The objectives of this revised renewal proposal (RO1 CA 102431A2) are to determine whether protein kinase C epsilon (PKC5) signals ultraviolet radiation (UVR)-induced development of squamous cell carcinoma (SCC) via increased expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF1) and granulocyte colony-stimulating factor (G-CSF) which may directly promote the proliferation of hair follicle stem cells, the targets of SCC. Chronic exposure to UVR is the most common etiological factor linked to the development of SCC, a nonmelanoma form of skin cancer that can metastasize. PKC5 is among the six isoforms (1, 4, 5, 7, 5, 6) expressed in both human and mouse skin. To determine the in vivo functional role of PKC5 in mouse skin carcinogenesis, we generated PKC5 transgenic mouse (FVB/N) lines 224 and 215 that overexpress approximately 8- and 18-fold respectively PKC5 protein over endogenous levels in basal epidermal cells and cells of the hair follicle. We reported that epidermal PKC5 level dictates the susceptibility of transgenic mice to the development of SCC elicited either by the repeated exposure to UVR or by the DMBA - TPA tumor promotion protocol. Our studies to elucidate mechanisms of PKC5-mediated development of SCC, either by DMBA-TPA or UVR, indicated a common converging point- elevated release of TNF1 and G-CSF. To conclusively determine whether TNF1 is essential for the development of SCC in PKC5 transgenic mice, we generated TNF1-knockout//PKC5 transgenic mice by crossbreeding TNF1 knockout mice with PKC5 transgenic mice. Deletion of both TNF1 alleles in PKC5 transgenic mice only partially inhibited (50%, p = 0.0136) the development of SCC by the DMBA-TPA protocol. Deletion of the TNF1 gene in PKC5 transgenic mice also partially suppressed UVR-induced skin damage and inflammation. These results indicate that, in addition to TNF1, other UVR-induced cytokines such as G-CSF may play essential roles in the development of SCC. We hypothesize that: 1) TNF1 cooperates with G-CSF in PKC5 signal transduction pathways to the development of SCC by UVR, 2) PKC5-induced TNF1 and G-CSF mediate induction of SCC through direct effects on stem cells in the mouse hair follicle and 3) PKC5 interacts with MAPK cascade to regulate UVR- induced TNF1 and G-CSF gene transcription. We propose three specific aims to test these hypotheses:
Specific aim #1 : To determine, using gene knockout approach, whether both TNF1 and G-CSF contribute to the development of SCC in PKC5 transgenic mice by UVR.
Specific Aim# 2: To define, in intact mouse skin in vivo, whether the roles of TNF1 and G-CSF in UVR-induced development of SCC involve the activation and proliferation of putative hair follicle stem cell. Hair follicle stem cell proliferation will be determined by quantitations of label retaining cells (LRCs) using BrdU and FACS analysis using cell surface markers (CD34, 16-integrin).
Specific Aim #3 : To define the mechanism, using gene-silencing techniques, by which PKC5 signals UVR-induced TNF1 and G-CSF gene expression.
Chronic exposure to ultraviolet radiation (UVR) is the most common etiological factor linked to the development of SCC, a nonmelanoma form of skin cancer that can metastasize. We propose to determine whether protein kinase C epsilon (PKC5) signals UVR-induced development of squamous cell carcinoma (SCC) via increased expression of pro-inflammatory cytokines tumor necrosis factor alpha (TNF1) and granulocyte colony- stimulating factor (G-CSF) which activate and promote the proliferation of hair follicle stem cells, targets of SCC.
|Hafeez, Bilal Bin; Meske, Louise; Singh, Ashok et al. (2016) Tissue-specific conditional PKCÎµ knockout mice: a model to precisely reveal PKCÎµ functional role in initiation, promotion and progression of cancer. Oncotarget 7:33069-80|
|Singh, Ashok; Singh, Anupama; Bauer, Samuel J et al. (2016) Genetic deletion of TNFÎ± inhibits ultraviolet radiation-induced development of cutaneous squamous cell carcinomas in PKCÎµ transgenic mice via inhibition of cell survival signals. Carcinogenesis 37:72-80|
|Singh, Ashok; Willems, Estelle; Singh, Anupama et al. (2016) Ultraviolet radiation-induced tumor necrosis factor alpha, which is linked to the development of cutaneous SCC, modulates differential epidermal microRNAs expression. Oncotarget 7:17945-56|
|Singh, Anupama; Singh, Ashok; Sand, Jordan M et al. (2015) Topically applied Hsp90 inhibitor 17AAG inhibits UVR-induced cutaneous squamous cell carcinomas. J Invest Dermatol 135:1098-107|
|Singh, Ashok; Singh, Anupama; Sand, Jordan M et al. (2013) Protein Kinase C Îµ , Which Is Linked to Ultraviolet Radiation-Induced Development of Squamous Cell Carcinomas, Stimulates Rapid Turnover of Adult Hair Follicle Stem Cells. J Skin Cancer 2013:452425|
|Sand, Jordan Marshall; Bin Hafeez, Bilal; Aziz, Moammir Hasan et al. (2012) Ultraviolet radiation and 12-O-tetradecanoylphorbol-13-acetate-induced interaction of mouse epidermal protein kinase CÃ½Ã½ with Stat3 involve integration with ERK1/2. Mol Carcinog 51:291-302|
|Aziz, Moammir Hasan; Sundling, Kaitlin Elizabeth; Dreckschmidt, Nancy Ellen et al. (2009) Protein kinase Cepsilon inhibits UVR-induced expression of FADD, an adaptor protein, linked to both Fas- and TNFR1-mediated apoptosis. J Invest Dermatol 129:2011-21|
|Aziz, Moammir H; Manoharan, Herbert T; Sand, Jordan M et al. (2007) Protein kinase Cepsilon interacts with Stat3 and regulates its activation that is essential for the development of skin cancer. Mol Carcinog 46:646-53|
|Aziz, Moammir H; Manoharan, Herbert T; Verma, Ajit K (2007) Protein kinase C epsilon, which sensitizes skin to sun's UV radiation-induced cutaneous damage and development of squamous cell carcinomas, associates with Stat3. Cancer Res 67:1385-94|
|Verma, Ajit K; Wheeler, Deric L; Aziz, Moammir H et al. (2006) Protein kinase Cepsilon and development of squamous cell carcinoma, the nonmelanoma human skin cancer. Mol Carcinog 45:381-8|
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