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Project 1: Intestinal Microbiota and Risk of Recurrent Adenomas

Co-Leaders: Temitope Keku, PhD and Robert Sandler, MD


Evidence from animal and human studies suggests that intestinal bacteria may contribute to the pathogenesis of colorectal cancer (CRC), a leading cause of cancer mortality in the United States. We propose to test the hypothesis that adherent colonic bacteria are linked with elevated risk of recurrent colorectal adenomas and that these bacteria modulate the association between diet, inflammation and colorectal adenomas. We propose that distinct patterns of commensal bacterial community composition or presence/absence of specific bacteria species will correlate with recurrent adenoma risk.

The specific aims are to 1) determine whether the adherent (mucosa-associated) bacterial community membership and structure at baseline persist over 3-5 years; 2) assess whether the adherent bacterial community membership and structure at baseline predict recurrent adenomas 3-5 years later; 3) evaluate whether systemic or local markers of inflammation (IL-23, IL-17, IL-8, IL-10, IL-6 and TNF-α) at baseline predict recurrent adenomas 3-5 years later.

Evaluation of the diversity of gut bacteria in relation to disease in humans is limited, in part, by the difficulty growing these organisms in culture. Recent advances in molecular methods have made it possible to assess the role of intestinal microbiota in diseases such as colon cancer. To test our hypothesis, we propose to use molecular-phylogenetic methods based on the highly conserved bacterial 16S rRNA gene to assess the contribution of intestinal microbiota to the development of colorectal adenomas. These methods include PCR amplification of the 16S rRNA gene and sequence analysis (454 pyrosequencer). Conducting the study in a longitudinal manner will minimize the confounding impact of inter-individual variation inherent in the human colonic microbiota.

The proposed study optimizes return on federal investment in cancer research by using specimens that have been collected, processed and analyzed through other NCI-funded studies directed by the SPORE investigators. Baseline biopsies, exposure information, and inflammatory markers will be obtained from an ongoing study of colorectal adenomas: Inflammation, Obesity, and Risk of Colorectal Cancer: The Diet and Health Study (R01 CA 44684-18). Baseline data on the adherent bacteria will be obtained from an ongoing study: “Intestinal Microbiota, Diet and Risk of Colorectal Adenomas” (R01 CA136887-01). The proposed study will prospectively obtain and analyze biopsies from follow-up procedures to evaluate the permanence of the microbiota and their role in recurrent adenomas.

Limited information exists on the role of gut bacteria in the development of adenomas. This study will provide critical insights on the composition and diversity of the microbiota and their association with recurrent colorectal adenomas and known risk factors. Using conservative figures, we estimate that the cost of using colonoscopic polypectomy to manage adenomas in the US each year is $1.6 billion. In addition to the discovery of important and unique biological information on colorectal carcinogenesis, the findings from this study could lead to the development of novel strategies for diagnostic and prognostic assessment, as well as therapeutic (prebiotic/probiotic) manipulations of the intestinal microbiota to prevent colorectal adenomas and cancer.

Project 2: Targeting GSK-3 and IKK in Pancreatic Cancer

Co-Leaders: Albert Baldwin, PhD and H.J. Kim, MD


Because of the difficulty in early detection, the aggressive phenotype of the disease, and resistance to standard forms of cancer therapy, pancreatic cancer exhibits extremely poor outcome. For these reasons, new therapeutic options for pancreatic cancer must be developed which must come from new insight into the pathways that are dysregulated in this disease. Activation of the transcription factor NF-kB is involved in inflammatory and immune responses and is strongly associated with oncogenesis and resistance to a variety of cancer therapies. Work from several groups, including our own, indicates that NF-kB is activated in pancreatic cancer cell lines and in human pancreatic tumors. Additionally, studies using pancreatic cancer cell lines show that NF-kB activation promotes chemotherapy resistance. Our published data indicates that the NF-kB pathway is important for growth/survival of several pancreatic cancer cell lines. Recently work from the Billadeau group indicates that the kinase GSK-3b is activated and nuclear in pancreatic cancer cells and in tumors, and that this kinase promotes the activation of NF-kB target genes. The use of a GSK-3b inhibitor blocked pancreatic cancer growth and induced apoptosis, potentially through the inhibition of NF-kB. We had earlier demonstrated an involvement of GSK-3b in controlling a subset of genes regulated by NF-kB. Our recently published work agrees with the observed effects of the GSK-3b inhibitor, but indicate that GSK-3a may be the more important kinase in controlling NF-kB in pancreatic cancer cells and in controlling cell growth/survival. Analyzing pancreatic cancer cells lines we show that some cells are more dependent on IKKa while others are more dependent on IKKb inhibition, with all cells being sensitive to GSK-3a/b inhibition. Preliminary data indicate that a GSK-3 inhibitor blocks growth of a human tumor xenograft, supporting the results described above.

Our goals are to: (i) determine how GSK-3 isoforms regulate NF-kB activity in pancreatic cancer cells focusing on control of the TAK1 and Notch1 pathways, (ii) determine the differential involvement of IKKa vs. IKKb in pancreatic cancer cells and analyze whether nuclear localization of IKKa or IKKa/b phosphorylation and/or an IKK subunit specific gene signature predicts relative activity and sensitivity to inhibition, (iii) analyze human pancreatic tumors for detection of markers of IKK, TAK1, and GSK-3 activation and for NF-kB-dependent gene expression signatures, and (iv) determine if IKKa or IKKb, and GSK-3a/b pharmacologic inhibitors (alone or in combination approaches – including chemotherapy) will function as therapies for pancreatic tumors developing in a genetically engineered murine model of pancreatic cancer and in nude mice transplanted with tumor tissue from human patients. Furthermore, we will determine if a hedgehog inhibitor will augment responses to GSK-3 or IKK inhibition. The goals are to develop a new understanding of the molecular pathways dysregulated in pancreatic cancer and to validate these findings using pharmacologic inhibitors. The ultimate goal is to initiate clinical trials based on the findings derived from these studies.

Project 3: ERBB3 as a Therapeutic Target against Colorectal Cancer

David Threadgill, PhD and Bert O’Neil, MD


Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and accounts for the second largest number of cancer deaths in Western societies (American Cancer Society, 2008 estimates). One of the major molecular targets to arrise over the last decade is the epidermal growth factor receptor (EGFR), a member of the ERBB family of receptor tyrosine kinases and a major mitogenic signal transducer used by many epithelial cell types, including those of the gastrointestinal tract. Although in vitro studies suggested that EGFR inhibitors should be efficacious, clinical response to EGFR inhibitor monotherapy is rare. During the previous grant period, we used several approaches to demonstrate that a subset of CRCs arises independently of EGFR activity. Using mouse models of CRC with genetically-defined EGFR sensitivities, we identified a unique gene expression signature that can distinguish CRCs based upon their dependency on EGFR and sensitivity to EGFR inhibitor therapy. The gene expression signature was found to be constitutionally present within cancers before therapeutic intervention and the genes comprising the signature lead to the hypothesis that another member of the ERBB family, ERBB3, is more important for CRC development and may contribute to the resistance of human CRCs to EGFR inhibitor therapy by heterodimerizing with other ERBB family members. This hypothesis was validated by genetically ablating Erbb3 in mouse models of CRC and human CRC cell lines. In this renewal, therapeutic inhibition of the naturally kinase dead ERBB3 using blocking monoclonal antibodies will be investigated as a new therapy against CRC by 1) Examining the sensitivity and specificity of ERBB3 pharmacological inhibition in vivo, 2) Investigating the potential for ERBB3 combinatorial therapies, and 3) Evaluating the efficacy of ERBB3 inhibitor therapy in patients.

The proposed research is paricularly relevant to improving the outcome from CRC. Clinical trials to date using EGFR inhibitor therapies have resulted in inconsistant and sometimes contradictory results. Our previous data suggest that EGFR is not an optimal target for CRC. Rather, the related kinase dead receptor ERBB3 appears to be a critical signal medator for CRC growth. Since ERBB3 does not have a functional kinase, blockade by small molecule inhibitors is not possible. Consequently, the translational experiments proposed in this renewal project, based upon strong preclinical data, will directly evaluate the utility of ERBB3 inhibitor therapy using a novel antibody-based therapeutic. The resutls of the proposed studies should lead to a new therapy for CRC that has greater efficacy than currently available therapies.

Project 4: Targeting KRAS Effector Signaling for Colorectal Cancer Treatment

Co-Leaders: Channing Der, PhD and Jen-Jen Yeh, MD


In this revised proposal we return to our previous focus to target Ras effector signaling for CRC treatment. This return is prompted by [1] our validation of the RalGEF-Ral effector pathway in CRC, [2] our establishment and acquisition of new mouse models for the study of KRAS-driven CRC, [3] our recent acquisition of clinical candidate inhibitors for the Raf and PI3K effector pathways, and finally, [4] reviewer concerns that we had prematurely abandoned our CRC studies. Only recently has KRAS mutation status been recognized as a biomarker of resistance to anti-EGF receptor therapies. Therefore, given the validation from recent genome-wide analyses of CRC that the KRAS oncogene is the most frequently mutated oncogene in CRC (50%), there is a considerable need to develop anti-Ras-targeted therapeutic strategies for CRC treatment. Currently, the most promising directions for anti-Ras drug discovery are focused on targeted inhibition of downstream effector signaling. However, this issue is complicated by the multitude of effector signaling pathways that Ras may utilize to promote oncogenesis. Is there any one effector pathway that is the “best” therapeutic target? Will concurrent inhibition of multiple effector pathways be required for effective inhibition of Ras? With the recent availability of clinical candidate inhibitors of each component of the Raf-MEK-ERK and PI3K-AKT effector pathways, and with our validation of a third effector pathway (RalGEF-Ral) in CRC, we now hypothesize that pharmacologic targeting of the PI3K-AKT or RalGEF-Ral signaling networks, either alone or in combination with Raf-MEK-ERK inhibition, will provide effective anti-Ras therapy for CRC. We propose four new specific aims to: [1] determine the specific role for the RalGEF-Ral effector pathway in KRAS-dependent CRC growth, [2] determine the contribution of five key Ras effector pathways in KRAS-dependent CRC growth, [3] identify therapeutic approaches for blocking Ral-mediated oncogenesis in CRC, and [4] determine if coordinate inhibition of the Raf, PI3K and RalGEF effector pathways will be effective for CRC treatment. Our overall goal to rapidly translate our findings to the clinic will be accomplished through the use of state-of-the-art mouse models that will better reflect patient response, including patient-derived xenografts and a KRAS-driven CRC GEMM, in these aims. Therefore lead candidates and combinations identified from our studies in these mouse models will direct our design of Phase II clinical trials for CRC at the completion of this grant period.

Project 5: Pharmacogenetic Dissection of Colorectal Cancer Therapy

Co-Leaders: Howard McLeod, PhD and Richard Goldberg, MD


A big obstacle to preventing death from colorectal cancer is the development of clinical resistance, of which there is a paucity of mechanistic information. While the development of new drugs for metastatic colorectal cancer offers hope for the next decades, there are also opportunities now to understand and improve the use of current therapies. Most of our understanding of the mechanisms of drug resistance has been derived from preclinical experiments, primarily in human cancer cell lines. This has taught us some important lessons about the process by which a cell can escape the effects of cytotoxic drug therapy. While a continued effort in preclinical systems is useful, there is still the concern that we are missing the most relevant mechanisms of drug resistance if we don’t anchor our efforts in findings from real patients. Therefore, we are pursuing an ‘enriched’ approach for genomic discovery, whereby we focus our initial efforts on clinically resistant tumor from surgically resected tumor tissue. This project will use cutting edge genomic approaches to discover somatic mutations predictive for clinical resistance to commonly used therapy, developing an adequate characterization of the mechanisms of clinical resistance in metastatic colorectal cancer patients.

This project will apply innovative translational studies and state-of-the-art genomics technology to evaluate genetic mechanisms of clinical resistance in metastatic colorectal cancer.

Specific Aim 1: Discover somatic mutations associated with clinical resistance to first line chemotherapy in patients with colorectal liver metastases.

Our hypothesis is that whole transcriptome gene sequencing of colorectal liver metastasis from recently treated patients will identify novel mutations that are associated with resistance to FOLFOX therapy.

Specific Aim 2: Determine the mutation spectrum in 1400 patients with metastatic colorectal cancer.

Our hypothesis is that the presence of resistance mutations will predict for inferior outcome after FOLFOX chemotherapy for advanced colorectal cancer.

Specific Aim 3: Discover somatic mutations associated with generalized clinical resistance/sensitivity to chemotherapy and biologic therapy in patients with colorectal liver metastases.

Our hypothesis is that tumors that are resistant to multiple rounds of chemotherapy develop common somatic mutations that lead to a generalized clinical resistance.

Without an understanding of the obstacles to success, one can never truly succeed. This project is a moderate risk, high yield effort to ascertain the genomic context of clinical resistance to the current therapy for metastatic colorectal cancer. This proposal has a rare combination of being highly achievable and clinically relevant. The information will enable the design of novel therapeutic strategies to overcome resistance. It will also help to develop molecular pathology prognostic tumor profiles, for use in therapy selection. However, the most important output from this proposal will be a clearer understanding of the process for developing key information on drug resistance for all therapies for metastatic colorectal cancer. Sharing the lessons and success of this proposal will make the path more apparent for others trying to overcome the practical obstacles to improve therapy for metastatic colorectal cancer.