findings indicate that cell non-autonomous determinants such as tissue architecture affect chromosome segregation. Understanding the molecular mechanisms underlying this regulation will not only be critical for understanding how chromosome segregation is controlled by tissue context but may also shed light on the origins of genome instability so frequently found in cancer. Loss of tissue architecture is a hallmark of solid tumors and could thus be a cause of chromosome instability in tumor cells.
Effects of Aneuploidy on Cell Physiology and Tumorigenesis
What happens to cells in which the mechanisms that ensure accurate chromosome segregation fail? How does the result of a chromosome mis-segregation event, known as aneuploidy, impact cells and organisms? In all organisms analyzed to date, aneuploidy is frequently associated with disease. In humans, aneuploidy is the leading cause of miscarriages and mental retardation. It is also a key characteristic of cancer. More than 90 percent of all solid human tumors are aneuploid. To begin to understand how aneuploidy causes diseases, we analyzed the effects of aneuploidy on normal cell physiology in yeast and in mammals.
We created two types of aneuploidy models. In one class of models, the constitutive models, cells harbor defined chromosome gains. In the other class, the chromosome instability models, cells harbor mutations that increase chromosome mis-segregation continuously creating random aneuploidies. Their analysis revealed that aneuploidy is deleterious at the cellular level, causing cell proliferation defects in both yeast and mammals. Perhaps most exiting was our discovery that aneuploid yeast and mammalian cells share a number of phenotypes known as the aneuploidy associated stresses that include but are not limited to proteotoxic and energy stress as well as genomic instability (Figure 4). Our studies further showed that these phenotypes are caused by changes in relative ratio of gene copy number. In aneuploid cells, changes in gene copy number largely, but not universally, cause a corresponding change in gene expression. These imbalances in gene expression cause the myriad of phenotypes observed in aneuploid cells including the aneuploidy-associated stresses. Currently, we are studying how aneuploidy elicits the aneuploidy associated stresses, focusing on the origins of proteotoxic stress and genomic instability. These studies will shed light on the origins of the pathological conditions associated with diseases caused by chromosome gains or losses such as Down Syndrome.
We also study how aneuploidy affects tumorigenesis. We have developed cell culture and mouse models to probe how defined aneuploidies as well as mutations that cause genomic instability affect oncogene induced tumorigenesis. Our results indicate that while aneuploidy interferes with tumor cell proliferation it appears, under some circumstances, to promote other aspects of the disease such as survival under sub-optimal conditions and chemotherapy resistance. Our findings that aneuploidy causes multiple forms of genomic instability further suggests that the gain or loss of whole chromosomes per se could fuel the evolution of the malignant state. Together, our findings led us to formulate the following hypothesis about the relationship between aneuploidy and tumorigenesis. Aneuploidy generally impairs cell proliferation but rare favorable variants exist allowing cells to survive especially under stressful conditions that cancer cells experience during metastasis or chemotherapy. Genomic instability caused by the aneuploid state allows these rare favorable variants to evolve quickly leading to the development of aggressive disease.
The observation that aneuploidy impairs rather than promotes cell proliferation postulates that cancer cells must overcome the adverse effects of aneuploidy in order to outgrow euploid cells and take advantage of potential benefits that arise from the aneuploid condition. We have begun to identify genetic alterations that suppress the proliferation defects of aneuploid yeast and mouse cells. In yeast, this approach identified mutations in the proteasomal degradation system. The characterization of the genes, which when inactivated improve the proliferative ability of specific or multiple different aneuploid yeast strains, will not only provide an understanding of the defects underlying the aneuploid condition but are likely to shed light on the evolution of tumors.
We are also exploring the possibility that aneuploid cancers have retained some of the vulnerabilities of the aneuploid state. With this hypothesis in mind we are identifying genetic alterations that exhibit synthetic lethality with the aneuploid state either by exaggerating the adverse effects of aneuploidy and/or by interfering with pathways essential for the survival of aneuploid cells. These genetic alterations could provide the basis for the development of new cancer treatments.
This work is supported in part by a grant from the National Institutes of Health and the Paul F. Glenn foundation for Medical research.
As of February 25, 2016