Christopher Weghorst - US grants

The objective of this R29 proposal is to understand what role inactivation of the p16 tumor suppressor gene plays in the multi-stage development of oral cancers, with an emphasis on premalignant lesions. The studies described herein address the hypothesis that a subset of the genetic changes commonly present in end-stage oral squamous cell carcinomas (SCC) will have already occurred in premalignant (leukoplakia, erythroplakia and proliferative verrucous leukoplakia) lesions. The p16 gene has previously been shown to be inactivated through a variety of mechanisms in 80 percent of SCC of the head and neck. In contrast, a critical evaluation of the role of p16 inactivation in premalignant oral lesions has yet to be explored. Therefore, we will focus our studies on this critical cell cycle regulatory gene in premalignant cells. The experimental approach involves a combination of genetic and biochemical methodologies. Studies in Specific Aim 1 are designed to determine the incidence and mechanism of p16 inactivation (including deletions, insertions, single nucleotide substitutions, and gene hypermethylation events) in premalignant oral lesions. Secondary to our evaluation of premalignant lesions, SCC will be screened for p16 mutations in an effort to identify a larger number of missense mutations for subsequent studies in Specific Aims 2 and 3. Once identified, premalignant and malignant oral lesions exhibiting p16 mutations will be assessed at the level p16 transcription and translation for altered p16 expression compared to patient-matched normal tissues (Specific Aim 2). These expression studies will be performed as a means of determining a precise level of p16 deregulation. In addition, specific P16 mutant proteins will be evaluated in Specific Aim 3 for their degree of biologic activity compared to the wild-type P16 protein. Mutant P16 proteins will be constructed by site-directed mutagenesis and evaluated for their ability to bind to CDK4 and inhibit CDK4 kinase activity in vitro. The in vivo functional activity of specific mutant P16 proteins will also be evaluated indirectly by determining the levels of phosphorylated and unphosphorylated RB proteins in samples derived from individual premalignant and malignant oral lesions. Overall, it is anticipated that the results from these studies will not only broaden our understanding of the molecular mechanisms of oral cancer development, but may also aid in the identification of specific causative agents, intermediate endpoint biomarkers, and the development of successful intervention strategies which target premalignant disease.

The objective of this proposal is to understand what role inactivation of the TGF-beta receptor complex plays in the multi-stage development of oral cancers. As the functional unit which transduces the inhibitory signal directed by TGF-beta, this heteromeric receptor complex is composed to two serine/threonine kinases (TbetaR-II and TbetaR-I). Working in obligatory cooperation, TbetaR-II and TbetaR-I indirectly regulate the phosphorylation of the retinoblastoma tumor suppressor gene product (pRB) and subsequent progression of the cell cycle. Inactivation of either TbetaR-II or TbetaR-I gene may lead to the deregulation of cell cycle and ultimately uncontrolled proliferation. The studies described herein address the hypothesis that a subset of the genetic changes commonly present in end-stage oral squamous cell carcinomas (SCC) will have already occurred in premalignant (leukoplakia, erythroplakia and proliferative verrucous leukoplakia) lesions. We have previously shown the TbetaR-II gene to be mutated (21%) and down-regulated (87%) in end-stage SCC of the head and neck. However, other potential mechanisms of this down- regulation, as well as a detailed evaluation of the TbetaR-I gene component in premalignant and malignant oral lesions have yet to be explored. Our experimental approach involves a combination of genetic and biochemical methodologies. Studies in Specific Aim 1 are designed to determine the incidence and mechanism of TbetaR-II and TbetaR-I gene inactivation (including deletions, insertions, single nucleotide substitutions, and hypermethylation events) in premalignant and malignant oral lesions. Once identified, oral lesions exhibiting TbetaR-II or TbetaR-I mutations will be assessed at the level transcription and/or translation for altered expression compared to patient-matched normal tissues (Specific Aim 2). These expression studies will be performed as a means of determining a precise level of TGF-beta receptor complex deregulation. In addition, specific TbetaR-II and TbetaR-I mutant proteins will be evaluated in Specific Aim 3 for their degree of biologic activity compared to the wild-type proteins. Mutant proteins will be constructed by site-directed mutagenesis and evaluated for their ligand binding, phosphorylation and trans-binding abilities in vitro. Overall, it is anticipated that the results from these studies will not only broaden our understand of the molecular mechanisms of oral cancer development, but may also aid in the identification of specific causative agents, intermediate endpoint biomarkers, and the development of successful intervention strategies which target premalignant disease.

DESCRIPTION (provided by applicant): Cancer chemoprevention is defined as the use of natural or synthetic agents, in combination or alone, to inhibit, delay, or reverse the carcinogenic process and is recognized as a very promising area of cancer research. Numerous epidemiological studies have shown the strong correlation between frequent consumption of fresh fruits and vegetables and a decreased cancer risk. With a food-based approach to cancer chemoprevention, emphasis is placed on the potential for complex mixtures of preventive agents in whole food to inhibit multiple processes of carcinogenesis. In support of this approach, several preclinical animal studies have been conducted that demonstrate the remarkable chemopreventive activity of black raspberries on chemically-induced aerodigestive tract tumor development, including oral, esophagus, and colon. While this chemopreventive phenomenon is striking in animal models of cancer, the chemopreventive potential of black raspberries on human cancer development remains unstudied. The present proposal is fundamentally translational in nature and strives to extend the chemopreventive assessment of this fruit to the problem of human oral cancer. Preliminary studies by us have shown that an extract of lyophilized black raspberries (LBR) dramatically inhibits the in vitro proliferation of cells derived from a human oral squamous cell carcinoma, suggesting that the chemoprevention of oral cancer by LBR components in vivo is possible. We therefore hypothesize that LBR will modulate the expression of critical genes associated with key biological pathways which may ultimately result in the inhibition, delay or reversal of the carcinogenic process. We will first identify changes in the global gene expression profiles in response to black raspberry treatment of human oral cancer cells in vitro (Specific Aim 1) and then determine the ability of black raspberries to modulate similar gene expression profiles in primary human oral cancer cells in vivo through a Phase 1B study (Specific Aim 2). These novel studies will be the first to examine the chemopreventive and molecular effects of a natural food product on human oral tumorigenesis. Additionally, these data will provide a suitable foundation from which future chemopreventive studies using LBR or other chemopreventive agents that target responsive genes will be conducted in post-surgical patients or those presenting with premalignant lesions.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Cancers of the oral cavity account for 2.5% of cancers in the United States and are the sixth most common cancer worldwide with a five year survival of approximately 50%; the number of deaths per year has not significantly changed in the past 3 decades. Because of this high mortality rate and since present treatments are relatively ineffective, intervention methods for oral cancer must be developed and tested in animal models. An animal model of oral cancer, the hamster cheek pouch (HCP), has shown that cell and tissue changes that are involved in the cancer process are similar between humans and hamsters. Because of these similarities, we have used the hamster oral cancer model to demonstrate chemopreventive activity of black raspberries and have translated this research into Phase Ib-IIa human clinical trials. The current application expands an ongoing project funded by the California Strawberry Commission in which lyophilized strawberries (LS) are being used to inhibit formation and development of oral cancers. In the current proposal, we will evaluate the combined effect of LS and selenium on the chemoprevention of oral cancer using strawberries that have selenium incorporated during formation of the fruit. Three different protocols will be used. In the first, hamsters will be given a diet of AIN-76A pellets containing 0% or 10% powdered LS or LS-selenium (LS-Se) beginning one week prior to treatment of the HCP with a chemical carcinogen (3X/wk for 8 wks); LS and LS-Se feeding is continued through 13 wks (Complete Chemoprevention Protocol). In the second and third, HCP will be initiated with chemical carcinogen (3X/wk for 6-7 wks) and beginning 48 hrs after the last dose of carcinogen, hamsters are given a diet of AIN-76A pellets containing 0% or 10% LS or LS-Se or treated topically 3x/week with 0% or 10% LS or LS-Se (Post-initiation Protocols). Control animals receive only AIN- 76A pellets or solvent vehicle. The Post-initiation experiments will mimic a condition similar to that seen in former tobacco users. We anticipate that the strawberry powder will inhibit tumor formation when given in the diet before, during, or after carcinogen treatment, as we have observed with black raspberries. Further, based on studies showing chemopreventive activity of selenium alone, we posit that treatment with LS-Se will result in a significant enhancement of chemopreventive activity and will inhibit progression of dysplastic lesions into overt tumors. A successful outcome of the post-initiation experiments will lead to human clinical trials in high risk populations using LS or LS-Se in a vehicle compatible with human consumption. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Numerous epidemiological studies have shown a strong correlation between consumption of fresh fruits and vegetables and a decreased risk of oral cancer. Several preclinical studies demonstrate the remarkable chemopreventive activity of two berry fruits, black raspberries and strawberries, that inhibit human oral cancer cell proliferation in vitro and development of chemically-induced aerodigestive tract tumors in animals. A pressing, yet unanswered, question remains "What types of polyphenols in fruit are primarily responsible for these activities?" Prior studies have employed lyophilized powders or extracts of ripe fruits containing a complex mixture of berry polyphenols of which a major group is the anthocyanins. Epidemiological evidence suggests that diets rich in anthocyanins (colored fruits and vegetables) reduce the overall risk of cancer and laboratory evidence illustrates the inhibition of proliferation of tumor cells in culture and prevention of tumors in animal models. However, there are numerous other fruit phenolics in berries that have demonstrated antioxidant, antiproliferative, and chemopreventive activity in both cell cultures and animal models of cancer. Among these are the phenolic acids, (gallic, procatechuic, p-hydroxybenzoic, caffeic, p- coumaric, ferulic, chlorogenic, and ellagic acids), the flavonols (quercetin, kaempferol, mycetrin, and isorhamntin), and complex polyphenols (lignans, ellagitannin, and gallotannin). To examine the chemopreventive capacity of these latter berry phenolics, separate from their combined activity with anthocyanins, we propose to evaluate green berry fruits (with minimal anthocyanin content) and ripe berry fruits (with high levels of anthocyanins) for inhibition of oral tumorigenesis in an animal model and to determine their effect on modulation of cancer-associated genes. Hypothesis: We hypothesize that bioactive phenolics found in green strawberries and black raspberries, independent of the anthocyanins, will inhibit tumorigenesis just as effectively as ripe berries in an animal model of oral cancer and will modulate the expression of critical genes associated with oral cancer. Methods: Hamster pellets will be prepared using 5% and 10% green and ripe strawberries and black raspberries. Beginning 7 days after commencement of feeding with berry pellets, cheek pouches will be painted 3x/week for six weeks with the chemical carcinogen 7,12-dimethylbenz(a)anthracene. Berry pellets will be given during, and for six weeks after, carcinogen treatment. At 12-13 weeks, cheek pouches will be evaluated for total lesions and tumors and for modulation of caner- associated gene expression. Significance: The novel studies outlined herein will provide a practical assessment of the chemopreventive potential of berries that are essentially devoid of anthocyanins, as well as an opportunity to determine their ability to modulate specific molecular targets in oral cancers. The outcome will provide a suitable foundation for in-depth studies designed to elucidate the chemoprevention potential for oral cancer of various bioactive berry components.

Rationale: A food-based approach to cancer chemoprevention emphasizes the potential for complex mixtures of preventive agents in whole foods to inhibit multiple processes of carcinogenesis. Supporting this, several preclinical animal studies have been conducted that demonstrate the remarkable chemopreventive activity of lyophilyzed black raspberries [LBR] on tumor development, including oral cancer. Preliminary data by us demonstrate that short-term in situ delivery of LBR can modulate the expression of key regulatory genes in cancer cells and at-risk normal oral mucosa of pre-surgical cancer patients that may favor the prevention of cancer development. Hypothesis: Long-term daily exposure of LBR by post-surgical head and neck [HN] cancer patients will modulate expression of genes critical in the development of oral cancer. This modulation may predict the preventive potential of LBR in human oral carcinogenesis. Experimental Approach: Phase IBIIA post-surgical clinical trials will be conducted in 210 individuals following surgical resection of stage I or II HN cancers. Daily LBR doses (0, 4, 8 gm) in two delivery systems will be given for a period of six months. Oral cavity scrapings, blood, urine, and saliva will be collected at selected time points and evaluated for outcomes of adherance/exposure, safety/toxicity, and capacity to modulate specific gene expressions associated with carcinogenic progression. Specific Aims: Aim 1. Determine the adherence of post-surgical patients to clinical trial design expectations and define tolerability and, potential adverse effects of long-term LBR administration. Aim 2. Determine the interrelationships between exposure, dose and delivery vehicle, and uptake of LBR components in oral tissues. Aim 3. Evaluate the ability of LBR to modulate gene expression in ¿high-risk¿ oral mucosa of post-surgical oral cancer. Aim 4. Analyze for the persistence of modulation genes following cessation of LBR treatment. This novel translational study will provide essential data establishing the scientific foundation for experimental and design parameters to be included in a future Phase III randomized clinical trial and is the first to examine the molecular effects of long-term LBR administration on ¿at-risk¿ oral mucosa in post-surgical cancer patients. It will also provide valuable insights related to the modulation of critical genes that might lead to oral cancer prevention.

Invasive cervical cancer (ICC) is the most common cause of cancer death worldwide, and while decreasing in prevalence in the majority of the developed world, this disease continues to disproportionately affect certain populations in the United States (US). In particular, the rate of ICC incidence and mortality in Appalachian women is the highest in the US. While many risk factors are known to influence ICC development, little is known about the role of hereditary and genetic susceptibility factors. The transforming growth factor beta (TGF-B) is a universal critical regulator of various cellular functions, including cell proliferation, via its binding with a receptors complex on the cell surface. Cancer cells frequently avoid the inhibitory influence of TGF-B on cell proliferation via somatic inactivation of key components of the signaling pathway, including the ligand and receptor complex. Additionally, germline polymorphisms in the ligand and receptors have been associated with cancer development and increased cancer susceptibility. In this proposal, we hypothesize that the increased incidence of ICC observed in Appalachian women over their non-Appalachian counterparts is due in part to inherited and somatic alteration of the TGF-B ligand and receptor complex that can be further potentiated in association with various environmental, behavioral, and socioeconomic risk factors. Specifically, we will determine prevalence of inherited polymorphic and somatically acquired variants of key TGF-B pathway components in a large cohort of Appalachian ICC patients compared to healthy Appalachian women. Furthermore, we will determine whether these genetic alterations contribute individually or in combination with other known environmental (Human Papillomavirus, Epstein-Barr Virus), behavioral (smoking), and social (stress, social networks) risk factors, to the increased susceptibility of Appalachian women to ICC development.

DESCRIPTION (provided by applicant): Epidemiological data demonstrate a strong correlation between frequent consumption of fresh fruits and vegetables and a decreased risk of oral cancer. In support of these data, preclinical studies have demonstrated the remarkable ability of black raspberries (BRBs) to prevent the development of aerodigestive tract tumors in animals, including oral (hamsters), esophagus (rat), and colon (rats). Furthermore, our ongoing Phase I clinical trials have demonstrated the ability of BRBs to modulate inflammatory biomarkers of molecular efficacy in a manner that supports a chemopreventive strategy for oral cancer. From these human clinical trials, we have established a novel molecular signature of clinically relevant, oral mucosa-derived, BRB-responsive biomarkers. Efforts to elucidate the underlying basis of BRB-mediated oral chemoprevention and predictive efficacy to prevent human oral cancer development will require further employment of a suitable animal model with sufficient molecular reagents. While the hamster cheek pouch model has a history of successfully identifying chemicals and foods with cancer preventing activity (including BRBs), exploratory and comparative molecular studies are hampered by the lack of hamster genome information and subsequent lack of reagents and tools for in depth systems biology investigations. Therefore, another suitable animal model of oral cancer must be employed. The rat tongue/4NQO system of oral carcinogenesis represents such an established and well-studied model for the investigation of black raspberry-mediated oral chemoprevention. The present proposal will explore the ability of BRBs to inhibit oral cancer using the rat tongue model and compare the molecular efficacy of BRBs in the rat tongue to that which has been established in human oral tumors and high-at-risk mucosa. We hypothesize that the bioactive components in BRBs will inhibit tumorigenesis and decrease the expression of clinically relevant, pro-inflammatory molecular biomarkers associated with oral carcinogenesis. The following Specific Aims uniquely combines aspects of nutrition, molecular efficacy, and cancer prevention to address the proposed hypothesis. Studies proposed in Specific Aim 1 will establish the ability of dietary BRBs or a single BRB bioactive component (ellagic acid; EA) to inhibit tongue lesion formation during 4NQO-induced oral carcinogenesis in F344 rats. Studies proposed in Specific Aim 2 will determine the comparative molecular efficacy of BRBs and EA on rat tongue carcinogenesis using a clinically-relevant transcriptional profile of genes previous shown to be modulated by BRBs in tumor and high at-risk oral mucosal cells of oral cancer patients. Following the successful completion of the above Specific Aims, our novel studies will (i) define the chemopreventive potential of dietary BRBs in a rat tongue model of oral cancer, (ii) validate a signature of BRB molecular efficacy in rat oral mucosa that is directly relevant to human oral cancer and (iii) provide a foundation for future mechanistic investigation of oral cancer chemoprevention.

DESCRIPTION (provided by applicant): Oral cancer affects nearly 300,000 Americans each year and is a serious health concern due to its poor prognosis and high recurrence rate. Moreover, the worldwide tobacco epidemic is leading to thousands of new cases of oral cancer being diagnosed each year. Targeting preventive strategies for those at highest risk for oral cancer due to tobacco exposure is thus a high priority. Our ultimate goal is to employ a food-based cancer preventive strategy for high-risk populations that allows excellent long-term adherence and efficacy. The oral cavity is an open microbial ecosystem that plays host to over 700 species of bacteria that form health- compatible communities called biofilms. We have previously demonstrated that these biofilms are rapidly enriched for pathogenic bacteria in smokers, resulting in an early hyper-inflammatory response. Furthermore, smoking cessation reverses this pathogenic bacterial recolonization, demonstrating that smoking has a direct effect on the oral microbiome and may increase the risk for oral carcinogenic processes by disturbing normal host-bacterial interactions. Therefore, our central hypothesis is that a critical bi-directional interaction exists between oral bacteria and phytochemical-rich black raspberry food products, which ultimately results in attenuation of inflammation and amelioration of disease. First, we will determine the effect of black raspberry phytochemicals (BRBs) on community dynamics within oral biofilms by combining a longitudinal clinical study design and a novel BRB delivery system (BRB nectar) with the resolution provided by shotgun metagenomic sequencing and computational bioinformatics. Next, we will examine the effect of oral bacterial communities on metabolism of BRBs in current and never smokers. Targeted HPLC-MS/MS analysis will be used to identify known phenolics and metabolites, while untargeted UHPLC Q-TOF metabolome analysis will identify novel compounds. Finally, we will evaluate the efficacy of BRBs and their metabolites in reversing the effect of smoking on oral host-microbial interactions by combining a longitudinal clinical study design with a novel, high-throughput dual RNA-Seq methodology to simultaneously quantify both oral bacterial and oral mucosal transcriptome changes before and after exposure to BRB nectar. Following the successful completion of these Specific Aims, we will demonstrate for the first time the ability of BRBs to mitigate pathogenic metabolic process and gene expression patterns induced by cigarette smoke in a manner that supports oral carcinogenesis prevention strategies.

1.0 PROJECT SUMMARY The Ohio State University Comprehensive Cancer Center (OSUCCC) proposes to establish a postdoctoral training program for cancer prevention and control that will prepare fellows to be impactful and independent research scientists in academia, government or industry. This will be done by providing focused transdisciplinary state-of-the-art research training. The overall goal of this training program is to reduce the risk of cancer and improve the lives of cancer patients. The research areas for the postdoctoral fellows may focus on carcinogenesis, behavior, cancer risk and survivorship. The training and research experience can be laboratory-, clinic- or community-based. The transdisciplinary training includes a core curriculum, varied research seminars, and emphasis on dual mentorship with biostatistical/bioinformatics advisors. The participating faculty consist of the 2 program directors and 22 mentors, all of whom have track records for peer- review funding, most participate or have participated in multi-investigator programmatic grants and are almost all experienced senior leaders and hold the rank of Professor. The research areas and scientific disciplines encompassed by the mentors included here are represented by their individual affiliations with four OSUCCC Research Programs, while the faculty come from 6 OSU colleges. The OSUCCC and its research programs have undergone the peer-reviewed Cancer Center Support Grant (CCSG) process, and received an ?Exceptional? descriptor (impact score of 10) for its state-of-the-art research, its research environment and the ability to foster transdisciplinary research. Now, the OSUCCC is seeking to expand its training programs with a specific focus on cancer prevention and control, one of our strategic priorities. While this is a new T32 training program, the OSUCCC has a strong track record for postdoctoral training, such as a 39-year history of a T32 general oncology training program that has trained 154 postdoctoral fellows in basic and translational cancer research by a cadre of outstanding NIH-funded participating faculty. While the OSU has numerous T32 training programs, there are only four that have a cancer focus, and these have little to no cancer prevention activities. Thus, there is little overlap with this newly proposed training program, which will have a large applicant pool locally and nationally. This application details a specific recruitment, training and career development plans, along with evaluation methods for the fellows and the overall program. There is strong institutional support and senior leaders from the OSUCCC and several colleges have leadership roles in this application. This new application seeks support for 2 trainees in year 1 and 3 trainees in years 2-5.

Summary: Oral cancer affects nearly 300,000 Americans each year and is a serious health concern due to its poor prognosis and high recurrence rate. Moreover, the worldwide tobacco epidemic is leading to thousands of new cases of oral cancer being diagnosed each year. Targeting preventive strategies for those at highest risk for oral cancer due to tobacco exposure is thus a high priority. Our ultimate goal is to employ a food-based cancer preventive strategy for high-risk populations that allows excellent long-term adherence and efficacy. The oral cavity is an open microbial ecosystem that plays host to over 700 species of bacteria that form health compatible communities called biofilms. We have previously demonstrated that these biofilms are rapidly enriched for pathogenic bacteria in smokers, resulting in an early hyper-inflammatory response. Furthermore, smoking cessation reverses this pathogenic bacterial recolonization, demonstrating that smoking has a direct effect on the oral microbiome and may increase the risk for oral carcinogenic processes by disturbing normal host-bacterial interactions. Therefore, our central hypothesis is that a critical bi-directional interaction exists between oral bacteria and phytochemical-rich black raspberry food products, which ultimately results in attenuation of inflammation and amelioration of disease. The proposed Administrative Supplement is in response to PA-17-447: Validation Studies of Analytical Methods for Dietary Supplements and Natural Products. We aim to validate a UHPLC UV-Vis/QTOF MS method for quantifying urolithins and protocatechuic acid in biological specimens. Successful completion of this aim will specifically support a primary objective of U01CA188250 ?Interactive Omics: Black raspberry metabolites and the oral microbiome in smokers?. The aim is designed to examine the effect of oral bacterial communities on metabolism of BRBs in current and never smokers. The validated method will focus upon the targeted HPLC-MS/MS analysis will be used to identify known phenolics and metabolites. The validated method will be disseminated through publication and will support reproducibility in future research of putative biologically active botanical constituents, indicators of bioavailability, and of potential metabolic states.