Researchers funded for the
2001-2003 funding cycle

MICHAEL A VOGELBAUM MD PhD
THE CLEVELAND CLINIC FOUNDATION

Regulation of Apoptosis in Glioma Primary Cell Cultures

Malignant brain tumors remain one of the most formidable foes in our battle against cancer. In spite of the tremendous advances seen recently in the treatment of such cancers as leukemia, breast, lung, and colon, cancer of the brain remains an almost invincible killer. The treatments that exist for brain tumor patients are, unfortunately, effective for a only small number of patients in the long-term. It is unlikely that we will ever be able create a true cure for brain cancer through surgical techniques, radiation therapy, or traditional forms of chemotherapy. The ultimate cure for brain cancer lies rather in learning how to attack these tumor cells at their fundamental, biological level. This requires knowledge of how tumor cells are biologically different than other cells, how we can use these differences to specifically identify tumor cells, and finally, how to use these differences to destroy tumor cells.
Normally, cells that acquire damage to their genetic code that cannot be repaired eliminate themselves (to help prevent a cancer cell from developing) by a process of cell suicide called "apoptosis." Cancer cells have often found a way to avoid apoptosis, which can make them resistant to the DNA-damaging treatments that we most often use to treat cancer. Brain tumors, in particular, are resistant to DNA-damaging treatments, and hence continue to grow despite the aggressive use of radiation therapy and chemotherapy.
Our laboratory focuses on understanding the particular mechanisms by which brain tumors have eliminated their ability to undergo apoptosis, with the goal of identifying treatments that can specifically overcome their mechanisms of defense. It is the goal of this project to determine whether the mechanisms that we identify in the laboratory by which tumor cells block apoptosis are found also in tumors obtained from the operating room.

ZHONGWU GUO
CASE WESTERN RESERVE UNIVERSITY

Glycoengineering Cancer Cells for Selective Immunotargeting of Cancer

Treatment of cancer patients using cancer vaccines is regarded as ideal therapy for cancer. Traditionally, cancer vaccines are prepared from the molecules that are special on cancer cells. The vaccines are used to provoke the patients' immune system to recognize the special molecules and then eliminate cancer from the body. Therapeutic cancer vaccines are specific and effective.
The aim of this research is to develop new cancer vaccines based on polysialic acid (PSA) which is a unique structure on several tumors including leukemia, small cell lung cancer (SCLC), neuroblastoma and Wilms' tumor. However, the problem with PSA is that it is tolerated by the immune system and thus cannot provoke specific immune responses. This research designs a new strategy to overcome the problem.
Thus, a vaccine will be made from a chemically modified PSA that is more likely to induce specific immune responses than PSA itself. The vaccine will be applied to cancer patients for stimulating their immune system. Once a specific immune response to the modified PSA is established, the patients will be treated with an agent that can induce cancer cells to produce the modified PSA. Then, the provoked immune system will recognize the specially marked tumor cells and remove them from the patients.
In particular, this research will investigate the properties of vaccines made of modified PSAs, the modification of cancer cells, and the efficiency of the new vaccines to cure cancer. These studies are important for the design of new, effective therapies for cancer.

ARUNA BASU PHD
METROHEALTH MEDICAL CENTER

Functional Analysis of LKB1 Gene Linked to Peutz-Jeghers Syndrome

Peutz-Jeghers syndrome (PJS) is an inherited cancer syndrome of the gastrointestinal tract. Patents with PJS syndrome are at an increased risk for developing tumors. The gene responsible for PJS encodes a protein kinase called LKB1/STK11 with the addition of a phosphate by virtue of its own enzymic activity (autophosphorylation). The phosphorylation states of a protein often determine its function. In many PJS patients, autophosphorylation activity of LKB1 is impaired by mutation of this gene. In view of this, it is quite likely that LKB1 functions in cells as a tumor suppressor.
Consistent with this finding, the introduction of LKB1 into tumor cells with severely reduced level of expression of LKB1, could suppress cell growth accompanied by cell cycle arrest. However, little is known in regard to the mechanism by which the activity of LKB1 is regulated in cancer cells.
We hypothesize that the autophosphorylation of LKB1 might be linked to the delicate mechanism of "Programmed Cell Death" or "Apoptosis". Our hypothesis stems from the prevailing concept that growth arrested cells at any checkpoint of cell cycle are usually eliminated by apoptosis. Apoptosis is the flip side of cell proliferation. Normally, a healthy individual maintains a critical balance between cell growth and cell death processes. Too little cell death can potentially contribute to the emergence of different categories of cancer. The clear comprehension of the regulatory mechanism, by which this potential tumor suppressor gene executes its biological function, is critical and eventually helpful for developing effective therapy for cancer.

MICHAEL E WEINSTEIN PHD
THE OHIO STATE UNIVERSITY

Functions of Smad2 in Mesodermal Differentiation and Tumor Progression

Mouse geneticists use embryonic stem cells (ES cells) to analyze the genetics of cancer. These very special cells can form every tissue in the body, and may be very useful in future medical applications.
One way to test the function of a tumor suppressor, a gene important in cancer, is to knock it out in ES cells and see how it affects their growth and their ability to form many tissues. This has been done for the Smad2 gene, which transmits the signals of an important signaling molecule called TGFbeta. The ES cells that lack Smad2 undergo a remarkable transformation in their growth, called a epithelial to mesenchymal transition, which resembles a transformation that occurs when a benign, harmless cancer becomes malignant and deadly. ES cells can be used to form tumors, and the ones that lack Smad2 form much bigger ones with more rapidly multiplying cells.
The purpose of this study is to confirm that the Smad2 gene is actually responsible for this transformation. We would also like to see how well these cells form specific tissues, such as nerves, muscle, and blood. Lastly, we want to see how the lack of Smad2 causes some cells to speed up their growth.

XINHUA LIN
CHILDREN'S HOSPITAL RESEARCH FOUNDATION, CINCINNATI

Role of Dally-like, a Drosophila Glypican in Cell-Cell Signaling

Wnt and Hh proteins are two large families of signaling molecules involved with both normal development and cancers. Mutations in molecules involved in Wnt or Hh signaling have been identified in human cancers. Thus, a clear understanding of the molecules involved in the regulation of Wnt and Hh signaling will provide new insights into the mechanisms of development of human cancers. Fruit fly protein Wingless (Wg) and Hh are the counterparts of human Wnt and Hh proteins. Fruit flies provide a great model system to study cancers mediated by Wnt and Hh proteins.
We are interested in studies of the regulation of Wnt and Hh functions by a group of cell surface molecules called HSPG. Glypican is a major class of cell surface HSPG. In humans, mutations in Glypican 3 cause Simpson-Golabi-Behmel Syndrome (SGBS) that is associated with high risk for the development of embryonic tumors including Wilms' tumor (kidney cancer) during early childhood.
In this proposal, we will investigate the function of a fruit fly counterpart (Dly) of humna Glypican 3. We will analyze the role of Dly in both Wnt and Hh signaling, both of which play critical roles in development and cancers.
The genetic studies in fruit fly have revealed many insights into the processes related with human cancers. The results obtained from this research will ultimately lead to interventions by the development of specific drugs for the prevention and treatment of human cancers that are associated with Glypican as well as Wnt and Hh signaling.

SUSAN WALTZ PHD
CHILDREN'S HOSPITAL RESEARCH FOUNDATION, CINCINNATI

Supported by
William Hughes Roberts

Ron in Skin Cancer

The long-term objective of this proposal is to define the role of a protein referred to as Ron in human cancers. Ron is a receptor tyrosine kinase in the cell membrane whose function is to transmit signals from outside the cell into inside the cell..
Numerous tyrosine kinases have been shown play a role in human cancers. Ron is a pivotal protein that can induce cells to increase growth rates, induce cell-cell detachment and induce cellular motility-all factors that are critical for tumor growth and metastasis.
To date, Ron has been found to be overproduced in human tumors, in cells from human tumors, and in cells which resemble skin carcinomas. In order to determine if Ron plays a role in tumor formation and metastasis, our laboratory has engineered cells that overproduce Ron. We have shown that the cells that over-produce Ron develop tumorigenic characteristics in vitro and form solid tumors in vivo.
A primary goal of this proposal is to determine the role of Ron in skin cancer progression. We intend to use mice that either lack Ron or over-produce Ron in the skin. These mice will be subjected to carcinogens that mimic the initiation, promotion and progression phases of skin cancer. The role of Ron in each well-defined stage of progression to skin cancer will be determined. In conclusion, these studies should shed significant light on the role of this protein in the formation and spread of tumors.

ZALFA A ABDEL-MALEK
THE UNIVERSITY OF CINCINNATI

Supported by the
You and the Night and the Music
Star Award Event

Oxygen Radicals Mediate the Mutagenic Effects of UVA on Melanocytes

The incidence of melanoma, the deadliest form of skin cancer, is increasing at an alarming rate. Melanoma represents a challenge to clinicians because of its resistance to most forms of therapy. The best hope to conquer the disease is by prevention and early detection.
Sun exposure is the main cause for skin cancer. However, the way by which sun exposure alters the normal behavior of the melanocyte (the cell responsible for producing pigment in the skin) and transforms it into a melanoma tumor cell is not clear.
We are proposing to study the effects of the most prevalent form of solar ultraviolet radiation, namely UVA, on human melanocytes that we culture in my laboratory from normal skin with different extent of pigmentation. There is sufficient evidence for a role of UVA in melanoma formation and stimulation of melanoma tumor growth. Melanin, the pigment in the skin, is thought to protect against the cancerous effects of sun rays. Individuals with fair skin that contains a low amount of melanin tend to have a high risk for skin cancer, including melanoma. We plan to explore how UVA affects the melanocyte, and to what extent melanin that is synthesized by melanocytes contributes to the response of those cells to UVA radiation.
The ultimate goal of this study is to design new ways by which the skin can be protected from the cancerous effects of UVA exposure. This can possibly be achieved by the use of antioxidants that inhibit the putative mechanisms by which UVA may lead to the transformation of melanocytes into melanoma.

ANDREW M LOWY MD
UNIVERISTY OF CINCINNATI

Tcf/beta catenin transactivation in gastric cancer

Supported in part by
Discover Financial Services, Inc.

Stomach cancer remains a major cause of death in the United States and around the world. Dr. Lowy's research has demonstrated that in stomach cancer, a gene known as beta-catenin may be inadvertently switched on. This gene has been shown to be important in the development of cancers such as cancer of the colon and liver. Dr. Lowy will investigate how switching on the beta-catenin gene may influence the growth of stomach cancer cells itself and through its action of switching on other cancer causing genes.

SHIUH WEN LUOH MD
UNIVERSITY OF CINCINNATI

HER-2/NEU Amplification In Human Breast Cancer

Breast cancer is the most common cancer in women. About 175,000 new cases of breast cancer are diagnosed every year in the US, constituting 30% of all cancers in women.
Breast cancer is the result of accumulation of molecular genetic alterations in breast epithelial cells. Some alterations, such as BRCAl and BRCA2, are inherited. Most alterations contributing to breast cancer, however, are acquired during a woman's life.
One such alteration known to play an important role in the progression of breast cancer is the amplification of a small region on human chromosome 17 containing the HER2/Neu gene. Gene amplification means that many more copies of a particular segment of chromosome are present than the normal complement of two (one inherited from each parent). An increase of the number of copies of a target gene may result in an increase in the amount of protein product encoded by that gene. In the case of HER2/Neu gene, amplification of HER2/Neu gene leads to overexpression of HER2/Neu protein.
Since gene amplification typically involves a string of genes from the same region, altered production of protein encoded by multiple resident genes is expected. Breast cancer with HER2/Neu amplification is a more virulent disease. It tends to be more spread out when discovered and come back more quickly after treatment. Patients with this disease tend to require more aggressive form of chemotherapy and respond less favorably to hormone treatment. Despite this clinical relevance, the complexity and informative content of the amplified segment have not been fully appreciated. The nature of the genes and mechanisms that contribute to the aggressive nature of these tumors has not been discovered.
We plan to examine thoroughly the molecular genetic alterations resulting from HER2/Neu gene amplification and identify and characterize all the genes from the amplified segment. Our work should help us understand why breast cancer with HER2/Neu amplification is a more aggressive disease. Our work may also reveal novel and rational targets for therapeutic intervention.

HAN-FEI DING MD PHD
MEDICAL COLLEGE OF OHIO

Capase-8 and p53 in N-Myc-Induced Sensitization to Apoptosis

Tumor therapy often kills cancer cells by provoking the cell to commit suicide. p53 is a protein that plays a critical role in initiating this suicide process. This project aims to find the proteins that help p53 to do its job so better treatments for cancers can be developed.
We have recently found that a protein called caspase8 can help p53 to trigger the suicide program in test tubes and to kill mouse tumor cells. In this study, we want to see if caspase8 can also help p53 to kill human tumor cells from neuroblastoma, a cancer that most commonly occurs in children.
This study may lead to a more effective and less toxic approach to the treatment of this deadly childhood cancer.

CYTHINA M SMAS DSC
MEDICAL COLLEGE OF OHIO

Function of the HLH Protein Id-1 in Prostate Cancer

The genes expressed in a cell determine the functions and actions of that particular cell type. An important class of genes are those that are able to "turn on" and "turn off' other genes. Such "transcriptional regulatory genes" may direct the proliferation and differentiation fate of the cell.
If these genes are misregulated, then the actions of the cell will be affected and in some cases abnormal cell growth and cancer will result. One family of transcriptional regulators is the basichelixloophelix (bHLH) group. These proteins have related features that allow them to combine in pairs with each other to bind DNA and "turn on" and "turn off' other genes.
The Id1 gene belongs to this family and due to the structure of the Id1 protein, Id1 can bind to other members of the bHLH family to block their function. Id1 expression is increased in a number of cancers and is generally thought keep cells in an undifferentiated, cancerous state, although in some instances Id1 expression increases in mature differentiated cells. Id1 expression in prostate cancer is not known. Studies from this laboratory indicate that in human prostate cells Id1 is present and that it increases when these cells differentiate to a neuroendocrine phenotype. Determining genes that govern the neuroendocrine phenotype in prostate cancer is important for two reasons. If we can force prostate cancer cells to differentiate, they will no longer be able to divide and therefore no longer considered tumorigenic. Increased numbers of neuroendocrine cells often heighten the severity of prostate cancer that occurs when prostate cancer cell growth is no longer controlled by androgens, a male sex hormone. Because the most common form of treatment for prostate cancer is androgen deprivation, by controlling the formation of neuroendocrine population, we may be able to decrease the severity of prostate cancer by allowing for effective androgen deprivation therapy.

VLADIMIR V POPIK PhD
BOWLING GREEN STATE UNIVERSITY

Development of Two-Photon Photoactivatable Enediyne Antibiotics

This project plans to address a major problem of modern anticancer chemotherapy: lack of selectivity. The majority of anti-tumor drugs work by attacking and damaging DNA molecules. This prevents transfer of the genetic information in the cell replication process and eventually leads to the death of a cell. Unfortunately, conventional anticancer drugs destroy DNA tumorous cells and other rapidly dividing tissues, such as bone marrow, hair bulbs, etc., thus causing serious toxic side-effects.
This project proposes to address this problem by incorporating a photo-triggering device of natural anti-tumor antibiotics of the enediyne family in place of the chemical trigger. These substances should be non-toxic in the dark but selectively activated in the target tissue by irradiation with red laser. Since the simultaneous presence of both photoactivatable antibiotics and light are required to develop toxicity, damage to healthy tissues can be minimized.
The results of this investigation are expected to serve as a basis for the development of new non-invasive anticancer therapy. It is envision that such therapy would consist of administration of photoactivatable enediynes antibiotics to the cancer patients followed by through-the-skin irradiation of cancerous tissues with focused pulses of ultrafast red laser. Extremely cytotoxic enediyne are then generated only in irradiated tissues, eventually destroying the tumor.

LAKSHMIDEVI PULAKAT PhD
BOWLING GREEN STATE UNIVERSITY

AT2-Mediated Regulation of the ErbB2/3 in Breast Cancer

A major difference between the development of a fetus and a tumor is that the cell-growth in a fetus is under tight regulation whereas the regulation of cell-growth in a tumor fails.
It has been shown that when cells of tissues such as breast and ovary convert themselves into cancer cells, certain protein molecules called ErbB2 and ErbB3 that instruct the cells to grow are overproduced and overactivated so that they continuously instruct the cells to grow. This uncontrolled growth results in tumors. Therefore, it is reasonable to assume that if overproduction and overactivation of the ErbB2 and ErbB3 molecules can be regulated, it is possible to regulate the development of tumors.
We have been working with a protein molecule called the Angiotensin II receptor subtype AT2 that has the ability to exert anti-growth effects on normal cells. Recently, we have identified a direct interaction between the AT2 and the ErbB2 and ErbB3 molecules using yeast cells for our studies. Interestingly, the region of the AT2 that interacts with the growth promoting ErbB2 and ErbB3 molecules is actually the region of the AT2 shown to be important for its growth-inhibitory effects. This interesting observation leads us to hypothesize that this interaction may mean that the AT2 can control the overproduction and overactivation of the ErbB2 and ErbB3 molecules seen in cancer cells and as a result can regulate the growth of breast cancer cells.
This idea is novel and previously untested. This proposal seeks funding to test this hypothesis, namely, whether overproduction and overactivation of the ErbB2 and ErbB3 molecules and the resulting uncontrolled growth of breast cancer cells can be regulated by induced co-expression and activation of the AT2 receptor in these cells.

LIRIM SHEMSHEDINI PhD
UNIVERSITY OF TOLEDO

Isolation of Repressors of the Androgen Receptor

Prostate cancer is the second leading cause of cancer deaths among men. This cancer, like all cancers, results from the interplay of multiple proteins that act on the cell cycle and cause it to become uncontrollable.
Many of these proteins carry out this activity by modulating gene expression, the process that leads from a gene to a functional protein. One such protein is the androgen receptor. This protein binds to and is activated by androgens (male hormones which are necessary for the normal sexual development of males) and thus mediates the physiological actions of androgens. The prostate is a major target tissue of androgens, and the proper development of this tissue and its transformation to a cancerous tissue are dependent on both the androgen receptor and gene expression that is regulated by this receptor. The importance of the androgen receptor in the development of prostate cancer is demonstrated by the success of antiandrogen therapy at the early stages of prostate cancer. While antiandrogen therapy does not work in recurring prostate cancer, when it most aggressive and deadly, the tumor cells still have large amounts of functional androgen receptor, strongly suggesting that receptor activity is important for not only the development but also the progression of prostate cancer.
The androgen receptor is under the control of various accessory proteins, the coactivators and corepressors. Coactivators stimulate the gene expression activity of the androgen receptor and similar receptors, while corepressors inhibit their activity. While a number of proteins have been demonstrated to enhance the androgen receptors, few have been shown to have inhibitory effects. Of those proteins that can inhibit the androgen receptor, and are known to have other functions in the cell, none appears to be involved in prostate cancer. Similarly, none of the many known coactivators has been shown to be directly involved in prostate cancer.
If none of these proteins that are known to affect androgen receptor activity is important in prostate cancer, then what is? We hypothesize that an undiscovered repressor of the androgen receptor is important in prostate cancer. Our specific hypothesis is that the activity of such a repressor is reduced or gone in prostate cancer, resulting in higher androgen receptor activity.
To find repressors of the androgen receptor, we will use a system in yeast cells that we call a functional screen. Since these cells do not contain the androgen receptor, we can put into these cells a modified version of the androgen receptor that may allow us to identify specific repressors of the androgen receptor. Yeast cells and various systems in these cells have been used widely and successfully to study the activities of many mammalian proteins. In fact, our lab has successfully used the yeast functional screen to identify a novel repressor of cJun, another protein that plays an important role gene expression and cancer. The repressors of the androgen receptor that are isolated in the yeast screen will be studied in prostate cells to determine what activity they have on the androgen receptor and how this affects the behavior of the cells.
Thus, this work can result in the identification of proteins that are involved in prostate cancer which may serve as diagnostic markers for this disease and be potential targets of future cancer therapy.