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Novel regulatory mechanisms for lung cancer growth

Institution: University of California, San Diego
Investigator(s): Randolph Hastings, M.D., Ph.D.
Award Cycle: 2000 (Cycle 9) Grant #: 9RT-0037 Award: $100,000
Subject Area: Cancer
Award Type: Research Project Awards
Abstracts

Initial Award Abstract
Parathyroid hormone-related protein (PTHrP) is a protein that was discovered in the blood of a patient with lung cancer. PTHrP is responsible for causing high levels of calcium in the blood of such patients and patients with other types of cancer. PTHrP is made by the lung cancer cells and has been detected in cells from all of the types of lung cancer. Many investigators have studied the role of PTHrP in causing elevated blood calcium levels in cancer patients, but PTHrP may also have other effects. For example, PTHrP stimulates many lung cancer cells to grow and proliferate. Thus, PTHrP produced by the cells might stimulate the growth of lung tumors or spread of the cancer to other parts of the body. This project fits with the research priorities of the TRDRP because it will study the processes important in growth of lung cancer, a disease caused by tobacco use or exposure, and might lead to development of new treatments for lung cancer. The first aim is to learn whether PTHrP stimulates proliferation of lung cancer cells by being released from the cells and acting at sites on the cell surface, or by being transported inside the cell to the nucleus, the site containing the cell’s DNA. This knowledge is important for understanding the cancer disease process and will be significant for designing therapies related to PTHrP. The experiments will involve genetically modifying lung cancer cells to make PTHrP that is released from the cell or PTHrP that is transported to the nucleus. The different cells will be grown in plates and the rate of cell proliferation will be measured and compared. The second aim is to learn whether PTHrP stimulates lung tumors to grow in the same way it stimulates the cancer cells in culture plates to grow. Tumors will be formed by implanting the cells into the lungs of mice that have compromised immune systems and cannot reject the foreign cells. The cells will be modified as described above to make PTHrP that is either released from the cells or transported to the nucleus. The cells will also be modified to be fluorescent. After six weeks, lung tumor growth will be evaluated by inspecting the lungs, measuring the fluorescence, or examining the lungs microscopically. The third aim is to determine if lung tumor growth can be slowed by treatments that reduce the effects of PTHrP on lung cancer cells. Using an animal model, we will contrast the effectiveness of antibodies against PTHrP, drugs that counter the effects of PTHrP, or substances that prevent the cells from making PTHrP. Lung tumor growth will be measured as described for the second aim. The results of these studies will improve our understanding of how lung cancer grows, and may lead to new therapies for lung cancer.

Final Report
Parathyroid hormone-related protein (PTHrP) is a protein that was discovered in the blood of a patient with lung cancer. PTHrP was responsible for causing high levels of calcium in the blood of this patient and has been implicated in causing similar problems in many patients with lung cancer and other types of cancer. PTHrP is made by lung cancer cells and has been detected in cells from all of the types of lung cancer. Many investigators have studied the role of PTHrP in causing elevated blood calcium levels in cancer patients, but PTHrP may also have other effects. For example, PTHrP stimulates many lung cancer cells to proliferate. Thus, PTHrP production might stimulate the growth of lung tumors and could be important in progression of the cancer and metastasis. The overall goal of this project was to develop and test several techniques that could be used to study the effects of PTHrP on growth of lung cancer cells and progression of lung cancer. The specific aims were (1) to clone genes, known as plasmids, that could be introduced into lung cancer cells to cause them to produce various forms of PTHrP; (2) to introduce these plasmids into lung cancer cells (called transfecting) to obtain cell lines that produce these forms of PTHrP; (3) to characterize the form of PTHrP produced by these cell lines; and (4) to test whether the cell lines can form lung tumors in mice with compromised immune systems.

Progress has been made in all four aims. For the first aim, we have now constructed over 30 plasmids that code for different forms of PTHrP. Many of the PTHrP forms are shortened versions of the molecule. Various parts of the protein that may be important for the effects of PTHrP in lung cancer cells have been removed. In other forms, mutations have been introduced that may alter the effects of PTHrP forms. Alternatively, some of the forms have been changed so that they cannot be released from the cell like normal PTHrP. Finally, some of the forms have been modified so they are fluorescent. The fluorescence will help us track the PTHrP to the part of the cell where it has its effect. For the second specific aim, we have successfully introduced some of the PTHrP forms into A549 lung cancer cells and BEN lung cancer cells and all of the forms into COS cells. The COS cells do not make PTHrP on their own, and are useful in studying how well the plasmids work and whether they are producing the PTHrP form that we expect. We achieved the third aim by determining the DNA base sequence of the plasmids compared to the expected sequence and by measuring the PTHrP produced by the COS cells with assays that are specific for small portions of the PTHrP molecule. For example, COS cells transfected with only the first half of the PTHrP molecule made PTHrP that could be detected with an assay specific for the first half of the molecule, but PTHrP levels were not detectable with an assay for the second half. We also characterized where the PTHrP was found in the cell. We discovered that mutations in the middle of the molecule prevented PTHrP from entering the nucleus, as expected from research by other people. We discovered that mutations within the last 23 amino acids also prevented nuclear localization, a new finding. Finally, for Aim 4, we have introduced A549 cells and BEN cells into lungs of mice with poorly functioning immune systems. Both types of lung cancer cells form tumors. We have completed one research paper in this area and have submitted a second manuscript for consideration for publication.

The next steps will be to study the effects of different forms of PTHrP on growth and death of lung cancer cells in culture and then to translate this work into studies of the effects of PTHrP on progression of lung tumors.in animals. The results will improve our understanding of the progression of lung cancer and may ultimately lead to new therapies.
Publications

Parathyroid hormone-related protein regulates growth of orthotopic human lung tumors in athymic mice
Periodical: Cancer Index Medicus:
Authors: Hastings RH, Burton DW, Quintana RA, Biederman E, Gujral A, Deftos LJ ART
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