Doris M. Benbrook - US grants

This proposal is to study regulation of gene expression by interaction with transcription factor proteins which bind certain specific sequence elements in the DNA. Two classes of transcription factors have been described: AP1 and CREB. In order to bind DNA, the proteins must first dimerize. One of the AP1 proteins, cJUN, can form homodimers and respond to the TRE sequence element, or can form heterodimers with any of three CREB proteins and bind to the CRE sequence element. Each of these complexes has a unique trans- activation potential. All potential dimer complexes will be trans- lated from cDNA clones in reticulocyte lysates, and binding to oligonucleotides of known sequence will be determined by gel retardation analysis. Constructs of a plasmid containing the reporter gene luciferase coupled to the various oligonucleotides will be used to determine transactivation potential at various points in the cell cycle. %%% This proposal is to study proteins which enhance and promote the transcription of DNA, the genetic material. Two kinds of proteins (a total of four) will be used. The proteins contain two interesting structural features: DNA binding regions and "leucine zippers", which allow the proteins to form pairs of identical or different proteins. Each dimer binds to a unique sequence in DNA; the sequences will be determined and compared. The importance of the stage of the cell cycle for one type of dimer or another will be determined using luciferase from firefly tails as a reporter molecule.

DNA damage; retinoids; neoplasm /cancer nutrition therapy; combination cancer therapy; ovary neoplasms; cervix neoplasms; p53 gene /protein; retinoid binding proteins; cis platinum compound; cell growth regulation; metastasis; neoplasm /cancer radiation therapy; apoptosis; cell line; athymic mouse; flow cytometry; neoplasm /cancer transplantation; nutrition related tag; antisense nucleic acid;

DESCRIPTION (provided by applicant): Organotypic cultures are tissue culture models that mimic in vivo tissue architecture through manipulation of epithelial and stromal cells within and on top of an extracellular matrix. These models incorporate aspects of cell-matrix and epithelial-stromal interactions that cannot be evaluated in monolayer cultures. They can be used to evaluate monoclonal colonies, whereas individual xenograft tumors in animals are established from millions of cells. Colonies in organotypic culture therefore, more accurately mimic tumorigenesis than animal xenografts. Organotypic cultures can be more readily genetically and chemically manipulated than animal models, allowing increased numbers of experiments and less cost. They cannot replace animal models however, because they do not incorporate metabolic, physiologic and immunologic effects. Thus organotypic cultures can potentially be used to screen and improve chemoprevention agents prior to testing in animal models. In addition, they provide opportunities to evaluated hypothesis driven research on carcinogenesis and chemoprevention. We developed organotypic models of normal human endometrium, benign human ovarian tissue, and human borderline ovarian tumors of low malignant potential (LMP). The objective of this project is to further develop these organotypic models into models of carcinogenesis that can be used to study the processes of carcinogenesis and chemoprevention. The experiments proposed will evaluate the interaction of aromatic hydrocarbons, estrogen, progesterone, tamoxifen, differentiation and adult stem cells in endometrial and ovarian carcinogenesis. Our future goals will be to validate these models and to use them in our efforts to develop low-toxicity retinoids, heteroarotinoids, for chemoprevention. This research will enhance our understanding of the process of carcinogenesis and chemoprevention, which could ultimately translate into strategies and pharmaceuticals for prevention of cancer.

women's health; tissue resource /registry; disease /disorder etiology; prevention; therapy; neoplasm /cancer; fertility; diabetes mellitus; Alzheimer's disease; prognosis; heart disorder; diagnosis; sample collection; human tissue; clinical research;

[unreadable] DESCRIPTION (provided by applicant): SHetA2 is a novel anti-cancer compound that regulates growth and differentiation similar to retinoids, but does not directly activate RARs or RXRs. SHetA2 inhibited the growth of ovarian cancer xenografts without evidence of toxicity. Additional animal models have demonstrated that SHetA2 does not induce teratogenicity or skin irritation. Thus, SHetA2 exhibits an improved therapeutic ratio over retinoids capable of activating the retinoid receptors. Because SHetA2 is currently in pre-clinical development through the Rapid Access to Intervention and Development (RAID) program of the National Cancer Institute (NCI), additional basic science research, which is not supported by RAID, is needed to understand the mechanism of action of this compound before it is tested in humans. The hypothesis is that SHetA2 directly interacts with the mitochondria resulting in generation of reactive oxygen species, mitochondrial membrane depolarization, and inhibition of NF-kappaB activity and expression of thymidine phosphorylase and thrombospondin-4 gene expression. In endothelial cells this pathway inhibits the development of capillaries. The hyperactive metabolic state of ovarian cancer cells, increases the sensitivity of their mitochondria to the SHetA2 perturbations with the ultimate outcome being the induction of the intrinsic pathway to apoptosis. The more stable mitochondrial state of normal cells makes them more resistant to SHetA2-induced apoptosis. SHetA2 induces glandular differentiation through activation of hepatic nuclear factor-4, which regulates genes involved in glycoprotein metabolism, Galactosamine (N-acetyl)-6-sulfate sulfatase and UDP-galactose-4-epimerase and the TSP-4 glycoprotein. The specific aims are to decipher the SHetA2 molecular pathways of: 1) differential apoptosis in cancerous versus normal ovarian cells, 2) glandular differentiation and 3) inhibition of endothelial cell capillary formation. The results of these studies will provide mechanistic information on SHetA2 required for appropriate design of clinical trials and improved compounds, and scientific information on apoptosis and differentiation. The elucidation of the proteins involved in the SHetA2 pathwayswill provide potential biomarkers for ovarian cancer diagnosis, prevention and treatment strategies. [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Ovarian cancer is the most deadly of gynecologic cancers because it is most often detected at late stage and little is known of its etiology. A current theory is that high grade serous, the most common ovarian cancer, originates in the fallopian tube. We propose to study pharmaceutical and chemoprevention activity of our non-toxic oral chemoprevention agent, NSC 721689 (SHetA2) in fallopian tube tissue and cells. Our drug interferes with the function of a molecular chaperone called mortalin leading to mitochondrial swelling and mitophagy that transitions to apoptosis in cancer cells, while normal cells are resistant to these effects. A clue to understanding the reason for this differential effet is an altered form of mortalin that appears in NSC-721689-treated normal epithelial cell cultures and is gradually lost as the cultures undergo stasis, immortalization and transformation. We observed mortalin overexpression in the ovarian cancer tumor microenvironment. NSC 721689 also causes G1 cell cycle arrest through phosphorylation, ubiquitination and degradation of cyclin D1; events that occur in both cancer and non- cancer cells in vitro, and in a murine chemoprevention model in vivo. Extensive preclinical studies of our drug conducted by the NCI demonstrated chemoprevention activity in vitro and in vivo, lack of mutagenic metabolites, mutagenicity or teratogenicity, no toxicity and a pharmacologic profile suitable for an oral chemoprevention agent. Our objectives are to study NSC 721689 in ovarian cancer chemoprevention producing data needed to: 1) bring this promising drug to clinical trials for ovarian cancer chemoprevention and 2) develop additional chemoprevention strategies that may be applicable to all cancers. Aim 1 will conduct a Phase 0 clinical trial to determine the number of oral NSC 721689 capsules needed to achieve micromolar drug levels in the blood of healthy volunteers and in fallopian tube tissues of patients scheduled for hysterectomy. Aim 2 will generate Dicer-Pten Double Knock Out [Dicerflox/flox Ptenflox/flox Amhr2cre/+] mice, which have been shown to develop fallopian tube tumors that spread to the ovary, and study the effects of oral NSC 721689 on fallopian tube tumor histology and multiplicity. Aim 3 will measure NSC 721689 effects on cell cycle regulatory proteins and mortalin in specimens from the clinical trial and animal model. Significant results will be validated in cultures of human fallopian tube secretory epithelial cells (hFTSECs) established from the clinical specimens. Aim 3 will also determine the identity of the NSC 721689-induced alteration in mortalin and will study how sensitivity to NSC 721689-induced autophagy and apoptosis is gained, while NSC 721689-induced mortalin alteration is lost, as hFTSECs are passaged, immortalized and transformed. Tissue microarrays (TMAs) and image analysis to separately evaluate cancer and stromal cells, reverse phase protein arrays (RPPAs) and hFTSEC/fibroblast co-cultures will be used to study the roles of mortalin and other NSC 721689 altered proteins in drug sensitivity.

? DESCRIPTION (provided by applicant): High risk human papillomavirus (HPV) causes cervical dysplasia which can progress to cervical cancer, the second leading cause of cancer-related deaths among women. Therapeutic treatments for cervical cancer are highly toxic and often negatively impact quality of life. Cervical cancer is prevented by detection and destruction of cervical dysplasia involving significant cost, discomfort and potential loss of fertility, althogh the majority of these lesions will not progress to cancer. We developed a non-toxic cancer drug that has a mechanistic profile suitable for application in cervical dysplasia and cancer. Our drug, called SHetA2 (NSC 721689) counteracts the cell cycle regulatory consequences of high risk HPV. Extensive preclinical studies of SHetA2 demonstrated cancer therapeutic and chemoprevention activity in vitro and in vivo, lack of mutagenicity or teratogenicity, no toxicity and a pharmacologic profile suitable for an oral therapeutic agent. A pre-Investigational New Drug (IND) meeting report from the US Food and Drug Administration (FDA) listed no obstacles for our moving forward to a Phase 0 Clinical trial of SHetA2 in cervical cancer. In this project, w hypothesize that SHetA2 causes G1 arrest and apoptosis in human and murine cervical tumors by counteracting the alterations in cyclin D1 and other cell cycle regulatory proteins caused by HPV. We will test this hypothesis by conducting a Phase 0 clinical trial in Stage IB2-IVB cervical cancer patients to determine if oral administration of SHetA2 capsules can achieve systemic and cervical tissue concentrations of SHetA2 known to regulate cyclins A, D1 and E, and RB phosphorylation events. Pre- and post-treatment cervical tumor biopsies and a time course of collected peripheral blood mononuclear cells (PBMCs) collected in this trial will be used to determine which Rb pathway defects found in cervical cancer are altered by SHetA2 treatment. In addition, we will compare oral capsule and vaginal suppository SHetA2 formulations for alterations of cell cycle regulatory proteins in association with reduction of cervical tumor formation in K14-HPV16 transgenic mice. Cell cycle proteins, such as cyclins A, D and E, that are counter- regulated by HPV and SHetA2 will be tested for their direct involvement in SHetA2 regulation of G1 cell cycle arrest and apoptosis. These studies will lay the groundwork for future clinical trials in cervical dysplasia and cancer by determining the pharmacokinetics (PK) and pharmacodynamics (PD) of SHetA2, and by determining whether oral or vaginal delivery will achieve higher tissue concentrations of SHetA2, greater alterations in cell cycle proteins and greater prevention of cervical cancer in HPV transgenic mice. Our mechanistic studies will develop candidate PD biomarkers for use in future clinical trials planned for cervical dysplasia and will provide increased knowledge of HPV associated cell cycle defects in cervical dysplasia and cancer.

GYNECOLOGIC CANCERS PROGRAM ? ABSTRACT The goal of the Gynecologic Cancers (GC) program at the Stephenson Cancer Center (SCC) is to raise the standard of care and improve clinical outcomes for all women with gynecologic malignancies, with an emphasis on addressing problems relevant to Oklahoma, the SCC catchment area. Program members accomplish this goal by conducting transdisciplinary, team-based research that advances discovery of molecular mechanisms into early- and late-phase randomized trials. The Specific Aims for the GC program are: (1) to discover and interrogate molecular mechanisms central to carcinogenesis; (2) to investigate mechanisms underlying cancer therapy response and resistance; and (3) to design and conduct early- and late-phase randomized clinical trials that advance novel discoveries and improve patient outcomes in the catchment area and nation. Highlights of the GC program include: 1) highly interactive basic discovery and clinical members focused on gynecologic cancers; 2) Specific Aims that organize these program members into focused translational teams, leading to multi-PI grants and an exceptional rate of intra-programmatic publications; 3) a gynecologic oncology clinical program that impacts the catchment area by providing multidisciplinary care and access to clinical trials (58% enrollment rate) for half the new GC cases in Oklahoma; 4) a large annotated biospecimen bank that supports GC discovery research; 5) active development of GC-focused investigator-initiated trials and high accrual of GC patients to early-phase novel treatment trials; and 6) national leadership in the design and conduct of practice-changing NCTN- and Industry-sponsored randomized GC trials. The GC program has 18 members, representing seven departments, four colleges and two institutions, which illustrates the transdisciplinary expertise among program members. The translational focus of the program is reflected in the balance of MD (9) and PhD (9) members. As of December 31, 2016, program members were awarded $3,865,290 in annual NCI and other peer-reviewed cancer-related support (direct cost). NCI funding ($3,181,558) accounts for 82.3% of the peer-reviewed funding, highlighting the program's cancer focus. Total annual GC program funding is $8,700,601 (direct cost). For the five-year reporting period (2012-2016), GC program members published 262 peer-reviewed articles, of which 34.4% included intra-programmatic, 11.8% included inter-programmatic, and 73.3% included inter-institutional collaborators. For 2016, inter-programmatic publications were 21.1%, highlighting the increasing number of collaborations underway with the Preclinical Translational Cancer Research (PTCR) and Cancer Prevention and Control (CPC) programs.