Mesalamine for Colorectal Cancer Prevention Program in Lynch syndrome (MesaCapp)

Colorectal cancer (CRC) has become the second leading cause of cancer death. Patients with Lynch syndrome (LS) have a high lifetime risk for the development of CRC. The incidence of LS in Europe is estimated between 1 in 370 to 1 2000 in individuals, a population of at least 250.000. LS individuals are defined as carriers of a deleterious mutation in at least one mismatch repair (MMR) gene as MLH1, MSH2, MSH6, or PMS2. Chemoprevention is a big hope for LS family members. Cancer development in LS occurs through a mutational mechanism called microsatellite instability (MSI). The genetic loss of MMR activity induces a mutator phenotype in which genetic deletions occur frequently in repetitive DNA sequences. In vitro, mesalamine (5-ASA), a well-tolerated drug that has been used for over 30 years in ulcerative colitis, reduces MSI via improvement of replication fidelity. In ulcerative colitis it seems to reduce the risk of CRC. In a genetic mouse model of LS, carrying a villin-cre specific conditional knock out of the Msh2 gene, 5-ASA reduced the tumor incidence, tumor multiplicity, and MSI. We could show similar effects for thymoquinone, the main active compound in the volatile Nigella sativa oil. Here, we propose a multicenter, multinational, randomized, 3-arm, double blind, phase 2, clinical trial with 2400mg 5-ASA, 1200mg 5-ASA or placebo in LS patients for 2 years. The applicant for the funding is the principal investigator of the clinical trial. In total 540 patients from Austria, Germany, The Netherlands, Poland, and Israel will be randomized for the study. Tumor free patients, assessed by colonoscopy, will be allocated to the study. After a 2-year intervention phase patients will be assessed for the presence of colonic neoplasia by colonoscopy. Second, we intent to test for synergistic effects of 5-ASA with thymoquinone or Nigella sativa oil and to verify the effect of further candidate substances such as broccoli-extract in vivo. Msh2 loxP/loxP Vill-cre mice will be fed 2 different concentrations of the natural substances with our without 5-ASA for 10 month. The primary endpoint of the study is the reduction of the tumor prevalence. Intestines will be analyzed for tumor multiplicity, localization and size. DNA will be isolated from microdissected normal intestinal, tumor and tail tissue and tested for MSI by fragment analysis using five microsatellite markers. Immunohistochemical staining of relevant pathways will be performed (e.g TUNEL, Ki-67, Nrf2, NQO1). Last, we intend to study the molecular effects of 5-ASA on activation of Nrf2, a protein that is involved in cellular stress response. 18 out of 20 MSI reducing agents also activate Nrf2 suggesting that Nrf2 or specific target genes may interfere with replication fidelity. We will study the role of Nrf2 upon 5-ASA treatment regarding cell cycle progression, activation of cell cycle check points, protein expression, chromatin binding and remodeling and replication fidelity by chemical inhibition of Nrf2, knockdown or overexpression of Nrf2. Also colonic tissue from patients receiving 5-ASA will be investigated for Nrf2 expression. Tumor progression in Nrf2 -/- in the context of MMR deficiency will be studied by crossing to MSH2-/- mice.

Patients with Lynch syndrome have a 50 % chance of developing colorectal cancer in their lifetime. It is assumed that 350 000 people in Europe may have this disease, for which currently no treatment exists. Prevention of this disease is a big hope for these people. In Lynch syndrome, the loss of a DNA repair gene leads to mutations in repetitive DNA sequences during replication, finally leading to colorectal cancer and other cancers. Treatment with mesalamine (5 aminosalicylic acid, 5-ASA) reduces these mutations in a cell based system and in a Lynch syndrome mouse model. Mice treated with 5-ASA show fewer tumors. Also, 5-ASA induces a protein, called Nrf2, which protects against oxidative stress in the cell. Within this study we could show that upon 5-ASA treatment not only Nrf2 is upregulated, but Nrf2 also moves into the cell nucleus, where it induces several downstream targets. These proteins are important for keeping the production of reactive oxygen species in check, which can otherwise lead to mutations in cells. Inhibition of Nrf2 diminishes the effect of 5-ASA, leading to higher mutation rates. Furthermore, we studied the effect of 5-ASA intake in patients with adenomatous polyps in a clinical study. Our data point to an activation of Nrf2 upon 5-ASA treatment in colonic tissue. Also patients with bowel inflammation have increased expression and activity of Nrf2 in inflamed cells and tumor tissue, supporting the importance of Nrf2 in oxidative stress response. Overall, these findings suggest that activation of anti-oxidant Nrf2 pathway by 5-ASA contributes to its molecular actions in reduction of oxidative stress. In the absence of functional DNA repair genes in Lynch Syndrome, reduced oxidative stress (upon 5-ASA treatment) could lessen the mutational load and hence impede tumorigenesis.