Chuanshu Huang - US grants

DESCRIPTION (provided by applicant): Although arsenic is acknowledged to be highly carcinogenic to human skin, its action at the cellular and molecular level is not well understood. It is thought that arsenite-induced cell proliferation is one of the central events involved in its carcinogenic activity. Our preliminary data have revealed that treatment of cells with arsenite results in the activation of phosphatidylinositol 3-kinase (PI-3K) and downstream kinases, protein kinase B (Akt) and p70 S6 kinase (p70S6k), as well as an increase in cyclin D1 induction, cell proliferation and transformation in human keratinocytes. Considering the essential role of PI-3K and its downstream kinases in cyclin D1 induction, cell proliferation and transformation, the main hypothesis of this proposal is that PI-3K and its downstream signaling pathways play a pivotal role in arsenite-induced cyclin D1 expression, cell proliferation and transformation in human keratinocytes. The long-term goal is to elucidate the molecular mechanisms by which arsenite causes human skin cancer development. The overall aim of this proposal is to test the effect of arsenite on the PI-3K signal transduction pathway and its role in the regulation of cyclin D1 expression, cell proliferation and transformation in human keratinocytes. Especially, we will investigate this issue in accordance with the following testable hypotheses and specific aims (SA): SA 1: To test the hypothesis that PI-3K is required for the activation of Akt and p70S6k in arsenite-treated human keratinocytes. SA 2: To investigate whether PI- 3K/Akt/p70 s6k pathway plays a role in arsenite-induced cyclin D1 expression and cell proliferation in human keratinocytes. SA 3: To test the hypothesis that PI-3K/Akt/p70S6k pathway plays a role in arsenite-induced cell transformation in human keratinocytes. SA 4: To test the hypothesis that AP-1 and/or NFkappaB are downstream transcription factor(s) in human keratinocytes response to arsenite treatment. SA 5: To test the hypothesis that activation of AP-1 and/or NFkappaB is required for induction of cyclin D1 expression, cell proliferation and cell transformation by arsenite in HaCat cells. The significance of the research proposed in this application is that the results derived from the proposed studies will greatly facilitate the understanding of the molecular mechanism of cancer development caused by arsenite in human skin. A better understanding of signal transduction pathways leading to the induction of cyclin D1 expression, cell proliferation and transformation, will provide valuable information needed for designing more effective agents for the prevention and therapy of cancers caused by arsenite. Such agents could interfere with signaling pathways leading to cyclin D1 induction, cell proliferation and cell transformation.

DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are ubiquitous complete carcinogens that are present in tobacco smoke. The majority of work in previous studies has focused on the mutations that are associated with the tumor initiation effect of these compounds. However, the tumor promotion effect of PAHs, which is thought to be mediated through regulation of signal transduction pathways leading to activation of transcription factors, remains unclear. Our preliminary studies suggest that benzo[a]pyrene diol-epoxide (B[a]PDE), an ultimate carcinogenic metabolite of benzo[a]pyrene (B[a]P), is a major compound responsible for activation of the transcription factor activator protein-1 (AP-1) in non-cytotoxic concentrations. Since growing evidence has shown that activation of AP-1 by carcinogens is required for tumor promotion in both cell culture models and animal experiments, the main hypothesis of this proposal is that signal transduction pathways leading to activation of AP-1 play a critical role in the tumor promotion effect induced by B[a]PDE. The overall aim of this proposal is to elucidate the molecular mechanisms by which PAHs induce tumor promotion. Especially, we will establish the signal transduction pathways leading to AP-1 activation by B[a]PDE in a well-characterized tumor promotion cell culture model, mouse epidermal C141 cells. We will then investigate whether the same pathway occurs in an in vivo model using an AP-1-luciferase reporter transgenic mouse model. Furthermore, we will determine the role of AP-1 activation in B[a]PDE-induced tumor promotion in a two-stage carcinogenesis mouse skin model using dominant negative mutant c-jun (TAM67) transgenic mice. We will investigate these issues in accordance with the following testable hypotheses and specific aims: 1) To elucidate early events involved in initiating the signaling pathways leading to AP-1 activation by B[a]PDE in mouse epidermal C141 cells; 2) To test the hypothesis that the PI-3K/Akt/p7086k pathway is required for B[a]PDE-induced AP-1 activation in mouse epidermal C141 cells; 3) To determine whether B[a]PDE is able to induce AP-1 activation in vivo by using AP-1-luciferase reporter transgenic mice and whether this activation is through the same signal transduction pathways as in vitro; 4) To test the hypothesis that AP-1 activation is essential in the tumor promotion effect of B[a]PDE in a two-stage carcinogenesis mouse model. The significance of the research proposed in this application is that the results derived from the proposed studies will greatly facilitate the understanding of the molecular mechanism of cancer development caused by B[a]P and B[a]PDE. A better understanding of signal transduction pathways leading to AP-1 induction may provide valuable information needed for designing more effective agents for prevention and therapy of cancers caused by cigarette smoke. Such agents could interfere with the signaling pathways leading to AP-1 activation.

[unreadable] DESCRIPTION (provided by applicant): The carcinogenicity of nickel compounds has been well documented by studies both in vitro and in vivo. However, the molecular mechanisms by which nickel compounds cause cancer are not well understood. It is accepted that the tumor promotion effects of nickel compounds occur through regulation of gene expression, which is mediated by activated transcription factors. A nuclear factor of activated T cells (NFAT) and nuclear factor-(B (NF(B) are transcription factors, which are believed to play an important role in cancer development. Our preliminary data show that nickel compounds are able to induce activation of NFAT and NF(B in human bronchoepithelial cells. Therefore, the main hypothesis of this proposal is that NFAT and NF(B play a critical role in nickel-induced tumorigenicity in human bronchial epithelial cells (HBECs). The overall goal of this proposal is to study the molecular mechanisms by which nickel compounds mediate carcinogenesis. In particular, this proposal seeks to identify the initiating signaling leading to activation of transcription factors NFAT and NF(B, and their roles in nickel-induced tumorigenicity of human bronchial epithelial cells. Thus, we will investigate this issue in accordance with the following specific aims: 1) To test the hypothesis that reactive oxygen species (ROS) are involved in activation of transcription factor NFAT and NF(B in HBECs in response to nickel compounds; 2) To elucidate the role of NFAT activation in nickel-induced tumorigenicity of HBECs; 3) To test the hypothesis that NF(B transactivation is an important player in nickel-induced tumorigenicity in HBECs; 4) To establish the in vivo effects of nickel compounds on NFAT and NF(B activation by using NFAT and NF(B-luciferase transgenic mice. The significance of the research proposed in this application is that the results derived from the proposed studies will greatly facilitate the understanding of the molecular mechanism of carcinogenic effects of nickel compounds. This study will also provide in vivo models to further investigate the role of NFAT and NF(B activation in nickel-induced carcinogenesis in vivo. Furthermore, it will also help us to determine whether we can use NFAT or NF(B as targets for chemoprevention of nickel-induced carcinogenesis. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Arsenic remains a top environmental concern in the United States as well as world-wide because of its global existence and serious health impacts. Epidemiological studies provide ample evidence that arsenite exposure is associated with the increased incidence of skin and lung cancers. The scope of the parent grant addresses the relation of reactive oxygen species (ROS) generation and skin carcinogenesis due to arsenite exposure in both the human keratinocytes and the mouse skin models. While the scope of current competitive revision seeks to evaluate the contribution and molecular mechanisms of linking the arsenite-induced chronic inflammation to lung carcinogenesis both in vitro and in vivo, this competitive revision seeks to expand the original scope of study from ROS generation in the mediation of skin carcinogenesis, to identification of the molecular mechanisms that link arsenite-induced chronic inflammation to lung tumorigenicity in vitro and in vivo in cellular as well as animal response to arsenite exposure. Although both the parent grant and the current competitive revision propose to study the mechanisms implicated in the carcinogenic effects of arsenite exposure, this revision is specifically intended to identify the contribution of key inflammatory mediators TNF-1 and COX-2 to arsenite-induced development of tumorigenicity of human bronchial epithelial cells (HBECs) in vitro and in vivo, as well as the molecular mechanisms involved in this tumorigenic process. The main hypothesis of this revision is that TNF-1 plays a central role in the formation and maintenance of sustained chronic lung inflammation and subsequently results in the induction of lung epithelial cell tumorigenicity due to arsenic exposure. The overall goal of this proposal is to determine role of TNF-1 and COX-2 in arsenite-induced HBECs'tumorigenicity, as well as the central role of TNF-1 in the maintenance of lung chronic inflammation during arsenic exposure both in vitro and in vivo. There are two Specific Aims proposed for this investigation: 1) To determine the contribution of TNF-1 and COX-2 to the development of tumorigenicity of HBECs due to arsenic exposure;2) To assess the central role of TNF-1 in arsenic-induced lung chronic inflammation and its mechanisms in vivo. Success of the revision will facilitate our understanding of the molecular mechanism(s) of the formation and maintenance of the chronic lung inflammatory microenvironment, and its role in lung cancer development induced by arsenic exposure. A better understanding of those issues may provide valuable information that is needed for the designing more effective agents for the prevention of and therapy for inflammation-associated lung cancers. Furthermore, funding of this competitive revision will save two jobs, one part-time for Dr. Jianxiu Yu and one for Dongyun Zhang, both of whom provide valuable expertise and are crucial for the proposed studies. The finding will also create new training opportunity for two postdoctoral fellows in the proposed research field. Since we have most of research tools, and research animals are either available in our laboratory or commercially, we believe that the proposed studies will be achieved within two years. PUBLIC HEALTH RELEVANCE: Arsenic remains the top environmental concern in United States as well as world wide, and the existence of a sustained lung chronic inflammatory microenvironment is thought to be a major driving force for the development of lung cancers due to arsenite exposure, however, the molecular mechanisms linking arsenite exposure to lung chronic inflammation are totally unknown. This application is to test the hypothesis that TNF-1 plays a central role in the formation and maintenance of sustained chronic lung inflammation and induction of lung epithelial cell tumorigenicity due to arsenic exposure. Success of the revision will facilitate our understanding of the molecular mechanism(s) of the formation and maintenance of the lung chronic inflammatory microenvironment, and its role in lung cancer development induced by arsenic exposure. A better understanding of those issues may provide valuable information needed for designing more effective agents for prevention and therapy of the inflammation-associated lung cancers.

DESCRIPTION (provided by applicant): This competitive renewal application will continue examining the molecular mechanisms by which nickel compounds mediate lung carcinogenesis. In particular, this proposal seeks to identify the molecular mechanisms linking nickel-induced lung sustained inflammation to tumorigenicity of human bronchial epithelial cells (HBECs) in vitro and in vivo. Although there is an association between nickel- induced sustained airway inflammation and lung cancer development, the molecular mechanisms linking nickel exposure to the sustained lung chronic inflammation are not understood yet. The studies obtained from the last funding period and preliminary studies demonstrate that nickel exposure results in the activation of the nuclear factor-:B (NF:B), which in turn mediates COX-2 induction. Our studies also show that nickel exposure enables activation of AP-1, which in turn mediates TNF-1 induction. Furthermore, we find that TNF-1 enables COX-2 induction through the NFAT-dependent pathway. In addition, we find that there is crosstalk between the NFAT and NF?B pathways during cellular response to nickel exposure. Thus, the main hypothesis of this renewal proposal is that the NFAT/ NF?B activation and the pro-inflammatory TNF-1 and COX-2 induction will form positive inflammatory feedback loops, which are responsible for the formation and maintenance of sustained chronic lung inflammation and the induction of lung epithelial cell tumorigenicity due to nickel exposure. We propose the following Specific Aims: 1), To test the hypothesis that the inflammatory positive feedback loops being formed by NF?B, NFAT, and TNF1 are responsible for the maintenance of sustained COX-2 induction due to nickel exposure in HBECs; 2), To determine the role of the positive inflammatory feedback loops in the development of tumorigenicity induced by nickel exposure in HBECs.; 3), To assess the central role of TNF-1 in nickel-induced chronic lung inflammation and its mechanisms in vivo. The overall goal of this proposal is to clarify the formation of positive inflammatory feedback loops among NFAT, NF?B, TNF-1 and COX-2, in nickel exposure both in vitro and in vivo, and to determine the role of the positive inflammatory feedback loops in nickel-induced HBECs' tumorigenicity, as well as the central role of TNF-1 in the maintenance of lung sustained chronic inflammation and lung carcinogenesis during nickel exposure in vivo. Success of the proposal will facilitate our understanding of the molecular mechanism(s) that lead to the formation and maintenance of a lung chronic inflammatory microenvironment, and its role in lung carcinogenesis due to nickel exposure. A better understanding of these issues may provide valuable information for the designing of more effective agents for the prevention and therapy of lung cancers. We believe that the proposed contribution of positive inflammatory feedback loop responsible for nickel-induced lung tumorigenicity is novel.

DESCRIPTION (provided by applicant): Urothelial carcinoma of the bladder (or bladder cancer, BC) is one of the most common cancers in the Western world. Because high-grade invasive bladder cancers (HGIBC) can progress to life threatening metastases, identifying a natural compound that specifically inhibits BC invasion and metastasis is of tremendous importance for potentially reducing mortality as a result of this disease. Isorhapontigenin (ISO) i a new derivative of stilbene, and isolated from a Chinese herb that has been used in China for treatment of BCs for hundreds of years without understanding of molecular mechanisms. Thus, the goal of this proposal is to determine the ISO potential therapeutic effect and the molecular mechanisms responsible for this anti-cancer activity. Our preliminary studies found that the X-linked inhibitor of the apoptosis protein (XIAP) was extremely highly expressed in all of the human invasive BC tissues that were tested, but it was barely detectable in all adjacent normal bladder tissues, and that XIAP expression level was also markedly elevated in BBN-induced invasive BC tissues in p53-/-/pRb-/- mice as compared with that from wild-type mice and oncogenic Ras- induced low-grade BCs. We also found that XIAP was significantly higher in cultured human BC cells derived from HGIBC than from those derived from low-grade papillary bladder tumors (LGPBT). Moreover, we showed that treatment of BC cells with ISO inhibited HGIBC T24T cell migration and invasion, was accompanied with specific inhibition of Sp-1 transactivation and XIAP downregulation at the transcription level without affecting cell proliferation at doses of 5-10 ¿M. Thus, we hypothesize that ISO is an effective therapeutic agent for the inhibition of BC invasion in vitro and BC invasion and metastasis in vivo via downregulation of the Sp-1/XIAP pathway. We will test this with the following aims: 1: To test the hypothesis that XIAP downregulation by ISO is responsible for its inhibition of BBN-induced BC formation in the mice lacking both p53 and pRb; 2: To evaluate the hypothesis that SP-1 is a major target for ISO downregulation of XIAP in BC in vitro and in vivo; 3: To define the molecular mechanisms whereby ISO inhibits the BC development in vitro and in vivo. Success of the proposal will facilitate our understanding of the molecular mechanism(s) responsible for the anti-cancer effects of ISO compound. This novel finding will provide valuable information for the design of more effective strategies for utilization of ISO or for the synthesis of other novel conformation- constrained derivatives for the treatment of BCs and other cancers. Taken together with the fact that BC is the most common malignant tumors, responsible for 336,000 new cases and 132,000 deaths annually worldwide, the studies should in turn help improve the clinical outcome of patients with BCs.

Bladder cancer (BC) is among the most common cancers in the Western world. Because high-grade invasive bladder cancers (HGIBC) can progress to life threatening metastases, understanding the molecular mechanisms underlying BC Invasion is of tremendous Importance for reducing the mortality of this disease. The X-linked inhibitor of apoptosis protein (XIAP) is a member ofthe inhibitors of apoptosis protein (lAP) family. In addition to its well-established role in apoptosis inhibition, we recently discovered a new role of XIAP in regulating cancer cell Invasion in vitro. First, knockout of XIAP decreased colon cancer cell migration and invasion in vitro and metastasis of these cells to the lungs in vivo. Conversely, resumption of XIAP expression in XIAP-depleted cancer cells restored their migration. Second, Rho dissociation Inhibitor (RhoGDI) is a principal downstream target of XIAP in regulating cancer cell migration. XIAP Interacted with RhoGDI and in so doing inhibited RhoGDI SUMOylation via its RING domain. Third, XIAP was highly expressed in human invasive BC tissues, but not in adjacent normal urothelial tissues. XIAP expression was also markedly elevated in N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-lnduced invasive BC tissues in p53- l-lpRb-l- mice, while it was almost undetectable in wild-type mouse urothelia and was low in oncogenic Ras- induced low-grade BCs. Fourth, knockdown of XIAP led to a marked decrease of HGIBC cell migration. Finally, XIAP was significantly up-regulated in the cells harboring p53 mutations, a genetic alteration highly prevalent in HGIBC. Our data from cell culture, transgenic mice and human tissues, demonstrating XIAP over-expression in the invasive BCs, prompt us to hypothesize that XIAP plays an important role in promoting/enhancing BC invasion. We will examine this hypothesis with three Specific Aims: 1: To test the hypothesis that p53 mutations activate XIAP expression via the upregulation of Spl and/or NFKB in BC cells; 2: To define the functional domain and molecular mechanisms whereby XIAP regulates BC cell invasion; 3: To examine the hypothesis that urothelium-specific over-expression of XIAP in transgenic mice can promote BC Invasion in vivo and that loss of XIAP in knockout mice or its RING domain in XIAPARNG knockin mice renders these mice resistant to invasive BC development. Our proposed studies will contribute in a major way to the understanding of the molecular basis of invasive BC. They will also pave the way for us to use XIAP as a novel prognostic biomarker and a therapeutic target for invasive BC. This should in turn help improve the clinical outcome of these patients. RELEVANCE (See instmctions): Although the invasive form of bladder cancer (BC) is responsible for all the deaths resulting from this disease, little is known biologically about what triggers BC invasion. We recently found a new role of XIAP previously known as an inhibitor of apoptosis, in cell migration and invasion. By studying the mechanisms leading to increased XIAP in BC cells and the role of XIAP in BC invasion using mouse models, we hope to determine whether XIAP can be used a prognostic marker and/or a therapeutic target of Invasive BC.

The three research projects that comprise this Program Project are all focused on understanding molecular and cellular events and mechanisms leading to bladder carcinogenesis of both major pathways of low-grade, non-invasive and high-grade, invasive bladder cancer. In addition t o t h e intellectual and experimental synergies that link the individual projects into a cohesive program, each ofthe three proposed research plans all depend upon a common set of critical reagents and services made available on a continuous basis for their success. These include but are not limited to a set of cultured cell lines that represent different stages of bladder cancer, different unique mouse models of bladder cancer developed by Dr. Xue-Ru Wu, recombinant proteins, nucleic acid reagents, antibodies and cDNA constructs. Obviously, it would not make sense, nor would It be an efficient use of time, money, and resources for each laboratory to Independently generate, procure, characterize, and maintain all these reagents. Instead, we will establish a centralized Reagent and Service Core, one of whose purposes is to serve as a central repository for key reagents and provide a central supply for these important research tools that can be shared by all involved in this Program Project. In addition to the cost-effectiveness, this approach offers the advantage of ensuring the quality of these shared reagents across the individual projects. Providing supplies of cell lines, tissues and animal models from a centralized core eliminates variability in reagent handling, facilitating data comparison amongst the three individual projects. Furthermore, it promotes additional interactions among the Pis and guards against accidental loss of Invaluable reagents in emergency situations such as power outages (which we last experienced in 2003). Finally, the Core will also provide pathology and statistical support for the individual projects. In this section ofthe Program Project application, we describe these reagents, how the Core functions to control quality and maintain databases, the contribution ofthe core to the individual research projects, and a plan for prioritizing usage of core services. The specific aims ofthe Core are: i) To maintain a centralized supply and distribute a common core set of reagents vital to support the research efforts of individual component projects; ii) To collect human and mouse specimens and provide the necessary pathology support service; and iii) To provide dedicated statistical support.

Project Summary Human bladder cancer is the sixth most common cancer in the United States. Discovery and evaluation of new alternative medications is of tremendous importance for reducing the bladder cancer mortality. Chinese herb Gnetum Cleistostachyun has been used for treatment of bladder cancers for centuries, but its bioactive components and anti-cancer mechanisms have been barely explored. Our recent studies discovered that Isorhapontigenin (ISO), a new derivative of stilbene compound isolated from this herb, exhibited multiple anti- cancer activities in human high grade invasive bladder cancer cells. Our preliminary studies provided strong evidence on ISO inhibition of tumor growth both in vitro and in vivo. In addition, the robust induction of SESN2 and BECN1, the important autophagy regulator and effector, have been shown as a critical event for ISO's anti-cancer activity in vitro, as depletion of either SESN2 or BECN1 significantly impaired the capacity of ISO to inhibit tumor cell growth. However, many questions, such as whether ISO affects cell invasion and tumor metastasis in vivo, and whether SESN2/BECN1 mediates ISO's in vivo activity, as well as upstream regulators being responsible for their upregulation by ISO, remain unknown. Therefore, in this application, three specific aims were proposed to address key events in SESN2/BECN1-mediated ISO tumor inhibition. The first Aim will target the potential upstream regulators and epigenetic mechanism that mediate ISO-induced upregulation of SESN2 and BECN1. The second Aim will employ both in vitro and in vivo approaches to address the functional relevance of SESN2 and BECN1 as well as new candidates obtained from Aim 1 in ISO inhibition of tumor invasion and metastasis. The last Aim will focus on in vivo role of SESN2 and BECN1 in BC development using BBN-induced mouse bladder carcinogenic model. The results obtained from the proposed studies will determine whether ISO specifically initiates the SESN2/BECN1/autophagy pathway to inhibit bladder tumor formation, invasion, and metastasis. The success of this proposal will facilitate our understanding of the molecular basis of ISO anti-cancer activity and will also provide new insights for developing a better therapeutic strategy for human high grade invasive bladder cancer.

Abstract Decades of both epidemiological research and in vitro and in vivo laboratory studies have consistently reported an association between environmental nickel exposure and lung cancer. As early as 1990, the International Agency for Research on Cancer (IARC) classified nickel compounds as Group 1 carcinogens: substances confirmed as carcinogenic to humans. Although the multiple mechanisms, including oxidative stress, epigenetic effects, and activation of signaling pathways that trigger differential gene regulation has been thought to be associated with nickel carcinogenic effect, there is no evidence that a single nickel- inducible factor can drive transformation of human bronchial cells, to the best of our knowledge. In our Preliminary Studies, we performed long noncoding RNA (lncRNA) deep sequencing using the Illumina HiSeqTM2000/2500 high throughout system to evaluate whether nickel affects the abundance of 5,929 known lncRNAs. Nickel treatment altered the levels of 24 of these lncRNAs in normal primary human bronchial epithelial cells (HBECs), while 16 of them were verified in Real-time PCR assay. With overexpression and/or knockdown analyses of the 16 lncRNAs, we further discovered that a reduced abundance of maternally-expressed gene 3 (MEG3) was sufficient for malignant transformation of HBECs. Our demonstration that MEG3 inhibition can independently transform HBECs provides the basis of our central hypothesis that MEG3 downregulation drives transformation and tumorigenecity of HBECs after nickel exposure. Here we propose to elucidate the molecular mechanisms that underlie our novel findings that MEG3 downregulation is a crucial driver for nickel-induced malignant transformation of HBECs with the following Specific Aims: 1, To test the hypothesis that p62 and C-Myc define an important MEG3-regulated axis that promotes transformation of HBECs upon MEG3 deficiency; 2, To test the hypothesis that the crosstalk between a p62/C-Myc cascade and the PHLPP/HIF-1? axis causes the MEG3-deficiency- correlated malignant transformation of HBECs; 3, To explore the biological significance of MEG3 deletion and its activated downstream molecules in lung tumoriginecity. Our novel Preliminary Findings suggest that MEG3 is downregulated by nickel exposure both in vitro and in vivo, that knockdown of MEG3 alone can transform HBECs, and that crosstalk between the putative signaling transducers downstream of MEG3 mediate the malignant transformation of HBECs caused by MEG3 deficiency. This proposal's strengths are the complementary use of cell culture models and novel conditional MEG3 knockout mouse models to examine integration of the molecular events that account for nickel-mediated lung carcinogenesis. Clarifying these issues will provide valuable insights regarding MEG3 as a prognostic biomarker and/or as a therapeutic target. Ultimately, both uses could improve clinical outcomes in lung cancer patients.