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Regulation of Growth in Normal Physiology and Disease
Summary: David Sabatini pursues two areas of research: the study of mammalian growth and how growth-related signaling pathways play a role in human diseases, and the development of new technologies to elucidate gene function in mammalian cells.
Since I began my lab I have been interested in the regulation of growth, the process through which organisms accumulate mass and increase in size. This broad topic can be tackled from many different angles. I chose to focus on the mammalian TOR (mTOR) pathway because of its emerging role as a central regulator of cell, organ, and organismal size and its deregulation in common human diseases such as cancer and diabetes. Our work on mTOR has yielded several surprising findings that have led us in directions I did not foresee a few years ago. Having started with work on mTOR signaling, we are now pursuing a diverse set of projects, including studies into the role of stem cells in solid tumors, of the liver in the physiological response to calorie restriction, and of energy and nutrient metabolism in cancer. In addition, we continue to participate in the development of new technologies, such as cell microarrays, pooled lentiviral RNAi (RNA interference) screening, and cell-based imaging.
During the past few years we discovered several new components of the mTOR pathway and their molecular and cellular functions. We now know that the mTOR protein kinase is the catalytic subunit of two distinct multiprotein complexes, called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). These complexes anchor interconnected signaling pathways that sense diverse signals, such as nutrients and insulin, to regulate many fundamental processes, including cell growth, survival, and division (see model in Figure 1). In ongoing efforts we have identified additional components of the pathway and are making progress toward understanding how mTORC1 and mTORC2 are activated and signal to downstream effectors.
One of the most interesting aspects of the mTORC1 pathway is that it is regulated by a variety of upstream signals. Many of these stimuli signal through the tumor-suppressor complex (TSC1/TSC2), but this is not the case for amino acids. We have identified the Rag family of GTPases as mTORC1-interacting proteins that are necessary and sufficient for signaling amino acid sufficiency to mTORC1. The Rag proteins affect mTORC1 signaling in an unexpected way. They do not directly stimulate the kinase activity of mTORC1 but rather mediate its translocation, in an amino acid–dependent fashion, to an intracellular compartment that contains the well-established mTORC1 activator, Rheb. This is an elegant system for cells to prevent mTORC1 activation in the absence of amino acids, the building blocks of much of the cellular mass. Even if cells growing in the absence of amino acids are exposed to progrowth stimuli, such as insulin, they cannot activate mTORC1 because it is not in the correct compartment to receive such stimuli.
We are also using our insights into mTOR signaling to study normal physiology in vivo in mice. For example, by activating mTORC1 in vivo under conditions when the pathway should be off, we are learning about the role of organ growth in the physiological response to calorie restriction.
Since my training as an M.D./Ph.D. student, I have wanted my research to have an impact on the understanding and treatment of human disease. With this goal, I am studying the roles of mTORC1 and mTORC2 in tumorigenesis, basing the work on our understanding of their biochemical functions within cells. A number of years ago we discovered that mTORC2 is the elusive "hydrophobic motif kinase" of Akt and thus a positive regulator of the PI3K (phosphatidylinositol 3-kinase)/Akt pathway (Figure 1), a signaling system that is hyperactive in cancer cells missing the PTEN tumor suppressor. Human tumors frequently have mutations that cause hyperactivation of PI3K and its downstream effector, Akt. For example, inactivation of PTEN, the main negative regulator of the PI3K/Akt pathway, occurs in up to 50 percent of prostate cancers and 60 percent of endometrial cancers. mTORC2-mediated phosphorylation of Akt is necessary for maximal but not basal Akt activity, suggesting that mTORC2 inhibition may selectively impair cells that depend on hyperactive PI3K/Akt signaling, such as those missing PTEN. Consistent with this, loss of mTORC2 function does not reduce the proliferation of primary fibroblasts in culture but does eliminate the capacity of PTEN-null prostate cancer cells to form tumor xenografts in mice. To expand on this finding, we generated mice in which rictor, a key component of mTORC2, is deleted in the prostate at the same time as PTEN. Our results indicate that mTORC2 function is necessary for the formation of invasive prostate adenocarcinoma, but not for the biogenesis of the normal prostate. To validate these results and generate tools to inhibit mTORC2 after mice already develop prostate cancer, we are collaborating with Nathanael Gray (Dana-Farber Cancer Institute) to identify small molecules that inhibit mTORC2.
There are a number of reasons to think that energy and nutrient metabolism in cancer cells in a solid tumor is likely to be different than in normal cells and thus a potential point of attack for cancer therapy. First, during the initiation and growth of a solid tumor, regions of the tumor may be poorly vascularized and thus lack the oxygen and nutrients necessary for normal cells to live. To survive, tumor cells must adapt their energy demands and intermediary metabolism to their environment. Second, the deranged signaling common to cancer cells can alter the expression and activity of metabolic enzymes, affecting metabolic processes in ways that may not occur in normal cells. Third, many cancer cells grow and proliferate at rates far higher than most other cells in an adult, creating a demand for the building blocks of macromolecules that is not shared by most normal cells. For these reasons, it is surprising that the literature contains relatively little information on the metabolic processes, besides glycolysis, that are necessary for tumor cell life.
We are undertaking efforts to identify systematically and understand the functions of the metabolic processes that are essential in tumor cells with common cancer-causing mutations. Our approach is to identify, in an RNAi-based loss-of-function screen of all the human metabolic genes, those genes that are necessary for cancer cells to survive in standard tissue culture conditions and in conditions that mimic oxygen-deprived regions of a tumor. This approach is possible because, as part of a consortium of labs in the Boston area, we generated a lentiviral RNAi library targeting all human genes and the methods for screening it in an arrayed high-throughput fashion. Using these libraries, we are also pursuing a number of additional questions. For example, in collaboration with William Hahn (Dana-Farber Cancer Institute) and David Root (Broad Institute), we are undertaking screens to identify genes necessary for brain cancer stem cells to maintain their stemness properties.
During the past few years our laboratory has also been developing additional technologies to systematically screen for gene function in mammalian cells. Our work first led to a "reverse transfection" system that generates microarrays of mammalian cells expressing defined cDNAs. In this system, cells are cultured on a glass slide printed in known locations with nanoliter volumes of a hydrogel containing cDNAs in expression plasmids. The cells that land on the spots printed with the DNAs take up the plasmids to form clusters of 80–200 cells that express the proteins encoded by the plasmids. Because each spot is only 150–250 μm in diameter, we can print 5,000–10,000 such spots on one standard microscope slide. More recently we have developed analogous systems in which we create microarrays of purified lentiviruses instead of cDNAs. Successful development of this latter system will allow us to screen our existing lentiviral RNAi libraries in the cell microarray format.
This research has been supported by the National Institutes of Health, W.M. Keck Foundation, Rita Allen Foundation, Pew Scholars Program, Department of Defense, Whitehead Institute for Biomedical Research, Broad Institute, and David H. Koch Institute for Integrative Cancer Research at MIT.
Last updated October 15, 2008
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