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Post-isoprenylation processing steps as anticancer targets

Institution: J. David Gladstone Institutes
Investigator(s): Stephen Young, M.D.
Award Cycle: 2000 (Cycle 9) Grant #: 9RT-0243 Award: $1,017,450
Subject Area: Cancer
Award Type: Research Project Awards
Abstracts

Initial Award Abstract
A large number of human cancers, including tobacco-related cancers, have been linked to mutations in genes that code for the Ras proteins. Ras proteins are located along the inner surface of the cell and transmit growth factor signals, triggering the proliferation of cells. Ras proteins can be activated by specific mutations, leading them to deliver growth signals to the nucleus in an unregulated fashion. Many human cancers contain mutant Ras proteins, and these are thought to be important in the development and progression of cancer.

After they are synthesized in the cells, the Ras proteins (and other related proteins) undergo three distinct processing steps. First, a specific lipid is added to the Ras protein (on a amino acid near the end of the Ras molecule). That process is termed isoprenylation. Following isoprenylation, there are two more protein processing steps (the “post-isoprenylation” processing steps). The last three amino acids of the Ras protein are clipped off of the isoprenylated protein (the endoproteolytic processing step). Finally, the last amino acid of the Ras protein is modified by the addition of a single-carbon methyl group (the carboxyl methylation step). All of these processing steps are carried out by specific cellular enzymes. Each of these three processing steps is essential for the proper localization and functioning of the Ras proteins within the cell.

The frequent involvement of abnormal forms of Ras in human cancers has prompted efforts to design drugs that interfere with the processing of the Ras proteins. For example, drugs that block the addition of the lipid to the Ras proteins have been developed. In experimental animals with Ras-induced cancers, these drugs retard the growth of tumors, and even cause remission of the tumors.

The objective of this project is to assess the potential of two new targets for anti-cancer drug therapy: the endoproteolytic processing step and the carboxyl methylation step. We have recently cloned the genes responsible for both the endoproteolytic processing step (Rce1) and the carboxyl methylation step (Icmt). We have gone on to generate mice lacking these genes. Mice lacking Rce1 die during embryonic development. However, studies with Rce1-deficient cells revealed that the nonprocessed Ras proteins are trapped inside the cell and do not reach their proper location at the surface of the cell. We have produced heterozygous Icmt-deficient mice but will have to wait 3 more months to determine whether the homozygous Icmt knockout mice survive embryonic development and are viable. In the meantime, however, we have been successful in generating cell lines that completely lack Icmt. Interestingly, the Ras proteins are similarly mislocalized in cells lacking Icmt. The mislocalization of the Ras proteins in the setting of absent endoproteolysis and absent methylation has buoyed our hopes that the enzymes carrying out these processing steps might be attractive targets for the treatment of tobacco-related malignancies.

During the next 3 years, we plan to use a variety of experimental approaches to determine whether the absence of endoproteolytic processing or carboxyl methylation would retard the growth of Ras-induced cancers. We have already used newer experimental techniques to produce viable adult mice that lack Rce1 in certain tissues. We have also been able to excise the Rce1 gene from the mouse cells that are actively growing in culture. We will determine whether the elimination of Ras endoproteolytic processing retards the growth of Ras-transformed cells in cell culture or the growth of Ras-induced cancers in mice. We also plan to use both cell culture and whole-animal experimental approaches to assess the impact of carboxyl methylation on cancer cell growth. We are optimistic that our experiments will address, in a definitive fashion, whether the elimination of the endoproteolysis or carboxyl methylation steps influences the growth of Ras-induced tumors. If tumor growth is retarded in the setting of these enzyme deficiencies, then those enzymes would become very attractive targets for the pharmacological inhibition of cancer cell growth. Our ultimate hope is that inhibitor drugs that would be useful in the treatment of lung cancers and other tobacco-related cancers.
Publications

Targeted inactivation of the isoprenylcysteine carboxyl methyltranferase gene causes mislocalization of a green fluorrescent protein-K-Ras4B fusion in mammalian cells
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Bergo MO, Leung GK, Otto JC, Casey PC, Young SG ART
Yr: 2000 Vol: 275 Nbr: Abs: Pg: 17605-17610

Isoprenylcysteine carboxyl methyltransferase deficiency in mice
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Bergo MO, Leung GK, Ambroziak P, Gomes AR, Seabra MC, Young SG ART
Yr: 2000 Vol: 276 Nbr: Abs: Pg: 5841-5845

The c-terminal polylysine domain and methylation of K-Ras are critical for the interaction between K-Ras and microtubules
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Chen Z, Otto JC, Bergo MO, Young SG, Casey PJ ART
Yr: 2000 Vol: 275 Nbr: Abs: Pg: 41251-41257

Biochemical studies of zmpste24-deficient mice.
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Leung GK, Schmidt WK, Bergo MO, Gavino B, Wong DH, Tam A, Ashby MN, Michaelis S, Young SG ART
Yr: 2001 Vol: 276 Nbr: Abs: Pg: 29051-29058

Absence of the CAAX endoprotease Rce1: Effects on cell growth and transformation.
Periodical: Molecular Biology of the Cell Index Medicus:
Authors: Bergo MO, Ambroziak P, Gregory C, George A, Otto JC, Kim E, Nagase H, Casey PJ, et al ART
Yr: 2002 Vol: 22 Nbr: Abs: Pg: 171-181

Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and golgi.
Periodical: Molecular Biology of the Cell Index Medicus:
Authors: Michaelson D, HAhearn I, Bergo M, Young S, Philips M ART
Yr: 2002 Vol: 13 Nbr: Abs: Pg: 3294-3302

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: 2003 Vol: 100 Nbr: Abs: Pg: 6529-6534

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

On the physiological importance of endoproteolysis of CAAX proteins: heart-specific Rce1 knockout mice develop a lethal cardiomyopathy.
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Bergo MO, Lieu HD, Gavino BJ, Ambroziak P, Otto JC, Casey PJ, Walker QM, Young SG ART
Yr: 2004 Vol: 279 Nbr: Abs: Pg: 4729-4736