This application requests support to evaluate the ability of benzylidenes and their boronic acid derivatives to prevent the incidence of prostate cancer via induction of phase II enzymes. Prostate cancer is the most frequently diagnosed cancer and is a leading cause of death in men in the United States. It is also the single most diagnosed non-skin cancer among African Americans. Our long term goal is to understand the detailed structure-activity relationships for cancer chemopreventive agents in the prostate and on that basis to produce nontoxic, prostate specific cancer chemopreventive agents. In this project we expect to (1) use Benzylidenes and their boronic acid derivatives to determine the structural features necessary to trigger the production of a potent amount of phase II enzymes in prostate cancer cells and (2) define the mode of action by which these Benzylidenes and their derivatives exert their chemopreventive effects using in vitro and in vivo models. Specifically, we propose to: (1) Prepare and characterize benzylidenes and their boronic acid derivatives; (2) Evaluate benzylidenes' ability to induce the phase 2 enzymes in DU145, LNCaP, and PC-3 prostate carcinoma cells and measure the cytotoxicity of these compounds in these cell lines; (3) Assess benzylidenes' ability to induce phase 2 enzymes and prevent prostate cancer in PTEN deleted mouse model. The preparation and characterization of the benzylidenes and their boronic acid derivatives will be completed at North Carolina A & T State University (NCAT) in the department of chemistry while the evaluation of those compounds in vitro and in vivo will be completed at Wake Forest University School of Medicine (WFUBMC). The co-Principal investigator and two (2) graduate students will benefit from training received at WFUBMC. At the end of the grant period, the co-Pi will use the training and information gathered from this project to complete and R01 grant application to further progress toward the pursuit of the completion of the long term objectives. ? ? Summary: This project will evaluate the ability of benzylidenes and their boronic acid derivatives to prevent the incidence of prostate cancer via induction of phase II enzymes. The applicant has proposed to complete this project by completing the following tasks: (1) by synthesizing benzylidenes and their boronic acid derivatives; (2) by evaluating the compounds' abilities to induce the phase 2 enzymes in DU145, LNCaP, and PC-3 prostate carcinoma cells; and, (3) by assessing the compounds' abilities to induce phase 2 enzymes and prevent prostate cancer in PTEN deleted mouse model. ? ? The preparation and characterization of the benzylidenes and their boronic acid derivatives will be completed at NC A&T, while the evaluation of those compounds in vitro and in vivo will be completed at WFUBMC. The detoxification of compounds in the body is generally considered a three step process, phase I metabolism includes oxidation, reduction, hydration, hydrolysis, generally increasing the chemical reactivity, followed by detoxification during phase II metabolism, which increases water solubility, and promotes excretion in phase III metabolism. ? ? There is considerable research evidence which notes that exposure of animal cells to low levels of electrophilic compounds (such as benzylidenes) results in the elevation and induction of phase 2 enzymes. Independently, other studies have indicated that boron, and boric acid have chemopreventive properties. The phase II enzymes to be tested include NAD (P) H quinone oxidoreductase (NQO1), GST (total), GST-mu, and GSTP1. Inducers of the phase 2 enzymes all belong to at least nine structurally diverse classes, but they all possess the ability to react with sulfhydryl groups, where reactivity correlates with potency. Other studies have shown that Benzylidenes and flavanoids are potent inducers of the NQO1 enzyme. Information regarding variation in the underlying mechanisms would have strengthened this section of the application. ? ? A series of 4 benzylidene compounds, and their boronic acid derivatives, will be synthesized and tested on cell lines (both androgen-dependent and androgen-independent). Phase II enzymes will be measured using western blot. Similar measurements will be made in prostate-cancer prone mice, when the tumors become palpable. For chemoprevention efficacy, one wonders if waiting until tumors are palpable is the most relevant time point. The integration of the two groups is another strongpoint, with monthly meetings of PIs and NC A&T students attending weekly lab meetings at WFUCCC. ? ? Information on the other boronic acid-benzylidene compounds which have also been shown to have anticancer effects was not adequately addressed in this application. These include the boronic acid-benzylidene compounds highlighted in reviews such as: Achanta G, Modzelewska A, Feng L, Khan SR, Huang P. Mol Pharmacol. 2006 Jul;70(1):426-33. A boronic-chalcone derivative exhibits potent anticancer activity through inhibition of the proteasome. The applicant mentioned that these would be the first. So the proposed innovation might be diminished somewhat, if these prior compounds are actually relevant. ? ? Innovation: The innovation relies primarily on the testing of relatively novel compounds in an excellent mouse model. ? ? Investigators: Drs. Franks, Torti and Chen are well suited to carry out this investigation, and their roles are clearly defined, and their strengths are complementary. Dr. Franks is a young investigator who performed his postdoctoral work with Dr. Torti. Dr. Chen has directly relevant experience working with the prostate cancer prone mouse model. Drs. Torti and Chen both have significant independent funding. ? ? Environment: The environment is excellent. ? ? PILOT PROJECT 2: Fra-1 expression regulates CD44 and C-MET expression in Glioblastoma Cells. ? (PI: Dr. Patrick Martin, NC A&T; Co-PI: Dr. Waldermar Debinski, WFUCCC). ? ? DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is the most lethal and malignant form of glioma known. Astroglial tumors are classified into four grades by the World Health Organization (WHO): a. pilocytic astrocytomas (Grade I, WHO classification); b. anaplastic astrocytomas (Grade ll-lll); c. glioblastoma (Grade IV). Glioblastomas differ from lower grades of astrocytic neoplasms; by being extremely aggressive, highly neovascularized and proliferative, as well as migratory/infiltrative. To compound the poor prognosis of patients with glioblastoma further, glioblastomas are typically insensitive to radiation therapy and chemotherapy. Although many different therapeutic approaches have been utilized, the overall median survival rate has not increased in the last twenty years. A better understanding of the molecular mechanisms that give rise to the invasive nature of malignant brain tumors is necessary in order to design more effective therapeutic regimens. The transcription factor, fosrelated antigen (Fra-1); glycoprotein CD44 and receptor tyrosine kinase c-Met are over-expressed in well characterized human GBM cell lines and human brain tumor specimens. Expression of Fra-1, CD44 and c-Met have been associated with increased glioblastoma migration. C-Met activation has also been associated with promoting inhibition to apoptosis. ? ? Summary: This application is focused on understanding the regulation of CD44 and c-Met expression by Fra-1 in high grade astrocytoma, especially glioblastomas. The proposed studies will also determine whether Fra-1 regulates invasive cell migration and also resistance to apoptosis induced by radiation via CD44 and c-Met respectively. ? ? Significance: Glioblastoma multiforme (GBM) is the most lethal and malignant form of glioma known. The long-term goals of these studies are to better understand the transcriptional regulation of glioblastoma cell migration as well as define biomarkers for malignant brain tumors. These experiments will answer important questions about glioblastoma biology, and will with high probability lead to publishable results, and have a similar high probability of serving as the foundation for applications by Dr. Martin for external funding. ? ? Approach: The proposed work will determine whether alterations in Fra- 1 expression: (1) regulates CD44 and c-Met expression; (2) regulates cell migration (via CD44 and c-Met); and, (3) regulates glioblastoma resistance to apoptosis. Lastly, the modulation by AKT of Fra-1 activity in glioblastoma cell lines will be explored. Additional studies will test whether Fra-1 expression regulates glioblastoma cell resistance to apoptosis. Fra- 1's ability to regulate CD44 and c-Met expression related cell activities will be by silencing targeted proteins with siRNA. The proposed studies will determine whether Fra-1 regulates invasive cell migration and also resistance to apoptosis induced by radiation via CD44 and c-Met respectively. ? ? Innovation: The innovation is primarily in the novelty of the anticipated results. Standard methodology is employed. ? ? Investigators: The combination of interests and backgrounds of both the mentor (Dr. Waldermar Debinski) and mentee (Dr. Martin) make this an excellent partnership. ? ? Environment: The environment is excellent. ? ? PILOT PROGRAMS: Training and Educational ? ? The current NC A&T science curriculum is not strong in cancer biology. With the support of the P20 WFUCCC faculty with work with NC A& T faculty to increase awareness of cancer research. These activities and plans will be implemented as follows: ? ? Pilot Program 1: Undergraduate Internship at WFUCCC. ? (Co-PI: Dr. Steve Akman, WFUCCC; Co-PI: Dr. Mary Smith, NC A&T). ? ? Pilot Program 2: Graduate Student Training in Cancer Research. ? (Co-PI: Dr. Patrick Martin, NC A&T; Co-PI: Dr. Marion Franks, NC A&T) ? ? ? Pilot Program 3: Genomics Education and Internships. ? (Co-PI: Dr. Xu, WFUCCC; Co-PI: Dr. Smith, NC A&T; Co-PI : Dr. Kelkar, NC A&T). ? ? Pilot Program 4: Computational Biology Training. ? (Co-PI: Dr. Goins, NC A&T; Co-PI : Dr. Jacqueline Fetrow, WFU). ? ? Pilot Program 5: Health Disparities Internship and Education. ? (Co-PI: Dr. Ronnie Bell, WFUSM; Co-PI : Dr. Kelkar, NC A&T). ? ? Pilot Program 6: Introduction to the Use of Animal Models in Cancer Research. ? (Co-PI: Dr. Tim Kute, WFU; Co-PI: Dr. V. Kelkar, NC A&T). ? ? The ultimate goal of these pilot programs is to lay the groundwork for a joint R25 submission between WFUCCC and NC A&T to increase minority training opportunities in Cancer Biology. ? ? Evaluation of Training and Educational Pilot Programs: ? ? Faculty Training: ? Dr. Patrick Martin, of the Department of Biology, will be mentored by Dr. Waldemar Debinski, who is also the Director of the WFUCCC Brain Tumor Center of Excellence. Dr. Marion Franks, Chemistry, will be mentored by Dr. Suzy Torti, who is Associate Professor of Biochemistry. Dr Franks completed her postdoctoral training at WFUCCC, and Dr. Yong Chen, Professor in the Department of Cancer Biology, the Director of Basic Science Center of Excellence for Prostate, and the Director of the Microarray Core, will also serve as a mentor. ? ? In addition, Drs. Martin and Franks will be further integrated into the WFUCCC in the following ways: ? 1) Through appointments as Associate Members of the WFUCCC. ? 2) By presenting a one-hour seminar each year in the weekly seminar series of the WFUCCC. ? 3) By participating in the monthly meetings of the Brain Tumor and Prostate Cancer Centers of Excellence of the WFUCCC. ? 4) By participating in the annual Department of Cancer Biology research retreat. ? ? Training of Students: Undergraduate summer internships: ? The Undergraduate summer internships at the WFUCCC will be provided for NC A&T juniors and seniors with outstanding academic records. Summer support for these students will be provided by existing NC A&T programs. ? ? Graduate student training in cancer research: ? Drs. Martin and Franks will recruit at least one NC A&T Master's student each into the collaborative pilot projects conducted jointly with WFUCCC faculty for the graduate student training in cancer research. ? ? Genomics Education and Internships: ? Jianfeng Xu, M.D., Dr.PH, Professor of Public Health and Professor of Cancer Biology and Director of Center for Cancer Genomics at Wake Forest University School of Medicine will provide an opportunity for NC A&T students to learn how genomics and proteomics tools are applied in cancer research. The Center utilizes state-of-the-art high-throughput molecular technologies and bioinformatics tools to evaluate millions of germline sequence variants in the genome among a large number of cancer patients and normal controls. They also integrate germline changes with somatic DNA changes and epigenetic changes in tumors to comprehensively interrogate genome for cancer related genes. ? ? Guest Lectures: ? Researchers from the WFUCCC Genomics Center will also guest lecture in the BIOL 640 Introduction to Bioinformatics and Genomics course at NC A&T for upper level undergraduate and graduate students. ? ? Computational Biology Training: ? Dr. Gregory Coins, Director of the BLEND program at NC A&T, will hold an awareness seminar for the BLEND undergraduate students to inform them regarding the opportunity of participating in computational cancer biology research projects ongoing at WFU under the mentorship of Dr. Jacquelyn Fetrow. ? ? Health Disparities Internships and Education: ? The Maya Angelou Research Center on Minority Health (MARCMH) at the Wake Forest University School of Medicine (WFUSM) will take a lead role in the development of research training and community outreach experiences for NC A&T students. Dr. Ronny Bell, Professor of Epidemiology and Prevention and MARCMH Director, and Dr. Melicia Whitt-Glover, Director of the MARCMH Community Outreach program, will work with Dr. Smith and other NC A&T colleagues in formulating the structure for the experience. ? ? Opportunities for research training will include internships and individual mentoring with MARCMH faculty affiliates, participation in MARCMH and other WFUSM seminars and conferences pertaining to health disparities and auditing courses in the WFUSM Master's of Sciences in Clinical and Population Translational Sciences Graduate Program. ? ? Additional Opportunities: Additional opportunities for community outreach will include participating in community initiated health-focused activities (health fairs, screenings, conferences, etc.) in the Triad area (Greensboro, Winston-Salem, High Point), particularly those targeting minority and low-income segments of these cities. ? ? Another opportunity is the course, titled, The introduction to the Use of Animal Models in Cancer Research. Dr. Tim Kute, Professor of Cancer Biology at WFU, will collaborate with Dr. Vinaya Kelkar at NC A&T to develop a teaching module on animal models of cancer to be integrated into the current experimental biology course (BIOL 703 Experimental Methods in Biology) that will be taught at NC A&T. ? ? Investigator/Personnel: Dr. Steve Akman, WFU Principal Investigator, is Professor of Internal Medicine and Cancer Biology at Wake Forest University. His research interests focus on DNA damage and mutagenesis, and non-canonical DNA structures. As a practicing medical oncologist, Dr. Akman has both a clinical and a research interest in breast cancer, a disease in which there is a clear disparity in outcome between African-Americans and other ethnic groups. In this regard, Dr. Akman serves as scientific director of the Breast Cancer Center of Excellence of the WFUCCC. As a member of the WFUCCC Internal Advisory Board, Dr. Akman will be in an excellent position to facilitate interactions between the WFUCCC and the P20. As Principal Investigator, Dr. Akman will have overall responsibility for the activities conducted under the auspices of the P20. He will work closely with Dr. Mary Smith, Co-PI from NC A&T. Dr. Akman will be responsible for communication with NCI program staff and submitting progress reports. He will draft the agendas and chair IAB meetings. Dr. Akman will devote considerable time and effort (25% effort) to the P20 during the initial planning and priority setting stages. ? ? Mary A. Smith, PhD will serve as the NC A&T Principal Investigator. She is an Associate Professor and Associate Chair of the Department of Biology. Dr. Smith is especially interested in mentoring and retaining underrepresented minority students in the sciences and increasing the number that pursue the PhD degree. She has become the new PI and Director of the MARC U* STAR grant at NC A&T, an avenue for transitioning talented students into cancer research PhD programs and careers. As associate Chairperson in the Department of Biology she has been involved in recruiting new faculty with a research focus in biomedical research and health disparities. She has led the effort to establish and sustain a scientific relationship between NC A&T and the WFUCCC since 2002. As coordinator of the multimillion dollar Research Infrastructure in Minority Institution Grant, Dr. Smith has been deeply involved in guiding the development of the biology research infrastructure, promoting and establishing a research culture with increasing emphasis on health disparities research. She is exceptionally capable and well positioned to foster the success of the P20 and develop a R25 training grant to increase the number of MS degree students who go on to earn terminal degrees in the biomedical sciences. ? ? ?
