AMPK as a molecular target for chemoprevention by apigenin in preneoplastic skin

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DESCRIPTION (provided by applicant): Over two million new cases of UVB-induced non-melanoma skin cancer are diagnosed yearly in the USA, making it the most common form of cancer in this country. Apigenin, a nonmutagenic, naturally occurring flavonoid found in a variety of fruits and leafy vegetables, inhibits UV-induced skin cancer. Our laboratory and others have shown that apigenin treatment of cells results in a wide variety of antitumorigenic and chemopreventive actions, including inhibition of angiogenesis, suppression of COX-2 expression, and induction of cell cycle arrest and apoptosis. However, the primary target(s) responsible for apigenin's chemopreventive function remains elusive. We have obtained preliminary evidence that apigenin activates AMP-activated protein kinase (AMPK) in keratinocytes (mouse and human keratinocyte cell lines, primary normal human epidermal keratinocytes (NHEKs) and mouse epidermis in vivo). The activation of AMPK results in inhibition of mTOR signaling, which relieves the block to the autophagy pathway, further inducing autophagy in human keratinocytes. Activation of the autophagy pathway holds promise as a chemoprevention strategy. Our preliminary results also show that a moderate AMPK activation was observed after UVB radiation, suggesting that there is a protective cellular response after UVB radiation alone to provide limited protection. More intriguingly, pretreatment of apigenin prior to UVB radiation enhances AMPK activation dramatically (both in vitro and in vivo), thus providing much stronger protection. In this proposal, we hypothesize that the AMPK/mTOR axis is involved in chemoprevention of UVB-induced skin cancer by apigenin, and mechanistically the activation of AMPK further induces autophagy to suppress UVB-induced tumorigenesis. We propose to test this hypothesis both in cell-based studies using human keratinocytes and in vivo using a UVB-irradiated mouse model. Aim #1 will investigate how apigenin activates AMPK and induces autophagy by using cultured human keratinocytes (both HaCaT cells and NHEKs). We will also determine how UVB plus apigenin treatment modulates AMPK activation, if UVB radiation by itself induces autophagy, and if pretreatment with apigenin enhances autophagy in UVB-irradiated keratinocytes. UVB-induced mutant p53 clusters of preneoplastic epidermal cells are an early morphologic change that has been demonstrated to be a very early event in UVB-induced skin cancer. Therefore, in Aim #2, we will use this model to investigate if topical apigenin activates AMPK in vivo, and if inhibition of AMPK activity will suppress apigenin's chemopreventive function against UVB-induced mutant p53 clusters of preneoplastic epidermal cells. Identifying AMPK as a key target of apigenin and targeting the autophagy pathway for cancer chemoprevention would provide a new target and strategy for the development of better treatment and prevention of skin cancer. PUBLIC HEALTH RELEVANCE: Chronic UV exposure is widely believed to be the cause of most non-melanoma skin cancer in the United States, and apigenin, a naturally occurring compound present in a wide variety of food sources, has been shown to inhibit UV-induced skin cancer. This proposal will test the innovative hypothesis that an enzyme - AMPK (AMP-activated protein kinase) is involved in chemoprevention of UV-induced skin cancer by apigenin. Identifying AMPK as a target for apigenin would have a significant impact in the skin cancer field and provide a new strategy for better treatment and prevention of skin cancer.