Nihal Ahmad - US grants

In American men, cancer of the prostate gland (CaP) continues to be one of the most frequently occurring malignancies accounting for approximately 29% of all new cancer cases. It is estimated that approximately 180,400 new cases of CaP will be diagnosed in the year 2000, and approximately 31,900 CaP-related deaths are predicted. Therefore, it is warranted to identify our efforts towards the development of novel approaches against CaP. Chemoprevention by naturally occurring or synthetic compounds could be a potential strategy for the management of cancer. Sanguinarine, derived from the root of Sanguinaria canadendid, is a benzophenanthridine alkaloid that is believed to posses anti-tumor properties. Recently, we showed that sanguinarine, at micromolar doses, caused apoptosis in human squamous carcinoma (A431) but not in the formal epidermal keratinocytes (NHEK) (Ahmed et al Clinical Cancer Research, In Press). Sanguinarine treatment also resulted in growth inhibition and apoptotic death of human prostate cancer cell viz. LNCaP, DU145 and PC-34. Further, we also found that sanguine treatment inhibited TNFalpha-mediated activation of NF- kappaB a pleiotropic transcription factor that has been shown to be associated with the development of cancer, in A431 and LNCaP cells. The current proposal capitalizes on these novel observations and has three aims. Firstly, employing a androgen-sensitive and insensitive human prostate carcinoma cells LNCaP and DU145 respectively, we will define the role of apoptosis during cell growth inhibition by sanguinarine. Secondly, we will investigate the involvement of NF-kappaB-pathway in sanguinarine-mediated apoptosis in these prostate cancer cells. Finally, employing athymic nude mice implanted with both types of human prostate carcinoma cells, we will investigate the relevance of the outcome of our in vitro studies to the in vivo situation. The central hypothesis of the work proposed is that sanguinarine will impart chemopreventive and possibly chemotherapeutic effects against prostate cancer via a modulation of NFkappa-B- pathway-mediated apoptosis. A corollary to this hypothesis that will be tested in the in vivo studies planned in this proposal is that sanguinarine treatment will reduce serum PSA levels that is regarded as A prognostic marker of prostate cancer burden in humans. Successful completion of this proposal will define i) the potential for sanguinarine against CaP, and ii) the molecular mechanism(s) associated with the biological response of sanguinarine.

DESCRIPTION (provided by applicant): In the United States, non-melanoma skin cancer that includes basal- and squamous-cell carcinoma is the most frequently diagnosed form of cancer and, according to an estimate, more than a million new cases of skin cancers are diagnosed annually in the USA. Therefore, it is warranted to intensify our efforts for the development of novel approaches for prevention and therapy of this cancer type. Chemoprevention by naturally occurring non-toxic compounds is a potential strategy to prevent the occurrence of the disease. Resveratrol (3,5,4'-trihydroxystilbene), a phytoalexin antioxidant found in grapes, red wines, berries and peanuts has been shown to afford cancer chemopreventive effects in chemically-induced murine skin carcinogenesis. The classical chemical carcinogenesis model of skin cancer is regarded to have little, if any, relevance to humans skin cancers because the excessive exposure to solar ultraviolet (UV) radiation is the major cause of human skin cancers. Our preliminary studies have shown that topical application of resveratrol prevents against chemically induced skin carcinogenesis in SENCAR mice. The molecular mechanism(s) of the biological effects imparted by resveratrol is not well understood. Our recent studies (Clinical Cancer Research, accepted for-publication), have demonstrated that resveratrol treatment to human epidermoid carcinoma (A431) cells results in an induction of the cyclinkinase inhibitor WAFl/p21 that inhibits cyclin (D1/D2)-cdk 6, cyclin (D1/D2)-cdk 4, cyclin E-cdk 2 complexes, thereby resulting in a G1-phase arrest of the cell cycle and an apoptotic death of the cells. We have also found that resveratrol treatment causes a modulation in refinoblastoma (pRb)-E2FDP machinery, during G1-phase arrest and apoptosis of A431 cells. The current application is based on these novel observations, and is designed to investigate the chemopreventive potential of resveratrol against UVB-mediated skin carcinogenesis, and the molecular mechanism(s) by which this food-based polyphenolic antioxidant imparts cancer chemopreventive effects. The central hypothesis of the work proposed in this application is that resveratrol will impart chemopreventive effects against photocarcinogenesis via modulating cki-cyclin-cdk network-mediated cell cycle regulation and apoptosis. In this study, we will first establish cancer chemopreventive potential of resveratrol against UVB-mediated damages including skin tumorigenesis in SKH-1 hairless mice. We will then conduct studies to assess the involvement of cki-cyclic-cdk network as a mechanism of chemopreventive effects of resveratrol during for UVB-mediated responses and skin tumorigenesis in SKH-1 hairless mice. Successful completion of this application will define i) the chemopreventive/therapeutic potential of resveratrol against skin cancer, and ii) molecular mechanism(s) of the biological effects of Resveratrol. This may pave the way for the development of novel strategies for prevention and possibly treatment of skin cancer and other epithelial cancers as well.

DESCRIPTION (provided by applicant): In the United States (US), non-melanoma skin cancer that includes basal- and squamous- cell carcinoma, is the most frequently diagnosed form of cancer and according to an estimate, more than a million new cases of skin cancers are diagnosed annually in the US. Therefore, it is warranted to intensify our efforts for the development of novel approaches for prevention and therapy of this cancer type. Chemoprevention by naturally occurring non-toxic compounds is a potential strategy to prevent the occurrence of the disease. Sanguinarine, derived from the root of Sanguinaria canadensis and other poppy-fumaria species, has been shown to possess anti-microbial, antioxidant and anti-inflammatory properties. In a recent study, the Principal Investigator of this application has shown that sanguinarine, at micromolar concentrations, imparts cell-growth inhibitory response in human squamous carcinoma (A431) cells via an induction of apoptosis. Recently, we found that sanguinarine treatment to human carcinoma keratinocytes (HaCaT) cells was found to induce apoptosis via modulations in Bcl-2 family proteins and other associated mitochondrial events. This application is based on these novel observations and our central hypothesis is that sanguinarine will impart chemopreventive effects against skin cancer by eliminating the damaged skin cells by an induction of apoptosis mediated via modulations in Bcl-2 family proteins- and associated mitochondrial events. Because most of the skin cancers in humans are regarded to be a result of excessive ultraviolet radiations from the sun, in the present study, we will employ the UVB-mediated murine skin tumorigenesis model of SKH-1 hairless mice to test our hypothesis. We will first establish cancer chemopreventive potential of sanguinarine against UVB- mediated damages including skin tumorigenesis in SKH-1 hairless mice. We will then study the effect of sanguinarine treatment on the modulations in i) Bcl-2 family proteins, specifically Bcl-2 and its phosphorylation, and, Bax, Bcl-xL, and ii) other associated mitochondria-events viz. cytochrome c, Apaf-1, caspases -3, -7, -8 and -9, during UVB-mediated responses and skin tumorigenesis in SKH-1 hairless mice. The outcome of this application will define i) the chemopreventive potential of sanguinarine against skin cancer, and ii) molecular mechanism(s) of the biological effects of sanguinarine. This may pave the way for the development of novel strategies for the management of skin cancer.

DESCRIPTION (provided by applicant): The use of complimentary and alternative medicine approaches is getting increasing popularity with patients of prostate cancer (CaP) that is the most common type of cancer in American men. In several clinical studies, melatonin (N-Acetyl-5-methoxytryptamine), a direct free radical scavenger and an antioxidant, has shown promise, either alone or in combination with traditional therapy, for the management of cancer. In vitro cell culture studies have indicated that melatonin possesses antiproliferative effects against human CaP cells. In our preliminary studies, we found that melatonin-treatment resulted in significant growth inhibition and a Gl-phase cell cycle arrest in both androgen-responsive LNCaP and androgen-unresponsive DU145 cells. Further, melatonin treatment to LNCaP cells resulted in a significant i) activation of its receptor MEL la, ii) decrease in the phosphorylation of mitogen activated protein kinase (MAPK), iii) decrease in the phosphorylation of mitogen activated protein kinase (MAPK), and iv) decrease in prostate specific antigen (PSA). Studies have shown the activation of melatonin-receptor modulates MAPK-pathway via suppression of cAMP that leads to a suppression of linoleic acid (LA) tumor uptake. This proposal capitalizes on these novel observations and the central hypothesis of the work proposed in this application is that melatonin will inhibit prostate cancer via melatonin-receptor mediated modulations in lipoxygenase-15- MAPK signaling. Employing a series of human CaP cells and normal human prostate cells, we will first assess the effect of melatonin on i) melatonin receptor, ii) 15-lipoxygenase and related events, and ii) MAPK-pathway, during its anti-proliferative/pro-apoptotic effects. Next, to validate the outcome of in vitro studies to in vivo situations, we will assess the anti-cancer effects of melatonin on prostate cancer development and its metastasis, employing transgenic adenocarcinoma mouse prostate (TRAMP) model that closely mimics human disease. The major advantage of this model is that metastatic CaP, in these animals, develops spontaneously without any chemical or hormonal treatment. The effect of melatonin-treatment will be evaluated on CaP development via monitoring tumor growth, tumor weight, volume and characteristics at the termination of the experiment. The effect of melatonin on CaP-metastasis will also be assessed. This will establish the anti-cancer effects of melatonin against CaP. We believe that a successful completion of this proposal will define i) the potential of melatonin against CaP, and ii) molecular mechanism(s) of the biological effects of melatonin.

[unreadable] DESCRIPTION (provided by applicant): Prostate Cancer (PCa), next only to lung cancer, is the second leading cause of cancer-related deaths in American males. At present, there are inadequate treatment options for the management of PCa. The public health impact from PCa has spawned tremendous interest in Chemoprevention trials. Preclinical, epidemiological, and phase III randomized, placebo-controlled clinical trials suggest that selenium and vitamin E could be effective in PCa prevention. Based on these studies, 'Selenium and Vitamin E Chemoprevention Trial (SELECT)' has been initiated. It is a randomized, prospective, double-blind study (7- 12 years) designed to determine whether selenium and vitamin E alone and in combination could reduce the risk of PCa among healthy men. This is an outstanding effort of its kind. However, several investigators are critical about the rationale whereas several others have arguments. Two popular questions in this direction are: (1) whether there are sufficient data on these supplements to justify this trial; and (2) whether more biomarkers (which could suggest the possible molecular mechanisms involved) should be included in SELECT trial. The present proposal, on a pre-clinical SELECT Trial in a well-established mouse model of prostate carcinogenesis, should provide answers to these questions. The hypothesis of this proposal is that a combination of vitamin E and selenium will provide synergistic chemopreventive effect against PCa via inhibiting oxidative stress and enhancing apoptotic and/or senescence response. Employing transgenic adenocarcinoma mouse prostate (TRAMP) mice, we propose to conduct a pre-clinical Chemoprevention/ intervention trial that mimics SELECT trial (in human) to determine whether selenium and vitamin E alone and in combination can inhibit PCa development and its metastasis. We will also study the involvement of oxidative stress, apoptosis and cellular senescence during PCa development and its inhibition by vitamin E and/or selenium. Finally, we will study the involvement of NF-kappaB pathway during the modulation in oxidative stress and apoptotic/senescence response of selenium and/or vitamin E treatments in TRAMP model. We believe that a successful completion of our study will provide important information regarding the possible outcome of SELECT trial in humans and may even provide novel information on which suggestions could be made for the modifications for the ongoing or future trials. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Studies have shown that resveratrol (trans-3,5,4'-trihydroxystilbene), a phytoalexin antioxidant found in grapes, red wines, berries and peanuts, imparts protection against several diseases. A plethora of pre-clinical studies have shown that resveratrol imparts chemopreventive as well as therapeutic response against several cancers. Further, resveratrol is being widely used as a supplement as well as a complementary and alternative medicine (CAM) approach for the management of several diseases/conditions. However, an important issue with resveratrol, especially in prevention settings (through diet or supplements), is believed to be its low bioavailability due to its rapid metabolism in mammals. It is known that during metabolism, the majority of resveratrol undergoes glucuronidation and sulfation. Studies have also shown that piperine, an alkaloid derived from black pepper (Piper spp.), inhibits glucuronidation in vivo. We believe that piperine will inhibit glucuronidation of resveratrol thereby enhancing its bioavailability leading to a superior chemopreventive response. This assumption is supported by our recent exciting preliminary data showing that piperine (20 mg/kg;oral gavage) enhances the bioavailability of resveratrol (500 mg/kg;oral gavage) in serum by ~175%. In this application, we propose to test the hypothesis that piperine will enhance the bioavailability of resveratrol leading to a superior chemopreventive and therapeutic response. In this application, we propose to conduct a clinical study (in human volunteers) as well as a preclinical trial (in a mouse model of prostate cancer. For our preclinical trial, we will conduct proof of principle study in a mouse model of prostate cancer (PCa). Our choice of PCa is based on the following point. PCa, next only to lung cancer, is the second leading cause of cancer related deaths in American males and because of its slow progression and long latency, is believed to be an ideal candidate disease for chemoprevention. Further, we have demonstrated that resveratrol imparts antiproliferative effects against human PCa cells, possibly via modulation of phosphatidylinositol 3'-kinase (PI3K)/Akt pathway and Bcl-2 family of proteins. Furthermore, recent studies have demonstrated that resveratrol suppresses PCa progression in TRAMP (transgenic adenocarcinoma of mouse prostate) and TRAP (Transgenic Rat for Adenocarcinoma of Prostate) models suggesting the potential of this agent for the prevention and treatment of PCa. The following two specific aims are proposed: 1) To determine the influence of piperine on the bioavailability of resveratrol in human volunteers;and 2) To determine if piperine coadministration enhances the bioavailability and chemopreventive effects of resveratrol on PCa development in TRAMP mice. We believe that a successful completion of this proposal may pave the way for the development of novel strategies to enhance the bioavailability of resveratrol. PUBLIC HEALTH RELEVANCE: Studies have shown that resveratrol, an antioxidant found in grapes, red wines, berries and peanuts, imparts protection against many diseases including certain cancers. Further, resveratrol is being widely used as a supplement as well as a complementary and alternative medicine (CAM) approach for the management of several diseases/conditions. However, an important issue with resveratrol, especially in prevention settings (through diet or supplements), is believed to be its low bioavailability due to its rapid metabolism in mammals. In this application, we will conduct a clinical study and a preclinical study to test the hypothesis that piperine (a chemical in black pepper) will enhance the bioavailability of resveratrol (via inhibiting its metabolism) leading to a superior chemopreventive and therapeutic response. We believe that a successful completion of this proposal may pave the way for the development of novel strategies to enhance the bioavailability and therefore preventive and/or therapeutic response of resveratrol.

? DESCRIPTION (provided by applicant): The objective of this study is to determine the interaction between Plk1 and B-Raf in melanocytic cells with the idea that this interaction plays a critical role in melanocytic transformation. Constitutively active oncogenic B-RafV600E mutations play an important role in melanomagenesis. However, B-Raf mutation alone are insufficient for melanocytic transformation, suggesting the involvement of other factors. Synthetic lethality, derived from the concept of `gene interaction', is gaining popularity towards developing new means and targets for certain diseases, including neoplastic conditions. Two genes are said to be synthetic lethal if mutation of either of them alone leaves the cell viable, while simultaneous modulations leads to a different fate for the cells. Thus, one gene buffers the effect of changes in the other to compensate for the effect of its deletion. This concept can help in identifying i) the buffering relationships among interacting genes, and ii) the malfunction and diseases that may occur when these relationships are altered. Exploring these relationships may lead to information how cellular processes work when the protein products expressed by two different genes have an effect together but not separately. Identification of interacting partner of B-Raf will provide a better understanding of mechanism of melanocytic transformation as well as identification of novel approaches to combat melanomagenesis. Polo-like kinase 1 (Plk1) plays pivotal roles in multiple aspects of cell division (mitosis). We have shown that compared to normal melanocytes, Plk1 is overexpressed in transformed cells and its targeted inhibition causes mitotic catastrophe and induction of apoptosis in melanoma cells, suggesting that Plk1 possibly plays an important role in melanocyctic cell proliferation. Significantly, our preliminary data show that melanocytic cells harboring B-Raf are hypersensitive to Plk1 inhibition than cells with wild type (WT) B-Raf, and that inhibition of B-RafV600E decreases cellular response to Plk1 inhibition. Based on our published and preliminary studies and available literature, in this application, we propose to challenge our central hypothesis that Plk1 acts as a synthetic lethality interaction partner of the oncogenic B- Raf. The following specific aims are proposed: 1) To determine the association between B-Raf mutation and Plk1 expression during melanocytic transformation; 2) To determine the extent to which the oncogenic B-Raf mutation enhances the sensitivity of the cellular response to mitotic stress, 3) To compare Plk1 loss-of-function phenotypes in melanoma cells with different B-Raf backgrounds; and 4) To assess the effects of Plk1 inhibition in a xenograft model with varying B-Raf. Outcome of this study is expected to enhance our understanding of mechanism of melanocytic transformation via dissecting interaction between Plk1 and B-Raf. This will be useful in designing novel strategies against proliferative melanocytic disorders, including melanomagenesis.

DESCRIPTION (provided by applicant): Novel mechanism-based approaches are needed for prostate cancer (PCa) management. A number of studies have suggested an important role of Zinc (Zn) in prostate biology. Zn exists in very high concentrations in the healthy prostate and is important for prostatic functions. Interestingly, in the cancerous prostatic tissue, the Zn level i significantly diminished and intracellular Zn levels have a strong inverse correlation with PCa progression. Therefore, Zn seems to play a critical role in PCa progression. During neoplastic transformation the normal prostate epithelial cells that are Zn-accumulating citrate producing cells seem to be metabolically transformed to citrate-oxidizing cells that lose the ability to accumulate Zn. Further, studies have suggested that a diminished expression of Zn transporter proteins (ZIPs), especially ZIP1, ZIP2 and ZIP3 may be associated with this metabolic transformation. Thus, apparently, down regulation of ZIPs in PCa tissue leads to low bioaccumulation of Zn. Interestingly, ZIP1 down-regulation in PCa was found to involve the overexpression of Ras responsive element binding protein-1 (RREB1). Restoration of adequate Zn levels in PCa cells has been shown to inhibit malignant potential. Further, Zn has been shown as a chemopreventive agent against PCa and supplementation with high dose of Zn may be useful. However, high dose of Zn is associated with many adverse effects. Further, malignant prostate cells in situ are incapable of accumulating high Zn levels from circulation. Therefore, novel means to enhance the bioaccumulation of sufficient Zn in the prostate cells via increasing ZIP-mediated Zn transport could be useful towards PCa management. Resveratrol, an antioxidant found in grapes and red wines, is shown to be capable of affording chemopreventive as well as therapeutic effects against PCa. A recent study has shown that resveratrol in combination with Zn dramatically increases the cellular Zn concentration in normal human prostate epithelial cells. Our preliminary data suggests that 1) a combination of resveratrol with Zn imparts a better anti-proliferative response in PCa cells, and 2) resveratrol-Zn increases ZIP proteins (ZIP1, ZIP2 and ZIP3) levels in PCa cells. Thus, based on published studies and our preliminary data, the hypothesis to be tested in this application is that resveratrol when combined with zinc will enhance its bioaccumulation, via RREB1 inhibition mediated increase in zinc-transporters (ZIP1, ZIP2 and ZIP3) in prostate, to impart a significantly superior chemopreventive and therapeutic response against PCa. Two specific aims are proposed: 1) To determine if a combination of resveratrol and Zn imparts superior chemopreventive and/or therapeutic response against PCa in vivo; 2) To determine the mechanism(s) of resveratrol-Zn combinatorial action. Outcome of our pilot study may provide useful information regarding the effectiveness of resveratrol-Zn combinatorial approach in PCa management, enabling us to design future in-depth studies including PCa clinical trial in human population.

DESCRIPTION (provided by applicant): The genetic dissection of a complex process such as melanocytic transformation is a daunting task. Indeed, different stages of differentiation of human melanocytic cells, such as normal melanocytes, nevi and melanomas representing different stages of disease progression, reflect distinct gene expression patterns. An in-depth knowledge of the genetic and epigenetic controls of cellular proliferation and cell cycle progression may provide critical information regarding the conversion of a normal melanocyte to its neoplastic phenotype. Over the past decade, SIRT1, a member of SIR2 family of sirtuin class III histone deacetylases, has garnered tremendous attention in neoplastic transformation and progression. A number of critical proteins, including p53 and FoxO transcription factors, have been identified as SIRT1 substrates. The roles and functions of SIRT1 in cancer have become extremely complex and are not well understood. Whereas a plethora of studies have shown a tumor promoter function of SIRT1, a number of studies have advocated its tumor suppressor role. It appears that SIRT1 plays dual functions in different tissue contexts depending on the spatial and temporal distribution and abundance of different SIRT1 downstream targets and factors that regulate SIRT1. The role and functional significance of SIRT1 in melanocyte biology, melanocytic transformation and progression is not clear. In our preliminary data, we have found that i) SIRT1 is expressed at much higher levels in melanoma cells and tissues, and ii) inhibition of SIRT1 results in a significant anti-proliferative response in melanoma cells. Further, the observed effects of SIRT1 inhibition were accompanied with transcriptional activation of p53. This is of particular interest because melanoma is one of the few cancers which rarely possess p53 mutations and this may provide an alternative route for altered p53 regulation without actual mutations. Thus, based on available literature and our exciting preliminary data, in this study we propose to test the hypothesis that SIRT1 plays a critical role in melanocytic transformation and melanoma survival via modulating its downstream regulation of p53, FoxO, and E2F1 transcription factors. The following aims are proposed: 1) to evaluate the expression profile of SIRT1 and its specific correlations with downstream regulators (viz. p53, FoxOs and E2F1 during melanomagenesis), and to determine if SIRT1 is involved in melanocytic transformation in an in vitro model; 2) to define the involvement of p53, FoxO and E2F1 transcription factors as downstream determinants of SIRT1 in melanocytic cells; and 3) to determine the therapeutic significance of SIRT1 inhibition in vivo in athymic nude mice implanted with melanoma cells. We expect that the outcome of studies proposed in this application may define the role and mechanism of SIRT1 in melanocyte biology, melanocytic transformation and melanoma progression. This may ultimately have therapeutic implications.

UWCCC Cancer Prevention and Control (CPC) Program Summary Co-Leaders: Hasan Mukhtar and Amy Trentham-Dietz PROJECT SUMMARY/ABSTRACT The Cancer Prevention & Control (CPC) Program spans basic, clinical, and population science to identify effective approaches to reduce the burden of cancer for patients, their families, and communities through improved prevention, early detection, and survival. In support of this overarching goal, the CPC program has defined three thematic aims. Theme 1: Identify promising chemopreventive targets and agents to test in clinical trials. Members build on a comprehensive integrated understanding of agents that decrease the incidence of cancer or its progression to clinically significant disease through drug development and clinical testing. Theme 2: Identify and implement effective approaches to reduce tobacco-related cancers. Members conduct intervention studies of innovative tobacco cessation approaches and lead observational cohort studies of real-world smoking patterns. Research focuses on underserved and minority populations since these are the same groups among whom the prevalence of smoking is greatest. Theme 3: Identify new strategies to improve the delivery of cancer care. Members identify effective approaches for delivering cancer screening and diagnostic tests and cancer therapies. CPC members are using innovative simulation modeling, bioinformatics, and multidisciplinary teams to improve the early detection of cancer; to leverage insights obtained through analysis of large databases to test approaches for improving access to guideline- concordant clinical care; and to incorporate patient-centered outcomes. CPC projects directly address the priorities present in the UWCCC catchment area and support reduction in cancer health disparities. The CPC Program has 33 members in 14 departments and 4 schools/colleges. Since 2012, CPC members have published 776 peer-reviewed manuscripts (25% intra-programmatic, 22% inter-programmatic). PIs were responsible for $11.1 million (direct costs) overall in peer-reviewed grant funding, with 60%, $6.7 million from NCI. Recent programmatic changes include new leadership, Dr. H. Mukhtar and Dr. A. Trentham-Dietz, and increased focus on interventions to reduce cancer and cancer risks in our communities. The CPC Program supports inter- and intra-programmatic interaction through pilot grants, seminars and retreats, a strong mentorship program, and collaborative working groups. Guided by strategic planning, priorities for the CPC program over the next 5 years include emphasizing cross-aim collaborations, building on existing momentum in health services research, and translational disparities research addressing needs in the UWCCC catchment area.