Andrew M Chan, PhD
- ADJUNCT ASSOCIATE PROFESSOR | Oncological Sciences
PhD, Institute of Cancer Research, University of London
National Cancer Institute, National Institutes of Health
Irma T. Hirschl Career Scientist Award
The Ras family of GTP-binding proteins, H-, N-, and K- ras, are frequently mutated in a large fraction of human tumors. Members of this class of small G- proteins are localized to the inner surface of the plasma membrane where complex signals are initiated and channeled into multiple signaling cascades in a highly coordinated fashion. Dysregulation of the Ras oncoproteins and the constitutive activation of downstream signaling events have been implicated in the genesis of certain human tumors. \r\n\r\n\r\n\r\n
One of the major kinase cascades controlled by Ras is the phosphatidylinositol 3-kinase (PI3-K)-dependent signaling pathway. Over the years, we have identified several related members of the RAS gene family, TC21, R-Ras, and R-Ras3, (1) which possess readily detectable transforming activity. These novel Ras-related oncogenes, together with the prototypic Ras utilize the PI3-K-dependent signaling pathways for their biological actions. More recently, the human PTEN tumor suppressor gene have been demonstrated to be a phosphatidylinositol-3-phosphate phosphatase. These observations imply that PTEN serves as a negative regulator of the PI3-K pathway and potentially impinges on the oncogenicity of Ras. Based on all these observations, our major goal is to elucidate the role of various Ras-related oncogenes in normal cell physiology. Also, we would like to delineate the interplay between Ras and PTEN in controlling tumor progression.\r\n\r\n
The three major areas of research are:\r\n\r\n
1. Signaling mechanisms responsible for R-Ras in promoting cellular transformation, adhesion and survival
\r\nThe Ras-related G-protein, R-Ras, has been shown to regulate cell adhesion by activating the affinity state of surface-bound adhesion molecules, integrins. In addition, R-Ras has been demonstrated to protect cells from programmed cell death (apoptosis) upon the withdrawal of vital growth factors or cytokines. Dysregulation of these functions in primary tumors has been implicated in the progressive transition to the malignant state. R-Ras, in sharp contrast to the prototypic Ras, is a weak activator of the mitogen-activated protein kinase (MAPK) pathway. However, its preferential activation of phosphatidylinositol 3-kinase (PI3-K)-dependent signaling cascades may play a crucial role in physiological states whereby adhesion and survival are preferred over proliferation (3). For example, R-Ras may be a critical factor in the maintenance of cells in their quiescent state during differentiation and senescence. Physiologically, the loss of cell adhesion in cultured epithelial cells and the associated cell death caused by anoikis are well documented. In addition, cell attachment to substratum is prerequisite for cell cycle progression from G1 to S phase. On the contrary, cells undergoing mitosis are usually characterized by cell rounding and a reduction in cell adhesion. To this date, the physiological functions of R-Ras still remained elusive. To address all these questions, mammalian cell lines with homozygous deletion of R-Ras alleles will be generated by the somatic cell knockout techniques. These cells lines will be valuable for assessing the role of R-Ras in cell adhesion during cell cycle, as well as responses to stimulations from both mitogenic and differentiation factors. In addition, somatic cell knockout of R-Ras in human metastatic tumor cell lines will provide us with interesting information regarding the role of this Ras-related G-protein in tumor dissemination.\r\n\r\n\r\n\r\n
2. Characterization of a brain-specific Ras-related G-protein, R-Ras3
\r\nR-Ras3 is a novel member of the Ras gene superfamily being expressed predominantly in the brain (2). Thisis in striking contrast to other Ras family members that show a ubiquitous pattern of expression. The major goal of this project is to test the hypothesis that diversity in G-protein family confers specificity in complex biological processes. By in situ hybridization, R-Ras3 transcripts were localized in abundance to the hippocampus and Purkinje cell layer of the rat brain. These data suggest that R-Ras3 may serve as a signaling molecule for such important biological processes as memory and motor functions. Initial studies have indicated that R-Ras3 efficiently induces neuronal differentiation in a rat phreochromocytoma cell line PC12. In addition, the ectopic expression of R-Ras3 in the same cell line protects it from cell death caused by the withdrawal of the trophic agent, nerve growth factor (NGF) (5). One working hypothesis is that R-Ras3 either acting alone or together with Ras in controlling neuronal differentiation and survival in the developing brain. It is possible that R-Ras3 and Ras response to specific extracellular neurotrophic factors and propagate signals for growth, differentiation, and survival. Efforts are initiated to search for neurotrophic factors that could specifically activate R-Ras3 but not Ras in selected neuronal cell lines. Independently, work has been initiated in the construction of mouse strains with homozygous deletion of the R-Ras3 gene locus. Altogether, these studies will contribute to the understanding of the role of Ras-related G-proteins in the development of the CNS.\r\n\r\n\r\n\r\n
3. Regulation of the human PTEN tumor suppressor protein in normal and tumor cells.
\r\nThe human PTEN tumor suppressor gene was isolated based on its localization to chromosome 10q23, a region frequently deleted in multiple types of human cancer. As expected, inactivating lesions in the PTEN gene are frequently found in the advanced stage of brain, breast, prostate, and skin cancer. Biochemically, PTEN encodes a phosphatase with specificity towards the D3 phosphate of phosphatidylinositides. This finding implies that PTEN serves as a negative regulator of the PI3-K signaling cascade. \r\n\r\n \r\n\r\n
The fact that the biological actions of most of the G-proteins being studied in our laboratory are PI3-K dependent, prompted us to investigate the potential interaction between Ras and PTEN in human cancer. This is of particular relevance since PTEN mutations have been reported in the endometrioid type of uterine tumors, which are associated with frequent activation of the K-Ras oncogene. Indeed, we have shown that PTEN potently inhibits H-Ras induced morphological transformation and anchorage-independent growth in NIH3T3 cells (4). This novel activity of PTEN is correlated more with its ability to suppress the PI3-K-dependent signaling cascade, but not the MAPK pathway. \r\nBy using a panel of truncation mutants, we have shown that the C-terminal 33 amino acids of PTEN are dispensable for the phosphatase activity as well as for suppressing Ras transformation. However, further truncations give rise to rapid degradation of the resulting molecules as manifested by their low levels of protein expression in cells. These data suggest that the C-terminal region of PTEN is required for the maintenance of protein stability. The presence of premature terminated PTEN gene products with presumably shorter half-life in human tumors may constitute a novel mechanism for the loss of tumor suppressor function. \r\n
Our future goal is to elucidate the regulation of PTEN functions in normal verses human tumor cells. We speculate that the C-terminal region of PTEN by interacting with regulatory proteins, play an important role in controlling PTEN biochemical functions, protein stability, and subcellular localization. The identification of binding partners, the elucidation of post-translational modifications of PTEN, and a detailed analysis of PTEN subcellular distribution are currently being studied.
Kimmelman A, Tolkacheva T, Lorenzi MV, Osada M, Chan AM. Identification and characterization of R-ras3: a novel member of the RAS gene family with a non-ubiquitous pattern of tissue distribution. Oncogene 1997 Nov 27; 15(22): 2675-85.
Osada M, Tolkacheva T, Li W, Chan TO, Tsichlis PN, Saez R, Kimmelman A, Chan AM. Differential roles of Akt, Rac, and Ral in R-Ras-mediated cellular transformation, adhesion, and survival. Mol Cell Biol 1999 Sep; 19(9): 6333-44.
Narla G, Heath K, Reeves H, Li D, Giono LE, Narla J, Glucksman M, Kimmelman A, Eng FJ, Chan AM, Ferrari A, Martignetti J, Friedman SL. Kruppel-like factor 6, a candidate tumor suppressor gene mutated in prostate cancer. Science 2001; 294: 2563-65.
Tolkacheva T, Boddapati M, Sanfiz A, Tsuchida K, Kimmelman AC, Chan AM. Regulation of PTEN binding to MAGI-2 by two putative phosphorylation sites at threonine 382 and 383. Cancer Research 2001 Jul; 61(13): 4985-89.
Chan TO, Rodeck U, Chan AM, Kimmelman AC, Rittenhouse SE, Panayotou G, Tsichlis PN. Small GTPases and tyrosine kinases co-regulate a molecular switch in the phosphoinositide 3-kinase regulatory subunit. Cancer Cells 2002 Mar; 1(2): 181-91.
Kimmelman AC, Nunez-Rodriguez N, Chan AM. R-Ras3/M-Ras induces Differentiation of PC12 cells through a Cell-Type Specific Activation of the Mitogen-Activated Protein Kinase. Mol. Cell. Biol. 2002 Aug; 22(16): 5946-61.
Kimmelman AC, Qiao RF, Narla G, Sanfiz A, Bos P, Banno A, Nunes-Rodriguez N, Lau N, Li D, Eng FJ, Beaven S, Benzeno S, Liang BC, Guha A, Martignetti JA, Friedman SL, Chan AM. Suppression of Glioblastoma Tumorigenicity by the Kruppel-like Transcription factor, KLF6. Oncogene 2004; 23: 5077-83.
Nunez Rodriguez N, Lee IN, Banno A, Qiao HF, Qiao RF, Yao Z, Hoang T, Kimmelman AC, Chan AM. Characterization of R-ras3/m-ras null mice reveals a potential role in trophic factor signaling. Mol Cell Biol 2006 Oct; 26(19): 7145-54.