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    The Role of KLF5 Transcription Factor Degradation in Breast Cancer

    Scientific Abstract:
    Background: KLF5 transcription factor plays an important role in embryo development, cell proliferation, differentiation, angiogenesis and carcinogenesis. Over 43% of breast cancer cells have genetic deletions at KLF5 locus on 13q21, which is one of the most frequent deleted regions in human cancer including breast cancer. 70% of breast cancer cells decrease KLF5 mRNA expression. Functional studies showed that KLF5 inhibits the T-47D breast cancer cell growth. These data suggest KLF5 could be a tumor suppressor in breast cancer. Recently, we found that KLF5 protein is extremely unstable. Proteasome specific inhibitors significantly suppress KLF5 proteolysis and increasing proteolysis is a novel mechanism to inactivate tumor suppressor function of KLF5. The transactivation domain (323-348) of KLF5 transcription factor plays a degron (degradation domain) role to determine KLF5 ubiqutination and proteolysis. At the same time, FLAG tag at N-terminus but not C-terminus stabilizes KLF5 protein implies that N-terminal ubiquitination may also contribute to KLF5 proteolysis. Furthermore, existence of several smaller bands below KLF5 protein suggests the degradation of KLF5 may undergo N-end rule. The degradation of KLF5 may be regulated by phosphorylation. Hypothesis: Increase of KLF5 proteolysis through ubiquitin-proteasome pathway promotes breast carcinogenesis. Specific Aims: 1) To characterize the major mechanisms of KLF5 proteolysis in breast cell lines. We will focus on three possible mechanisms: The transactivation domain as a degron, N-terminal ubiquitination, and N-end rule. We will map and confirm the degron through constructing a series of deletion and measuring its half-life in breast cell lines. 2) To test if the phosphorylation of KLF5 decreases KLF5 degradation. We will establish an in vitro cell free ubiquitination-degradation system using in vitro translated 35S-KLF5 proteins. Kinases, phosphatases or its inhibitors will be added to test the role of phosphorylation for KLF5 degradation. Then, we will confirm the results in vivo. The phosphorylation sites will be mapped. 3) To identify the E3 ligase of KLF5 by yeast two-hybrid system using the mapped degron as bait and investigate the alteration of KLF5 E3 ligase in breast cells. 4) To test the hypothesis that increasing KLF5 proteolysis promotes breast cancer progression. Cell proliferation and tumorigenesis will be examined after changing KLF5 proteolysis in breast cells. The result of this research may have implications for generating KLF5-based anti-tumor therapies.

    Lay Abstract:
    KLF5 gene plays an important role in several aspects of cancer progression. KLF5 gene is located within a region at chromosome 13, which is one of the most frequent deleted regions in human cancer including breast cancer. Our previous study suggests over 43% of breast cancer cells lost KLF5 gene at DNA level and 70% of breast cancer cells significantly decrease KLF5 mRNA expression. Functional studies showed that KLF5 suppresses breast cancer cell growth. These data suggest KLF5 could be a tumor suppressor in human breast cancer. Recently, we found that KLF5 protein is extremely unstable. The degradation of KLF5 is through ubiqtuitin-proteasome pathway (UPP) in cells. Ubiquitin is a small peptide that can be attached to protein by enzymes to form a polyubiquitin chain. The protein with polyubiquitin modification will be recognized rapidly and degraded by protein destruction machine proteasome. More importantly, we found increasing KLF5 protein degradation is a novel mechanism to inactivate tumor suppressor function of KLF5 in breast cancer. A small segment of KLF5 protein has been identified as degradation domain to determine KLF5 half-life. At the same time, fusing KLF5 protein with a short tag at N-terminus but not C-terminus protects KLF5 protein from degradation. Furthermore, existence of several smaller cut bands below KLF5 protein suggests the degradation of KLF5 may go through other mechanisms. Finally, we found the degradation of KLF5 may be regulated by another modification called phosphorylation. All these findings promote us to hypothesize that the increase of KLF5 degradation through UPP promotes breast cancer formation and development. We are going to test our ideas by four aims: 1) To figure out the major mechanisms of KLF5 degradation in breast cell lines. 2) To test if the phosphorylation modification of KLF5 decreases KLF5 degradation. 3) To isolate the enzymes that is responsible for KLF5 ubiquitin modification by yeast two-hybrid system because the enzyme is very important for stability of KLF5. We deduce that the activity of this enzyme may be over-activated in breast cancer cells so that it will provide a target for drug design in the future. 4) To test the hypothesis that increasing KLF5 degradation promotes breast cancer progression. We will change the KLF5 degradation speed by different methods and examine the breast cancer cell growth and potential of forming tumors on mice. After finishing this proposal, we hope to provide a basis for generating KLF5-based anti-tumor therapies.