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Regulation of MAP kinase signaling pathway in lung cancer

Institution: University of California, San Diego
Investigator(s): Renate Pilz, M.D.
Award Cycle: 2000 (Cycle 9) Grant #: 9IT-0069H Award: $84,671
Subject Area: Cancer
Award Type: Inno Dev & Exp Awards (IDEAS)

Initial Award Abstract
Lung cancer is the leading cause of cancer-related deaths for both men and woman in the United States and the majority (>80%) of lung cancers are attributable to tobacco smoke; overall one in nine smokers develop lung cancer in his or her lifetime. In the majority of patients, the lung cancer is detected at a stage when it has already spread to other organs by metastasis and cannot be cured by surgery. Unfortunately, most lung cancers are quite resistant to conventional chemotherapy agents, and better therapies are urgently needed.

There are two major kinds of smoking-induced lung cancers, called small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which develop from different types of lung cells and behave differently with respect to growth characteristics and spread in the patient. Growth and spread of both types of lung cancer is dependent on certain substances called growth factors; the cancer cells frequently produce their own growth factors which bind to the cell’s surface to stimulate growth. Growth factors send signals from the cell’s surface to the nucleus (the internal “headquarters” of the cell) to switch on growth-related programs. This signal transduction from the cell surface to the nucleus is accomplished by a number of signal transduction pathways which are often composed of many proteins called protein kinases. One such pathway, which we propose to study in detail, is called the mitogen- (or growth factor-) activated protein kinase (MAPK) pathway. Activation of this pathway by growth factors is necessary for SCLC and NSCLC cells to grow. Our project focuses on a small messenger molecule called cyclic AMP (cAMP) which can be made in response to drugs used for the treatment of common lung diseases such as asthma. This molecule can interfere with signal transduction through the MAPK pathway and can thereby inhibit cancer cell growth. We will study the mechanism by which cAMP regulates the MAPK pathway and inhibits cell growth in SCLC and NSCLC cells. These studies should help our understanding of how lung cancer cells use the MAPK pathway to grow and how one can interrupt this pathway to develop novel approaches for lung cancer treatment. Indeed, several different classes of drugs which block this pathway by different mechanisms are under development by the pharmacological industry and some show promising results in early clinical trials of cancer therapy.

Final Report
The mitogen-activated protein kinase (MAPK) pathway (including Ras or Rapl, Raf-1 or B-Raf and the MAPKs Erk-1 and Erk-2) is involved in the regulation of cell growth, differentiation and apoptosis of both small cell and non-small cell lung cancers (SCLC and NSCLC). We found that CAMP analogs inhibited MAPK activity in NSCLC, but CAMP treatment caused a prolonged increase in NIAPK activity in SCLCa; in both cell types, CAMP inhibited proliferation. The specific aims of the grant were: (i) to examine the effect of CAMP on basal and growth-factor-induced activities of Ras, Rap-1, Ra£ 1, B-Raf and Erk-1,2 in SCLC and NSCLC, to correlate the effect of CAMP on the MAPK pathway with the effect of CAMP on cell growth and viability, and to determine whether the MAPK pathway mediates the effects of CAMP on cell growth and viability; (ii) to examine the effect of 14-3-3 proteins on basal and growth factor-stimulated B-Raf and Raf-1 activity and regulation by CAMP in SCLC and NSCLC; and (iii) to examine the effect of activated Rapl on the activities of Raf-1, B-Raf and Erk-1,2 and on the growth and viability of SCLC and NSCLC cells.

During the first year of this exploratory grant, we completed the first specific aim. We found that Rap-1 is activated by CAMP in both SCLC and NSCLC cells, whereas Ras activity is not altered by CAMP. B-Raf kinase is activated by CAMP in SCLC but inhibited in NSCLC, which correlates with the effect of CAMP on Erk-1 and Erk-2 activity; in both cell types Raf-1 is inhibited by CAMP. Using different types of CAMP analogs, we found that the growth-inhibitory effects are variable, cell type-specific, and correlate more closely with the effect on A-kinase isoforms than with the effect on the MAPK pathway. For the other two specific aims, we had to optimize transfection efficiencies of SCLC and NSCLC cell lines. Transient transfection efficiencies were not high and reproducible enough to examine the interaction of B-Raf and 14-3-3 proteins, and our attempts to select stable transfectants expressing activated Rap l and the Rap l regulator A-GEF were initially unsuccessful because of negative effects of the transgenes on cell survival; however, work is continuing to establish stable lines using an inducible expression system.

The experiments have increased our understanding of MAPK pathway regulation in lung cancer, which is of particular importance as more pharmacologic agents which inhibit the Ras/MAPK pathway are being developed and enter clinical trials. Since about 80% of NSCLs and most of SCLCs are attributable to smoking, the work was directly relevant to tobacco-related diseases.

Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Qui W, Zhuang S, von Lintig FC, Boss GR, Pilz RB ART
Yr: 2000 Vol: 275 Nbr: Abs: Pg: 31921-31929

Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Qui W, Zhuang S, von Lintig FC, Boss GR, Pilz RB ART
Yr: 2000 Vol: 275 Nbr: Abs: Pg: 31921-31929