- ADJUNCT ASSOCIATE PROFESSOR Oncological Sciences
PhD, Institute of Cancer Research, University of London
Chester Beatty Laboratory
National Cancer Institute, National Institutes of Health
1996 - 2001
Irma T. Hirschl Career Scientist Award
ResearchThe 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.Members of the Ras subfamily of GTP-binding proteins, including Ras (H-, K-, and N-), TC21, and R-ras have been shown to display transforming activity, and activating lesions have been detected in human tumors. We have identified an additional member of the Ras gene family which shows significant sequence similarity to the human TC21 gene. This novel human ras-related gene, R-ras3, encodes for a protein of 209 amino acids, and shows approximately 60-75% sequence identity in the N-terminal catalytic domain with members of the Ras subfamily of GTP-binding proteins. An activating mutation corresponding to the leucine 61 oncogenic lesion of the ras oncogenes when introduced into R-ras3, activates its transforming potential. R-ras3 weakly stimulates the mitogen-activated protein kinase (MAPK) activity, but this effect is greatly potentiated by the co-expression of c-raf-1. By the yeast two-hybrid system, R-ras3 interacts only weakly with known Ras effectors, such as Raf and RalGDS, but not with RglII. In addition, R-ras3 displays modest stimulatory effects on trans-activation from different nuclear response elements which bind transcription factors, such as SRF, ETS/TCF, Jun/Fos, and NF-kappaB/Rel. Interestingly, Northern blot analysis of total RNA isolated from various tissues revealed that the 3.8 kilobasepair (kb) transcript of R-ras3 is highly restricted to the brain and heart. The close evolutionary conservation between R-ras3 and Ras family members, in contrast to the significant differences in its biological activities and the pattern of tissue expression, raise the possibility that R-ras3 may control novel cellular functions previously not described for other GTP-binding proteins.
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.Multiple biological functions have been ascribed to the Ras-related G protein R-Ras. These include the ability to transform NIH 3T3 fibroblasts, the promotion of cell adhesion, and the regulation of apoptotic responses in hematopoietic cells. To investigate the signaling mechanisms responsible for these biological phenotypes, we compared three R-Ras effector loop mutants (S61, G63, and C66) for their relative biological and biochemical properties. While the S61 mutant retained the ability to cause transformation, both the G63 and the C66 mutants were defective in this biological activity. On the other hand, while both the S61 and the C66 mutants failed to promote cell adhesion and survival in 32D cells, the G63 mutant retained the ability to induce these biological activities. Thus, the ability of R-Ras to transform cells could be dissociated from its propensity to promote cell adhesion and survival. Although the transformation-competent S61 mutant bound preferentially to c-Raf, it only weakly stimulated the mitogen-activated protein kinase (MAPK) activity, and a dominant negative mutant of MEK did not significantly perturb R-Ras oncogenicity. Instead, a dominant negative mutant of phosphatidylinositol 3-kinase (PI3-K) drastically inhibited the oncogenic potential of R-Ras. Interestingly, the ability of the G63 mutant to induce cell adhesion and survival was closely associated with the PI3-K-dependent signaling cascades. To further delineate R-Ras downstream signaling events, we observed that while a dominant negative mutant of Akt/protein kinase inhibited the ability of R-Ras to promote cell survival, both dominant negative mutants of Rac and Ral suppressed cell adhesion stimulated by R-Ras. Thus, the biological actions of R-Ras are mediated by multiple effectors, with PI3-K-dependent signaling cascades being critical to its functions.
Kimmelman A, Osada M, Chan AM. R-Ras3, a brain-specific Ras-related protein, activates Akt and promotes cell survival in PC12 cells. Oncogene 2000 Apr 13; 19(16): 2014-22.The GTP-binding protein, R-Ras3/M-Ras, is a novel member of the Ras subfamily of GTPases which shows highest sequence similarity to the TC21 gene. R-Ras3 is highly expressed in both human and mouse brain and ectopic expression of a constitutively active mutant of R-Ras3 induces cellular transformation in NIH3T3 cells. To gain further insight into the normal cellular function of R-Ras3, we examined the ability of R-Ras3 in activating several known intracellular signaling cascades. We observed that R-Ras3 is a relatively weak activator of the mitogen-activated protein kinase/extracellular-signal-regulated kinases (MAPK/ERKs) when compared to the H-Ras oncogene. On the contrary, both R-Ras3 and H-Ras activated the Jun N-terminal kinase (JNK) to a similar extent. Under similar experimental conditions, R-Ras3 significantly stimulated one of the phosphatidylinositol 3-kinase (PI3-K) downstream substrates, Akt/PKB/RAC (Akt), which has been extensively implicated in mediating cell survival signaling. The activation of Akt by R-Ras3 was most likely to be PI3-K-dependent since this biochemical event was blocked by the pharmacological inhibitors, Wortmannin and LY294002, as well as by a dominant negative mutant of PI3-K. More importantly, R-Ras3 affinity-precipitated PI3-K from cell extracts in a GTP-dependent manner, and associated lipid kinase activity was readily detectable in R-Ras3 immune complexes. The biological significance of R-Ras3 in inducing Akt kinase activity is evidenced by the ability of an activated R-Ras3 to confer cell survival in the rat pheochromocytoma cell line, PC12. As expected, this biological activity of R-Ras3 was also abrogated by the addition of LY294002. Thus, R-Ras3 represents a novel G-protein which may play a role in cell survival of neural-derived cells.
Tolkacheva T, Chan AM. Inhibition of H-Ras transformation by the PTEN/MMAC1/TEP1 tumor suppressor gene. Oncogene 2000 Feb 3; 19(5): 680-9.The human PTEN/MMAC1/TEP1 (PTEN) tumor suppressor gene encodes a phosphatase with specificity towards the D3 phosphate of phosphatidylinositides. PTEN mutations have been reported in the endometrioid type of uterine tumors which are associated with frequent activations of the Ras oncogenes. In this study, we report the ability of PTEN to potently inhibit H-Ras induced morphological transformation and anchorage-independent growth in NIH3T3 cells. This novel activity of PTEN was correlated more with its ability to suppress the phosphatidylinositol 3-kinase (PI3-K)-dependent signaling cascade, but not the mitogen-activated protein kinase (MAPK) pathway. To define the minimal region in PTEN protein that is responsible for this anti-oncogenic activity, a panel of carboxyl-terminal truncation mutants was generated. While deletions of 4 and 33 amino acids do not have marked effects, removal of up to 68 amino acids drastically reduced the ability of PTEN to inhibit Ras transformation. The propensity of these mutants to suppress Ras transformation is correlated with their relative ability to dephosphorylate inositol (1,3,4,5)-tetrakisphosphate in vitro, and to suppress Akt kinase activity in cultured cells. In addition, we have evidence to suggest that the C-terminal region of PTEN contributes to the stability of the encoded gene product.
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.
Solari F, Bourbon-Piffaut A, Masse I, Payrastre B, Chan AM, Billaud M. The human tumor suppressor PTEN regulates longevity and Dauer formation in Caenorhabditis elegans. Oncogene 2005; 24: 20-7.
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.
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