Development Project 2

Project 2
Diagnostic Imaging to Evaluate Inhibition of the NF-kB Pathway on Growth of Colon Carcinoma

Richard Gaynor, M.D., a medical oncologist with expertise in virology and molecular biology, will direct the biological aspects of the project. He has done pioneering work on the role of the NF-kB pathway in neoplastic and inflammatory disease. Orhan K. Öz, M.D., Ph.D., will coordinate and interpret the nuclear imaging studies. Dr. Constantinescu (radiochemist) will implement the labeling and ensure quality control. Jon Anderson, Ph.D. (medical physicist with special interest in nuclear imaging technology) will be responsible for the day to day function of clinical nuclear imaging devices and for consultation in any quantitative analysis of images.

Colon cancer affects 6 percent of the population in the US, and thus, is one of the most common cancers. Elegant molecular studies by Vogelstein have demonstrated the sequential development of such cancers. Adenomatous polyps develop an increasing number of genetic changes leading to the development of invasive carcinoma. A number of different cellular regulatory pathways are potentially subject to mutations that lead to the development of colon cancer. One critical pathway is the Wnt pathway, which includes the transcription factors b-catenin, lef-1 and the kinase GSK-3. GSK-3 phosphorylation of b-catenin results in the binding of the adenomatous polyposis colon gene product (APC), which facilitates the proteosomal degradation of b-catenin. Mutations in either the APC gene or b-catenin are found in a substantial subset of colon cancer. A second major pathway associated with the development of colon cancer is mutations in DNA mismatch repair genes. Mutations in several of these genes result in the development of a syndrome known as hereditary nonpolyposis coli in which patients develop colon cancer at an early age. Sporadic mutations of such genes are also frequently seen. Mutations in mismatch repair genes are frequently associated with mutations in the TGF-b receptor genes. Finally, mutations in a variety of dominant or suppressor oncogenes such as ras and p53 are frequently seen in colon carcinoma. Thus, mutations in a variety of different regulatory pathways can lead to the development of colon cancer.

Given the stepwise development of invasive colon cancer from polyps, it is clear that therapy should be directed at the earliest stages of colon dysplasia. Epidemiological studies indicate that prophylactic treatment of patients with NSAIDs such as aspirin or sulindac reduces the incidence of developing colon cancer from 50-70%. The mechanism of inhibition is unclear, but is thought to be due to inhibiting prostaglandin synthesis by the effects on the enzyme cyclooxygenase 2 (Cox-2). Cox-2 is overexpressed in the vast majority of colon cancers. Moreover, in mice with a predisposition to the development of colon cancer due to mutations in the APC gene, Cox-2 is markedly overexpressed and reductions in its level markedly reduces the development of colon cancer. These results indicate that Cox-2 overexpression with resultant increased synthesis of prostaglandins is involved in the pathogenesis of colon cancer. However, NSAIDs also inhibit growth of colon cancer cells even those cells that do not express Cox-2.

We recently demonstrated that there may be a second target for NSAIDs: the NF-kB pathway. NF-kB comprises a family of cellular transcription factors that are sequestered in the cytoplasm bound to inhibitory proteins known as IkB. A variety of cytokines can trigger the phosphorylation of IkB by increasing the activity of a kinase IKK. Following IkB phosphorylation, it is ubiquitinated and degraded by the proteosome. NF-kB then translocates to the nucleus, where it activates cellular gene expression including genes involved in preventing cellular apoptosis. We recently demonstrated that aspirin and sulindac can inhibit the activation of the NF-kB pathway by binding directly to IKK to inhibit its kinase activity for IkB. This inhibition of IKK occurs in a variety of colon cancer cell lines, including those that do not express Cox-2, and results in the induction of cellular apoptosis. Our recent data suggest that the role of Cox-2 overexpression in the pathogenesis of colon cancer is to increase prostaglandin synthesis and up-regulate the NF-kB pathway. The fact that aspirin and sulindac can inhibit the development of adenomatous polyps leading to apoptosis suggests that inhibition of both Cox-2 and IKKb may have therapeutic benefit for the treatment of established colon cancer. The major hypothesis that we wish to address is that NSAIDs, such as aspirin, sulindac, and the newly released selective Cox-2 inhibitor celebrex inhibit the growth of established cases of colon cancer. Our initial studies will evaluate the role of these agents in nude mice containing intra peritoneal solid tumor implants of different colon cancer cell lines. These studies will be extended to determine whether these NSAIDs prove to be effective in conjunction with chemotherapy for patients with metastatic or node positive resected colon cancer. We will also address whether sensitive imaging methodologies can be used to determine the response of colon tumors to NSAIDs. While there have been observations on the inhibitory effects of NSAIDs on preventing colon cancer, the mechanism underlying these effects is still unclear. Certainly, there has been no in vivo testing of our hypothesis. This collaboration makes possible the unique scientific opportunity of testing the effects of these agents in vivo in a non invasive serial fashion. Hitherto, such work has been primarily in vitro studies and relied on histological and gross anatomical effects of these agents. The collaboration will utilize non-invasive imaging techniques, which should accelerate the evaluation of NSAIDs as anti-proliferative drugs. The availability of both small animal and clinical imaging devices, as well as clinical units will facilitate the development of this therapeutic approach from “benchtop to viewbox” at a single institution. Moreover, it will facilitate close interactions of basic and imaging scientists and clinicians involved in cancer treatment and diagnosis.

1. Yin, M-J, Yamamoto Y, Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of IKB kinase. Nature 1998, 396, 77-80.

2. Yamamoto, Y, Yin M-J, Lin K-M, Gaynor RB. Sulindac inhibits activation of the NF-KB pathway. J. Biol. Chem. (in press).

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