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Enzymatic targets for anticancer therapy

Institution: J. David Gladstone Institutes
Investigator(s): Martin Bergo, M.D., Ph.D.
Award Cycle: 2002 (Cycle 11) Grant #: 11KT-0087 Award: $267,304
Subject Area: Cancer
Award Type: New Investigator Awards
Abstracts

Initial Award Abstract
Smoking tobacco is causally related to the development of several different types of cancer. Many types of cancers, including those caused by tobacco use, contain mutations leading to the activation of one of the Ras proteins. The Ras proteins are located along the inner surface of the plasma membrane and transmit signals from growth factor receptors to the cell nucleus, leading to the transcription of genes involved in cell growth. Activated Ras proteins transmit these growth signals in a continuous and uncontrolled fashion, leading to the development of cancer.

Controlling the unregulated Ras activity in Ras-induced cancers would be highly desirable because it would offer a potential way to treat these cancers. One potential method of interfering with Ras activity is to block the enzymes that normally modify the Ras proteins. The enzymatic modification of Ras proteins is important in allowing these proteins to reach their proper location along the plasma membrane.

After synthesis the Ras proteins undergo three sequential processing events. First, a lipid (prenyl group) is added to a cysteine amino acid residue four amino acids from the end of the protein. Second, the last three amino acids of the protein are clipped off. Third, the newly exposed, lipidated cysteine is modified with a methyl group by an enzyme called isoprenylcysteine carboxyl methyltransferase (Icmt). Each of these modifications assists in directing the Ras proteins to membrane surfaces within cells. The anchoring of the Ras proteins to the plasma membrane is crucial for their function.

Pharmaceutical drugs that block one of the “lipidation” enzymes (farnesyltransferase, or FTase) interfere with the membrane targeting of Ras proteins and interfere with Ras function. Unfortunately, K-Ras (a version of Ras most often implicated in human cancers) can be lipidated by another enzyme, raising doubts about whether those FTase inhibitor drugs will actually be effective. Remarkably, no one has ever used mouse genetic models to define the biological consequences of FTase deficiency. During the next 3 years, one goal of my project will be to define the consequences of complete absence of the FTase enzyme. I will generate genetically modified mice that harbor a conditional knockout version of the gene that encodes FTase. The development of this mutant mouse will make it possible to excise (knockout) the gene for FTase in specific tissues or in cells from the mice. Excision of the FTase gene will allow me to determine whether FTase deficiency has a significant impact on the growth of K-Ras-induced tumors.

A second goal of my project will be to explore the possibility that the absence of Icmt activity will result in a significant downregulation of Ras function and inhibition of Ras-induced tumors. During my postdoctoral fellowship, I generated mice and cell lines lacking the Icmt gene. The Ras proteins were profoundly mislocalized in Icmt-deficient cells, and only a very small percentage of the Ras proteins reached their proper location along the plasma membrane. Icmt-deficient fibroblasts displayed a substantial growth defect. Preliminary experiments have indicated that Icmt-deficient cells are resistant to cancerous growth by activated forms of the Ras proteins. These preliminary results were extremely intriguing. To pursue this issue further, I plan to generate another mutant version of the Icmt gene, one that will allow me to excise the Icmt gene from living cells and tissues, similar to the one described for FTase above. This experimental approach will make it possible to compare phenotypes of “normal Icmt expression” and “Icmt-deficiency” and to do so in the very same cell line. These gene-excision experiments will allow me to more precisely define the effects of Icmt deficiency on cancerous cell growth. Ultimately, my work could lead to the development of new ways to treat tobacco-related cancers.
Publications

Targeting Ras signaling through inhibition of carboxyl methylation: an unexpected property of methotrexate.
Periodical: Proceedings of the National Academy of Sciences of the United States of America Index Medicus:
Authors: Winter-Vann AM, Kamen BA, Bergo MO, Young SG, Melnyk S, James SJ, Casey PJ ART
Yr: 0 Vol: Nbr: Abs: Pg:

Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf.
Periodical: Journal of Clinical Investigation Index Medicus:
Authors: Bergo MO, Gavino BJ, Hong C, Beigneux AP, McMahon M, Casey PJ, Young SG ART
Yr: 2004 Vol: 113 Nbr: Abs: Pg: 539-550

On the physiologic importance of endoproteolysis of CAAX proteins: heart-specific Rcel knockout mice develop a lethal cardiomyopathy.
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Bergo MO, Lieu HD, Otto JC, Casey PJ, Walker Q, Young SG ART
Yr: 2004 Vol: 279 Nbr: Abs: Pg: 4729-4736

Carboxyl-terminal interactions of lamin B1 are dependent on Rcel-mediated endoproteolysis and subsequent methylation.
Periodical: Journal of Cell Biology Index Medicus:
Authors: Maske C, Hollinshead M, Higbee NC, Bergo MO, Young SG, Vaux DJ ART
Yr: 2003 Vol: 162 Nbr: Abs: Pg: 1223-1232

Methotrexate targets Ras signaling through inhibition of carboxyl methylation.
Periodical: Proceedings of the National Academy of Sciences of the United States of America Index Medicus:
Authors: Winter-Vann AM, Kamen BA, Bergo MO, Young SG, Melnyk S, James SJ, Casey PJ ART
Yr: 2003 Vol: 100 Nbr: Abs: Pg: 6529-6534

Prenylcysteine lyase deficiency in mice results in the accumulation of farnesylcysteine and geranylgeranylcysteine in brain and liver.
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Beigneux A, Withycombe SK, Digits JA, Tschantz WR, Weinbaum CA, Griffey SM, Bergo M, et al ART
Yr: 2002 Vol: 277 Nbr: Abs: Pg: 38358-38363

Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.
Periodical: Proceedings of the National Academy of Sciences of the United States of America Index Medicus:
Authors: Bergo MO, Gavino B, Ross J, Schmidt WK, Hong C, Kendal LV, Mohr A, Meta M, Genant H ART
Yr: 2002 Vol: 99 Nbr: Abs: Pg: 13049-13054