UNIVERSITY OF TEXAS M.D. ANDERSON CANCER CENTER, THE
Grant: $255,640 - National Institutes of Health - May. 8, 2009
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Award Description: (Also known as serine/threonine kinase 11, STK11) tumor suppressor is mutationally inactivated in Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder characterized by gastrointestinal polyps, mucocutaneous pigmentation, and a markedly increased risk for malignant tumors. Inactivating mutations in LKB1 are also found in cancer patients without PJS, including sporadic lung adenocarcinoma (common), ovarian cancer, breast cancer, pancreatic cancer, and biliary adenocarcinoma (rare). Although LKB1 encodes a protein kinase to which a variety of functions have been ascribed, as yet no unifying hypothesis has convincingly explained how loss of LKB1 function contributes to carcinogenesis. Therefore, the long-term goal of this project is to identify the molecular mechanism underlying the tumor suppressor function of LKB1, a prerequisite for targeting LKB1 function in cancer treatment. In the last decade, considerable evidence has accumulated suggesting that genomic instability plays an important role in tumorigenesis. Supporting a role in tumor suppression, our preliminary findings suggest that LKB1 plays a critical role in maintaining genomic stability. We found that LKB1 interacts with ATM, BRCA1, and other DNA damage response (DDR) proteins in vivo. LKB1-deficient cells exhibit a delayed DDR and reduced DNA repair after exposure to ionizing radiation (IR). LKB1 deficiency enhances intracellular reactive oxygen species (ROS) accumulation, which is a major cause of DNA damage and genetic instability. Ectopic expression of LKB1 reduces intracellular ROS levels. Based on these observations, we hypothesize that LKB1 suppresses tumorigenesis by safeguarding genomic stability through regulating DNA damage/repair response and inhibiting intracellular ROS production. We have designed the following specific aims to test our hypothesis. (1) Determine the role of LKB1 in IR- induced DNA damage/repair response. Hypothesis to be tested: LKB1 is a DDR protein that regulates the enzymes involved in DNA-damage repair and DNA replication. As a corollary, loss of LKB1 may lead to an accumulation of DNA damage, an increase in genomic instability, and augmented tumorigenesis. (2) Determine the role of ROS in the function of LKB1. Hypothesis to be tested: LKB1 protects the genome from ROS-induced oxidative stress by regulating antioxidant gene production. (3) Determine the protein signatures and signaling networks in IR- and H2O2- treated cells in the presence or absence of LKB1 and identify novel LKB1-interacting proteins under stressed conditions. Hypothesis to be tested: LKB1 interacts with specific proteins to mediate its role in the maintenance of genomic stability. Significance. Accomplishment of these objectives would suggest that LKB1 integrates different signals (DNA damage response, DNA repair, and antioxidant) and interacts with specific proteins in the maintenance of genomic stability. The findings should lead to a better understanding of the role that LKB1 mutation may play in the pathogenesis of PJS and tumorigenesis. These studies will also provide rationale for pharmacologic replacement or mimicry of LKB1 function in cancer prevention and treatment. PUBLIC HEALTH RELEVANCE: LKB1 is a serine/threonine kinase that is mutationally inactivated in Peutz-Jeghers syndrome and many cancers. It is estimated that 93% of PJS patients will develop a malignant tumor at an average age of 43 years 3. Also, more than a third of sporadic lung adenocarcinomas have been reported to have LKB1 mutations 5, 6. Although a variety of functions have been ascribed to LKB1, as yet no individual hypothesis has convincingly explained how loss of LKB1 function contributes to carcinogenesis.
Project Description: On the basis of our previous observations, we hypothesize that LKB1 may suppress tumorigenesis by safeguarding genomic stability through regulating DNA damage/repair response and inhibiting intracellular ROS production. With the support from the grant, in the past quarter, we have initiated the following work related to the project. (1) Establishment and validation of LKB1 stable cell lines and characterization of LKB1 antibodies: To identify the activities of LKB1 in vivo, we have established a series of stable cell lines expressing either wild-type or kinase-dead mutant (SL26) LKB1. Some cell lines were used in our preliminary study. Recently, we further characterized the cell lines by detecting the LKB1 protein expression, kinase activity in the cells, and activation of downstream kinases, such as AMPK. We also validated antibodies that have been used or will be utilized in our project. We believe these basic accomplishments will make our next step work go smoothly and secure stable and reliable results. (2) Construction of a stable cell line for the detection of homologous recombination (HR) DNA repair efficiency in LKB1-deficient cells. We were planning to use the DR-GFP HR substrate plasmid to detect the HR efficiency. This system was first designed by Dr. Maria Jasin in the Sloan Kettering Cancer Center. We fortunately obtained the plasmids from Dr. Jasin. We recently transfected the DR-GFP plasmid to U2OS cells and made a stable cell line of U2OS/DR-GFP. The cell lines have been successfully characterized and ready for the HR assay. (3)With the administration of r-H2AX foci formation and HR DNA repair assay, we further showed that DNA repair is insufficient in LKB1 deficiency cells. Our primary data showed that LKB1 may play an important role in DNA damage response, in particular in the HR DNA repair.
Jobs Summary: Our work is focused on the LKB1 tumor suppressor research. LKB1 is mutationally inactivated in Peutz-Jeghers syndrome and a variety of tumors. It represents the most frequently abnormal and the least studied gene in lung cancer, a major killer in the United States. Our preliminary data suggested that LKB1 may suppress tumorigenesis by safeguarding genomic stability through regulating DNA damage/repair response and inhibiting intracellular ROS production. Based on this hypothesis, we submitted the R01 proposal. According to our preliminary data obtained recently, it shows that LKB1 indeed plays an important role in DNA damage response and reduction of ROS levels. We believe our further work will definitely enhance the understanding of tumorigenesis mechanism and provide potential target for cancer prevention and cancer treatment. Funding of the project not only allows us to continue the important program and investigate the tumor suppression mechanism of LKB , but also maintains my laboratory and retains the scientists in the lab. We used the NCI grant to support the salary and laboratory consumable supplies for PI and a collaborator (both partially). We are recruiting 1-2 research scientists to join our group for the project very soon. We have interviewed candidates recently. Thus, we anticipate that there will be 2-3 positions created or retained in the next few months. Therefore, I must say that none of this could happen without the funding from the American Recovery and Reinvestment Act (ARRA) package and the support of great administrative teams in NCI and in the University of Texas M.D. Anderderson Cancer Center. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
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