Last Updated: 09/13/10
University of Colorado Cancer Center
Project 1: SMEA3F and ZEB1 in Lung Cancer: Therapy & Target Gene Discovery
Principal Investigators:
Harry Drabkin, MD
Robert Gemmill, PhD
Daniel Chan, PhD
A major component in the development / progression of lung cancer is loss of the tumor suppressor genes, E-cadherin and the secreted semaphorin, SEMA3F. We originally identified the SEMA3F gene and reported correlation of downregulation with advanced-stage disease. SEMA3F binds high affinity Neuropilin receptors on tumor and endothelial cells, which mediate its biologic actions together with plexin-A co-receptors. SEMA3F potently inhibits tumor cells in vitro and in vivo, and has additional anti-angiogenic effects on endothelial cells, where the Neuropilins function as co-receptors for VEGF. We reported that SEMA3F downregulates activated v3 integrin in tumor cells with loss of phospho-ERK, AKT and STAT3, and inhibitory effects on HIF and VEGF. Our current data indicate that SEMA3F also inhibits certain non-VEGF angiogenic factors. Thus, SEMA3F can block multiple pathways of neo-angiogenesis, emphasizing the therapeutic potential of this secreted tumor suppressor. However, it’s important to determine if local production of SEMA3F is required for these effects, or whether exogenously administered SEMA3F also affects tumor cells, endothelial cells, or both.
In lung cancer, we’ve shown that ZEB-1 is the major E-box transcriptional repressor regulating E-cadherin. E-box repressors are responsible for the epithelial-mesenchymal transition (EMT), which underlies the invasive / metastatic nature of epithelial cancers. We’ve also shown that ZEB-1 expression and E-cadherin loss confers resistance to EGFR inhibitors, which has led to three ongoing cancer trials combining erlotinib with SAHA, MS-275 or celecoxib. We’ve now discovered that ZEB-1 is linked to SEMA3F regulation. Furthermore, we are beginning to discover molecules that underlie the mechanism of EGFR inhibitor resistance observed with ZEB-1 over-expression.
Two Specific Aims are proposed. In Aim 1, we will: 1) determine whether constitutive expression of VEGF can overcome the anti-tumor activity of SEMA3F, 2) determine whether exogenously administered SEMA3F affects tumor cells, endothelial cells or both, and 3) define a signature of SEMA3F responsiveness in lung tumors. In Aim 2, we will investigate the mechanism of ZEB-1 induced resistance to EGFR inhibitors. In addition, we will determine if selected MAGE genes, which appear to be regulated by ZEB-1, can provide a complementary biomarker for the EMT process in tumors and pre-malignant lesions.
Project 2: Growth Factor Inhibitors for Lung Cancer Therapy and Prevention
Principal Investigators:
Paul A. Bunn, Jr., MD (Applied)
Daniel C. Chan, PhD (Basic)
The long-term goal of this project is to develop novel therapeutic and chemoprevention strategies for lung cancer through combinatorial inhibition of different autocrine / paracrine growth factor signal pathways. Previously, we developed a novel bradykinin-derived “biased agonist”, CU201, defined its spectrum of activity and pharmacokinetic and toxicity profiles. In the next grant cycle, we will precisely define the molecular mechanism of action of CU201 including the requirement for the bradykinin B2 receptor (BK2R) and specific downstream signal pathways. We will also complete late preclinical and early clinical development of CU201. During the past grant, EGFR-specific inhibitors emerged as therapeutic agents with activity towards a subset of lung tumors. Our studies defined predictive biomarkers for selection of patients for EGFR inhibitor therapy including a specific gene expression pattern indicative of more-differentiated epithelium as well as EGFR gene amplification. Importantly, the limited action of EGFR inhibitors, when employed as single agents, indicates that additional growth factor autocrine loops participate in lung tumorigenesis. Our preliminary studies, in fact, provide compelling evidence for the frequent activity of FGF and FGFR-mediated autocrine signaling in NSCLC and SCLC cell lines. Based on our published and preliminary studies, we hypothesize that multiple autocrine and paracrine growth factor signaling pathways driven by EGFR, FGFRs and BK2R participate in the pathogenesis of lung cancer. Moreover, we hypothesize that specific biomarkers and gene expression signatures can be identified that will predict the autocrine growth loops, either singly or combined, that are active in a given lung tumor. To explore these hypotheses, we will complete these specific aims.
Aim 1: Determine the role of bradykinin receptor (BK2R) and specific signaling pathways that dictate therapeutic responsiveness to the bradykinin receptor antagonist, CU201. 1A.Define the mechanism of action of CU201 and the role of BK2R in CU201 efficacy. 1B. Explore additive / synergistic interactions of CU201 with rationally selected agents. 1C. Determine the frequency and prognostic relevance of BK2R expression in our lung cancer TMAs and preneoplastic tumors. 1D. Conduct a phase I clinical trial of CU201.
Aim 2: Define specific FGFs and FGFRs as components of a novel autocrine signaling pathway in NSCLC and SCLC that can be targeted with existing FGFR inhibitors. 2A.Determine the correlation of FGF2, FGF9, FGFR1c and FGFR2c mRNA and protein expression in NSCLC and SCLC cell lines with response to FGFR TKIs (pazopanib) and an inhibitory FGFR1c mAb (IMC-A1) .2B.Test for additive/synergistic growth inhibition by rationally designed combinations.2C. Determine the frequency and prognostic relevance of FGF2, FGF9, FGFR1c and FGFR2c expression in our lung cancer TMAs and preneoplastic tissues.
Project 3: Prostacyclin and Peroxisome Proliferator-Activated Receptor in Lung Cancer
Principal Investigators:
Raphael Nemenoff, PhD (Basic)
Mark Geraci, MD (Applied)
Increased prostaglandin (PG) production has been associated with many types of cancer including lung cancer. Inhibition of cyclooxygenase (COX, PGH2 synthase) decreases PG production and prevents lung cancer in animal models. Prostacyclin (PGI2) is a PGH2 metabolite with anti-proliferative and anti-metastatic properties. Our laboratory has shown that targeted overexpression of PGI2 synthase or chemoprevention with the PGI2 analog iloprost reduced lung tumor multiplicity and incidence in mice, suggestingthat manipulation of the arachidonic acid pathway downstream from COX is a target for prevention of lung cancer. These studies led to a chemoprevention trial in which patients at risk for lung cancer are treated with iloprost. Studies performed during the previous funding period have shown that the anti-tumorigenic effects of PGI2 are not mediated through its cell-surface receptor (IP), but instead via activation of the peroxisome proliferator-activated receptor- (PPAR). Retrospective studies indicate that thiazolidinediones such as rosiglitazone or pioglitazone, which are specific PPAR activators, reduce the risk of lung cancer. We have shown that mice with targeted overexpression of PPAR are also protected against lung tumorigenesis. In human NSCLC cell lines, PPAR activation inhibits transformed growth and invasiveness, and promotes differentiation. These effects are associated with inhibition of COX-2 and decreased production of angiogenic cytokines. PGI2 and its analogs activate PPARin non-transformed epithelial cells but not in many NSCLC lines. We have demonstrated that the ability of PGI2 to engage PPAR in NSCLC is correlated with the expression of the Wnt family receptor Frizzled 9 (Fzd9). The goal of the current proposal is to examine the role of PGI2 and PPAR in the development and progression of lung tumors. Studies will use human NSCLC cell lines and mouse models to define molecular effectors and markers of response. These findings will be applied to analyze human samples from the iloprost trial and a new proposed pioglitazone chemoprevention trial, as well as samples from human lung tumors. Three specific aims are proposed. Aim 1 will use in vitro approaches to define biomarkers of iloprost and pioglitazone sensitivity in a panel of NSCLC, and to examine interactions between these agents and EGFR-TKIs. Aim 2 will use a chemical carcinogenesis mouse model to examine the effects of agent combinations, including iloprost plus pioglitazone and iloprost or pioglitazone with erlotinib. Xenografts of human NSCLC will examine the interactions between iloprost and Fzd9 and establish the basis for therapeutic trials using iloprost in combination with other agents. Aim 3 will use expression profiling of samples from chemoprevention trials to define expression signatures of dysplasia and response to iloprost or pioglitazone. Expression of molecules studied in Aims 1 and 2 will be examined in human tumors and correlated with clinical data. These studies will establish the role of this pathway in lung cancer initiation and progression, and help define new therapeutic targets.
Project 4: The Biology of Pulmonary Premalignancy: Application to Risk Assessment and Chemoprevention.
Principal Investigators:
York E. Miller, MD (Applied)
Tim E. Byers, MD MPH (Epidemiologist)
Fred R. Hirsch, MD PhD (Basic)
Marileila Varella-Garcia, PhD (Basic)
The overarching goal of this project is to reduce the lung cancer burden through the discovery and translation of knowledge regarding the biology of broncho-pulmonary premalignancy. Specifically, we aim to discover and validate clinically useful biomarkers of risk in sputum and to identify early events in pulmonary tissues that lead to lung cancer. Biomarkers of lung cancer risk in both sputum and bronchial tissues could have many clinical uses, including population screening for early detection, either alone or in combination with CT imaging, and to assist in clinical decisions regarding the management of patients found to have a pulmonary nodule of undetermined significance. We have established unique prospective cohorts of subjects in whom both prevalent and incident lung cancer is tracked, and we harvested and stored biological samples from them. We will build on our considerable preliminary evidence to further optimize and then validate a panel of biomarkers in sputum, including cytology, chromosomal aneusomy, and gene promoter methylation. We will also continue to develop tissue biomarkers of lung cancer, various types of biomarkers in bronchial alveolar lavage (BAL), and proteomic biomarkers in the blood.
Specific Aim 1. Identify and validate candidate biomarkers in sputum that may be useful for population screening and clinical decision making. We will test additional probes in chromosomal regions known to be amplified in lung cancer, and will assess additional methylated genes. We will then optimize panels of multiple biomarkers from both types of measures, as well as sputum cytology, for the prediction of lung cancer risk. We will subsequently assess the most promising set of biomarkers in a validation set of subjects sampled from the National Lung Screening Trial, an ongoing trial of CT screening. Analyses will determine the performance of this panel of sputum biomarkers for lung cancer screening, as well as for aiding clinical decisions regarding the management of pulmonary nodules of undetermined significance.
Specific Aim 2. Assess tissue biomarkers of lung cancer risk and evaluate tissue biomarkers as intermediate biomarker endpoints in chemoprevention trials. We will continue to identify and characterize biomarkers on the causal pathway to lung cancer from lung tissues, including bronchial biopsies and both cells and proteins found in BAL fluids. BAL is an understudied sample source that could well be very useful in the diagnosis of lung adenocarcinomas arising from for peripheral nodules. We will continue work tying molecular changes to bronchial histology, bronchial histology to lung cancer risk, and assessing biomarker modulation in past, current, and future chemoprevention trials.
Tissue Bank / Biomarkers Core
Directors:
Wilbur A. Franklin, MD
Fred R. Hirsch, MD, PhD
Since its establishment in 1992 the Colorado SPORE Tissue Bank and Biomarkers Core Laboratory (TBBC) has consistently aimed to 1. provide well characterized tissues and products derived from those tissues to SPORE investigators, 2. assess status of submitted specimens by histological, immunohistochemical and fluorescence in situ hybridization, 3. link specimens to clinical data including outcomes in rigid compliance with standards for maintenance of patient confidentiality and informed consent. The development of targeted treatments has engendered an urgent need to know the status of the targeted pathway, the most striking examples of which are tyrosine kinase receptor and arachidonic acid pathways. RNA, DNA and protein biomarker status can efficiently be analyzed using core equipment and standardized methods in a single core laboratory. The Core will accordingly perform standardized molecular tests including quantitative RT-PCR gene copy number by FISH and mutational analysis on extracts of tissue specimens obtained in support of individual SPORE research projects. Data from this testing is centrally tracked and distributed to SPORE clinical and basic science investigators who may then incorporate these data into hypothesis generation and testing. The expanded role of the Core in biomarker testing has prompted a name change for the core to Tissue Bank and Biomarkers Core Laboratory. The Core provides specimens and testing results for invasive tumors and is the central biorepository for unique and actively accruing early detection and chemoprevention trials for both the Colorado SPORE and the national SPORE program, collecting data and specimens from high risk patients without carcinoma at the time of enrollment. The Core is a unique source of biological materials that are used to investigate molecular changes that accompany and may predict invasive tumor. Remnant tissues and tissue products are made available to investigators on the basis of scientific merit, tissue availability and IRB approval after review by the Tissue Bank Committee. Specific services provided by the Core include consenting and enrollment of patients into tissue collection trials, preparation of kits for efficient sample collection and storage, sample procurement including but not limited to retrieval of tissue from operating and bronchoscopy suites, barcoding, accessioning and proper storage of SPORE specimens, histological sectioning and diagnosis of SPORE tissue samples, immunohistochemistry, and fluorescence in situ hybridization (FISH). In addition, specimens are prepared for RT-PCR, mutational analysis and oligonucleotide microarray studies required in the SPORE projects. Finally, specimens and data, including images (see Bronchial Map Project) are tracked through the central SPORE computer system and are available to SPORE investigators for outcome and clinicoepidemiological correlations.
Clinical Trials Core
Director:
Robert Keith, MD
The goal of the Clinical Trials Core is to provide support for clinical trials designed and implemented by SPORE project investigators. The support includes assistance with trial preparation, all regulatory issues, data safety and monitoring, auditing, conduct, and reporting. Our ever increasing understanding of the molecular basis of lung cancer has reinforced the need to continue conducting studies which involve the collection of both clinical data and specimens for molecular analyses. This translational approach has allowed us to investigate biological pathways of lung carcinogenesis in human tissue, and has acted as a powerful tool in the evaluation of novel, biologically-rationale strategies for the prevention, screening, early detection, and treatment of lung cancer. The Clinical Trials Core was created to encourage interactions between basic and clinical researchers to generate pivotal translational research clinical trials and provide the necessary infrastructure to develop and conduct clinical investigations. We offer expertise in clinical trial methodology, protocol writing, regulatory documentation and quality assurance measures. A sequel server clinical database has been established through the collaborative efforts of the Clinical Trials, Tissue Bank and Biostatistics/Informatics Cores, including the collection of common data elements (CDEs) that allows for the pooling of data from trials conducted at other investigational sites. We provide clinical research associates to accrue subjects and collect data/tissue samples for all SPORE-initiated trials. All data is entered into a web database designed to link the clinical information to the biological correlative studies for future analyses. Over the past five years there have been 10 clinical trials supported by the Core. These trials have enrolled 1,151 subjects, and additional trials are being planned. Currently, seven trials are actively accruing new subjects. A chemoprevention trial evaluating the activity of Iloprost, an oral prostacyclin analogue, was activated during the last grant cycle and should finish accruing subjects in early 2008. The success of the core has led to numerous publications. In the past grant year alone, 23 peer-reviewed manuscripts have been published. Several manuscripts have been published describing our ability to identify NSCLC patients who will respond to tyrosine-kinase inhibitor therapy and a seminal manuscript reported on promoter hypermethylation of multiple genes in sputum samples preceeding lung cancer development in our high-risk cohort. Basic researchers have reported on gene expression profiles predicting sensitivity to EGFR inhibitors, as well as a biomarker examination of dysplastic bronchial epithelium for VEGF and c-ErbB1/B2 to gain a better understanding of their potential role as chemopreventive targets. Manuscripts describing the association of Ki-67 labeling index with gender and smoking status, but not the presence of lung cancer or COPD, and summarizing our experience with sputum cytology and the development of lung cancer have recently been published. In preparation are manuscripts correlating bronchial dysplasia to patient characteristics (arising from numerous SPORE trials) and a manuscript summarizing the results of a chemoprevention trial of 13-cis retinoic acid with or without alpha-tocopherol. Overall, the Colorado Clinical Trials Core has been very productive in translating the science generated from each of the individual projects.
Biostatistics and Informatics Core
Core Directors:
Anna E. Barón, PhD
Jessica Bondy, MHA
The Lung Cancer SPORE Biostatistics and Informatics Core provides support in the areas of biostatistics, clinical informatics, and bioinformatics to SPORE investigators. This consultative and collaborative support is designed to increase the speed and efficiency with which lung cancer research is translated from the lab to the clinic. The Core is designed to provide support in study design, data management, data analysis, clinical informatics system creation, bioinformatics data management and interpretation. The Biostatistics group assists primarily with study and protocol design, and data analysis and interpretation. The Informatics group assists with data quality control, data sharing, designing and maintaining the SPORE database that captures clinical, pathologic, and laboratory data into a relational database. This group also designed the barcode system for collection and storage of all samples. The Bioinformatics group assists with computational evaluations of large databases, especially those created with genomic and proteomic analysis using gene expression and SNP arrays and proteomic profiles. All Core members participate in preparation of reports, presentations, and manuscripts. In addition to these services, Core members will continue their efforts to develop new approaches to improve the efficiency and outcomes of the process of translational research.
Biostatistics and Informatics Core members will assist SPORE investigators in the following areas:
- Experimental design: Design of both pre-clinical and clinical experiments that can provide useful answers to scientific questions of importance in lung cancer.
- Data collection/storage/retrieval/sharing: Creation and maintenance of a sound and user-friendly infrastructure for data collection, storage, quality assurance, retrieval, and sharing in support of SPORE trials and tissue banking.
- Data analysis and manuscript preparation: Structuring of data analyses to provide clear answers to questions, and to communicate those findings in reports and papers.
- Translational research methodology: Development and implementation of coherent methods that improve the efficiency and effectiveness of research across the wide spectrum from pre-clinical research to clinical studies, including work in the development of better understanding of the causes of lung cancer, early detection of lung cancer, biomarkers of lung cancer risk, and lung cancer therapeutics.
Administration Core
Core Director:
Paul A. Bunn, Jr., MD
Core Co-Director:
York Miller, MD
Core Administrator:
Donna Berrier
The goal of the administrative core of the University of Colorado Lung Cancer SPORE is to provide outstanding administrative and fiscal support for the entire program effort and to provide the scientific leadership for the program. The administrative core will oversee all administrative and scientific activities of the SPORE program, review and regulate financial expenditures, develop and prepare reports. The Administrative core consists of the two SPORE principal investigators, Drs. Bunn and Miller; administrator, Donna Berrier, a 40% grants manager/administrative assistant and 10% financial manager. This core will also develop and circulate research conference schedules, coordinate scientific review, schedule the monthly scientific meetings, aid project investigators in the preparation and publication of manuscripts, and maintain a record of all publications emanating from this grant. It will oversee the planning and evaluation efforts including the scheduling of visits by the external advisors, the planning and coordinating of the yearly internal retreats, yearly NCI SPORE meetings, the scheduling of meetings which include the Executive Committee, Developmental Research Committee and Career Development Committee and the SPORE advocacy program. The Administrative Core will coordinate and facilitate communication and travel with Project 1 principal investigators located at the Medical University of South Carolina. The Administrative Core works with the SPORE investigators and NCI program staff to insure compliance with all federal regulations and reporting requirements. It will coordinate activities with the Cancer Center and with other SPOREs to ensure that there is no redundancy, and to ensure joint projects are conducted in the most economical way. The Administrative Core will assist in community outreach efforts particularly with respect to public relations and community activities through the established Cancer Center mechanisms. This core provides support for the development and career development programs as well as the Visiting Scientist Program. The Administrative core oversees the functioning of the other three SPORE core resources.
