Ashok Singh

Plumbagin inhibits prostate carcinogenesis in intact and castrated PTEN knockout mice via targeting PKCε, Stat3 and epithelial to mesenchymal transition markers

Prostate cancer continues to remain the most common cancer and the second leading cause of cancer-related deaths in American males. The Pten deletions and/or mutations are frequently observed in both primary prostate cancers and metastatic prostate tissue samples. Pten deletion in prostate epithelium in mice results in prostatic intraepithelial neoplasia (PIN), followed by progression to invasive adenocarcinoma. The Pten conditional knockout mice [(Pten-loxp/loxp:PB-Cre4+) (Pten-KO)] provide a unique preclinical model to evaluate agents for efficacy for both the prevention and treatment of prostate cancer. We present here for the first time that dietary plumbagin, a medicinal plant–derived naphthoquinone (200 or 500 ppm) inhibits tumor development in intact as well as castrated Pten-KO mice. Plumbagin has shown no signs of toxicity at either of these doses. Plumbagin treatment resulted in a decrease expression of PKCϵ, AKT, Stat3, and COX2 compared with the control mice. Plumbagin treatment also inhibited the expression of vimentin and slug, the markers of epithelial-to-mesenchymal transition (EMT) in prostate tumors. In summary, the results indicate that dietary plumbagin inhibits growth of both primary and castration-resistant prostate cancer (CRPC) in Pten-KO mice, possibly via inhibition of PKCϵ, Stat3, AKT, and EMT markers (vimentin and slug), which are linked to the induction and progression of prostate cancer. Cancer Prev Res; 8(5); 375–86. ©2015 AACR.

Topically Applied Hsp90 Inhibitor 17AAG Inhibits UVR-Induced Cutaneous Squamous Cell Carcinomas

We present here that Heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG), when topically applied to mouse skin, inhibits ultraviolet radiation (UVR)-induced development of cutaneous squamous cell carcinoma (SCC). In these experiments, DMSO:acetone (1:40 v/v) solution of 17AAG (500nmol) was applied topically to mouse skin in conjunction with each UVR exposure (1.8 kJ/m2). The UVR source was Kodacel-filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). In independent experiments with three separate mouse lines (SKH-1 hairless mice, wild-type FVB, and PKCε overexpressing transgenic FVB mice), 17AAG treatment increased the latency and decreased both the incidence and multiplicity of UVR-induced SCC. Topical 17AAG alone or in conjunction with UVR treatments elicited neither skin nor systemic toxicity. 17AAG-caused inhibition of SCC induction was accompanied by decrease in UVR-induced: 1) hyperplasia, 2) Hsp90β-PKCε interaction, 3) expression levels of Hsp90β, Stat3, pStat3Ser727, pStat3Tyr705, pAktSer473 and matrix metalloproteinase (MMPs). The results presented here indicate that topical Hsp90 inhibitor 17AAG is effective in prevention of UVR-induced epidermal hyperplasia and SCC. One may conclude from the preclinical data presented here that topical 17AAG may be useful for prevention of UVR-induced inflammation and cutaneous SCC either developed in UVR exposed or organ transplant population.

Ultraviolet radiation-induced tumor necrosis factor alpha, which is linked to the development of cutaneous SCC, modulates differential epidermal microRNAs expression

Chronic exposure to ultraviolet radiation (UVR) is linked to the development of cutaneous squamous cell carcinoma (SCC), a non-melanoma form of skin cancer that can metastasize. Tumor necrosis factor-alpha (TNFα), a pro-inflammatory cytokine, is linked to UVR-induced development of SCC. To find clues about the mechanisms by which TNFα may promote UVR-induced development of SCC, we investigated changes in the expression profiling of microRNAs (miRNA), a novel class of short noncoding RNAs, which affects translation and stability of mRNAs. In this experiment, TNFα knockout (TNFα KO) mice and their wild type (WT) littermates were exposed to acute UVR (2.0 kJ/m2) and the expression profiling of epidermal miRNA was determined 4hr post UVR exposure. TNFα deletion in untreated WT mice resulted in differential expression (log fold change>1) of seventeen miRNA. UVR exposure in WT mice induced differential expression of 22 miRNA. However, UVR exposure in TNFα KO mice altered only two miRNAs. Four miRNA, were differentially expressed between WT+UVR and TNFα KO+UVR groups. Differentially expressed selected miRNAs were further validated using real time PCR. Few of the differentially expressed miRNAs (miR-31-5p, miR-196a-5p, miR-127-3p, miR-206-3p, miR-411-5p, miR-709, and miR-322-5p) were also observed in UVR-induced SCC. Finally, bio-informatics analysis using DIANA, MIRANDA, Target Scan, and miRDB algorithms revealed a link with major UVR-induced pathways (MAPK, PI3K-Akt, transcriptional mis-regulation, Wnt, and TGF-beta).

Abstract 2142: Plumbagin, a medicinal plant-derived naphthoquinone, inhibits the growth of docetaxel resistant prostate cancer cells via inhibition of the expression of P-glycoprotein and NF-kB activation

Prostate cancer (PCa) is one of the leading causes of death in men across the world. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy manifests by progression to castration resistant prostate cancer (CRPC), which is the end stage that accounts for the majority of PCa patient deaths. Docetaxel (DTX) is an approved drug for the treatment of CRPC. However, development of DTX resistance and toxicity are the major limitations in the use of DTX in the treatment of CRPC. Now we investigated to determine whether these limitations may be overcome by combining DTX with plumbagin (PL). PL is a quinoid constituent isolated from the root of Plumbago zeylanica L. The roots of Plumbago zeylanica L. have been used in Indian medicine for more than 2500 years for treatment of various ailments. We were the first to report that PL inhibits the growth and PCa metastasis. Now we present that PL: 1) sensitizes CRPC cells to DTX and thus reduces its effective therapeutic dose to prevent DTX resistance and toxicity, and 2) inhibits the viability and growth of DTX resistant cells (22Rv1R). In combination therapeutic experiments, the effects of different concentrations of PL (0, 1.0, 2.0, and 5.0 μM) alone or in combination with DTX (0.25 nM and 0.5 nM) on colony formation in 22Rv1 and C4-2 prostate cancer cells, was determined. Combined treatment of PL and DTX has more than additive inhibitory effects on colony formation. This synergism of PL and DTX on colony formation can be explained on the basis of unique mechanism of action of PL and DTX. PL-induced inhibition of PCa growth and metastasis involves inhibition of the expression of multiple targets including PKCe, which correlates with the aggressiveness of PCa and plays roles in both androgen independent androgen receptor (AR) activation and promotion of PCa cell survival. DTX targets microtubules, impairs AR signaling, and inhibits cell division. To determine the effects of PL on DTX resistant PCa cells, we established DTX-resistant 22Rv1 cell line (22Rv1R). 22Rv1R overexpresses multidrug resistant protein 1 (MDR1, also known as P-glycoprotein). MDR1 is overexpressed in many tumors and thus causing intrinsic drug resistant. Transcription factor NF-KB, which is linked to cell proliferation and survival, is also activated in 22Rv1R. PL inhibited the colony forming efficiency of 22Rv1R cells which accompanied inhibition of MDR1 expression and NF-KB activation. In summary, the results presented here and the ones reported by us previously with preclinical mouse models (TRAMP and PTEN knockout mice) of PCa indicate that PL has the potential to prevent as well as treat PCa. PL is a potential natural agent worthy of investigation for prevention and treatment of human PCa. (Support: NIH grant CA102431). Citation Format: Ashok Singh, Anupama Singh, Satyamedha Bathula, Saivenkateshkomal Bathula, Ajit K. Verma. Plumbagin, a medicinal plant-derived naphthoquinone, inhibits the growth of docetaxel resistant prostate cancer cells via inhibition of the expression of P-glycoprotein and NF-kB activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2142.

Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer.

PKCε is a transforming oncogene and a predictive biomarker of various human cancers. However, a precise in vivo link of PKCε to cancer induction, progression and metastasis remain undefined. To achieve these goals, we generated tissue specific conditional PKCε knockout mice (PKCε-CKO) using cre-lox technology. Homozygous PKCεLoxP/LoxP mice have normal body weight and phenotype. To determine what effect loss of PKCε would have on the prostate, the PKCεLoxP/LoxP mice were bred to probasin cre (PB-Cre4+) mice which express cre specifically in the prostate epithelium of postnatal mice. Western blot and immunohistochemical analyses showed reduced levels of PKCε specifically in the prostate of PKCε-CKO mice. Histopathological analyses of prostate from both PKCεLoxP/LoxP and prostate PKCε-CKO mice showed normal pathology. To determine the functional impact of prostate specific deletion of PKCε on prostate tumor growth, we performed an orthotopic xenograft study. Transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (TRAMPC1, 2×106) were implanted in the prostate of PKCε-CKO mice. Mice were sacrificed at 6th week post-implantation. Results demonstrated a significant (P<0.05) decrease in the growth of TRAMPC1 cells-derived xenograft tumors in PKCε-CKO mice compared to wild type. To determine a link of PKCε to ultraviolet radiation (UVR) exposure-induced epidermal Stat3 phosphorylation, PKCεLoxP/LoxP mice were bred to tamoxifen-inducible K14 Cre mice. PKCε deletion in the epidermis resulted in inhibition of UVR-induced Stat3 phosphorylation. In summary, our novel PKCεLoxP/LoxP mice will be useful for defining the link of PKCε to various cancers in specific organ, tissue, or cells.

Abstract 4570: Genetic deletion of TNFα in mice inhibits UVR-induced development of cutaneous squamous cell carcinomas via suppression of its own receptors and associated signals to epidermal cell survival

Skin cancer is the second most common malignancy encountered in the U.S. with an expected diagnosis of 1.3 million new cases of non-melanoma skin cancer each year. Cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the most common non-melanoma forms of human skin cancer. BCC is rarely life threatening because it is slow growing and is mostly localized. SCC, unlike BCC, invades the nearby tissue. Chronic exposure to ultraviolet radiation (UVR) is the most common etiologic factor linked to the development of cutaneous SCC. PKCe transgenic mice provide a unique model to investigate the molecular basis of the development of SCC. PKC is a major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). PKCe is among six isoforms (α, δ, e, η, μ, ζ) expressed in the mouse skin. To determine the in vivo functional specificity of PKCe in mouse skin carcinogenesis, we generated FVB/N PKCe transgenic mouse lines 224 and 215 that overexpress approximately 8- and 18-fold respectively PKCe protein over endogenous levels in basal epidermal cells. We observed that epidermal PKCe level dictates the susceptibility of transgenic mice to the development of SCC elicited by either the repeated exposure to UVR or using the DMBA-TPA tumor promotion protocol. Our earlier studies to elucidate mechanisms of PKCe-mediated development of SCC indicated an elevated release of cytokine TNFα. To conclusively determine whether TNFα is essential for the development of SCC in PKCe transgenic mice, we generated TNFα-knockout//PKCe transgenic mice by crossbreeding TNFα knockout mice with PKCe transgenic mice. We now present that deletion of both TNFα alleles in PKCe transgenic mice significantly (p = 0.0023) inhibited the development of cutaneous SCC by repeated UVR exposures. PKCe overexpressing mice deficient in TNFα elicited both increase in SCC latency (21 weeks) and decrease in SCC incidence (55%). Inhibition of SCC development in TNFα-knockout//PKCe transgenic mice accompanied by: 1) suppression of the expression levels of TNFα receptors TNFRI and TNFRII, 2) inhibition of the activation of transcription factors Stat3 and NF-κB, and 3) decreased expression levels of cell proliferation marker ornithine decarboxylase and metastatic markers MMP9 and MMP7. In summary, our results indicate that deletion of TNFα in mice inhibits UVR-induced development of cutaneous SCC probably via suppression of its own receptors and associated signals to epidermal cell survival pathways. (Support: NIH Grant CA102431) Citation Format: Ashok Singh, Anupama Singh, Samuel J. Bauer, Ajit K. Verma. Genetic deletion of TNFα in mice inhibits UVR-induced development of cutaneous squamous cell carcinomas via suppression of its own receptors and associated signals to epidermal cell survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4570. doi:10.1158/1538-7445.AM2015-4570

Abstract LB-137: Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer

Evidence from our laboratory and others indicate that PKCe is a transforming oncogene and a predictive biomarker of various human cancers including prostate, breast, head and neck, lungs, brain, bladder and cutaneous squamous cell carcinoma (SCC). However, a precise in vivo link of PKCe and its downstream signaling components to cancer induction, progression and metastasis remain undefined. To achieve these goals, we generated tissue specific conditional PKCe knockout mice using cre-lox technology. To do so, we generated a targeting vector in which Exon 4 of the PKCe gene was flanked by LoxP sites. This vector was used to generate mice carrying two floxed alleles of PKCe (PKCeLoxP/LoxP mice) by standard gene knockout methodology. Homozygous PKCeLoxP/LoxP mice have normal body weight and phenotype. To determine what effect loss of PKCe would have on the prostrate, the PKCeLoxP/LoxP mice were bred to prostate specific cre (PB-Cre4+). Western blot and immunohistochemical analyses showed inhibition of PKCe protein level in the prostate of PKCe-KO mice. However, no change in the PKCe protein level was observed in the spleen, liver and lungs of PKCe-KO mice. Also, PKCe deletion in prostate did not affect the levels of other PKC isoforms (PKCα, PKCβII, and PKCς). No significant difference was observed in the prostate weight of PKCeLoxP/LoxP and PKCe-KO mice. Histopathological analyses of prostate from both PKCeLoxP/LoxP and prostate PKCe-KO mice showed normal pathology in the PKCe-KO prostate. To determine the functional impact of prostate specific deletion of PKCe on prostate tumor growth, we performed an orthotopic xenograft study. In this experiment, TRAMP mouse tumor cells (TRAMPC1, 2×106) were implanted in the prostate. Mice were sacrificed at 6 weeks post-implantation. Results demonstrated a significant (P Citation Format: Bilal B. Hafeez, Louise Meske, Anupama Singh, Ashok Singh, Weixiong Zhong, Patricia Powers, Manorama John, Anne Griep, Ajit Verma. Tissue-specific conditional PKCe knockout mice: a model to precisely reveal PKCe functional role in initiation, promotion and progression of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-137. doi:10.1158/1538-7445.AM2015-LB-137

Abstract 2142: Plumbagin, a medicinal plant-derived 1,4-napthoquinone, inhibits prostate carcinogenesis in intact and castrated Pten knockout mice by modulation of tumor microenvironment

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnProstate specific Pten conditional knockout (PtenLoxP/LoxP:PB-Cre+4) (Pten-KO)) mice are unique preclinical model to evaluate agents for efficacy for both prevention and treatment of PCa. We present here for the first time that dietary plumbagin (PL) inhibits tumor development in intact as well as castrated Pten-KO mice. PL was mixed in an antioxidant free basal powdered diet and was given at two dose levels (200 and 500 ppm). Mice (4 wks old) were divided into three groups (Control (n=40), PL (200 ppm) (n=20), and PL (500 ppm) (n=40). Mice were sacrificed at 15 and 30 wks of age. To determine the prostate tumor volume, we carried out micro-PET/CT analysis of control and PL treated groups mice at 30 wks using a tumor selective radiopharmaceutical agent 124I-NM404. PL treatment (200 and 500 ppm) resulted in a dose-dependent decrease (p<0.01) in prostate tumor volume. PL treatment also resulted in a significant (P<0.001) decrease in weights of prostate tumors and urogenital apparatus at 15 and 30 wks. Histopathological analyses revealed inhibition of prostatic intraepithelial neoplasia (PIN) to invasive adenocarcinoma at 15 and 30 wks. PL treated mice also showed a significant (P<0.01) inhibition of small invasive adenocarcinoma with cystic change at 30 wks. To determine the effects of PL in castrated resistant prostate cancer (CRPC), we castrated 26 Pten-KO mice at 10 wks and divided into 2 groups. PL treatment was started one wk after castration and continued until 50 wks. At 50 wks, castrated control Pten-KO mice illustrated atrophy of prostate tumors. PL treatment (500 ppm) further resulted in a significant decrease of prostate tumors weight of castrated Pten-KO mice. Histopathological analysis of PL treated mice prostate tumors showed significant decrease of both invasive adenocarcinoma and cystic change. Biochemical analyses of Pten-KO mice prostate tumors elicited a constitutive activation of AKT, protein kinase C epsilon (PKCe), signal transducer and activators of transcription 3 (Stat3) and increased COX2 expression compared to wild type mice. PL treatment resulted in decreased expression of PKCe, Stat3 and COX2 compared to control mice. We also observed that PL targets tumor associated macrophages (TAM) in prostate tumors. Prostate tumors of PL treated mice showed an increased infiltration of M1 macrophages and inhibition of M2 macrophages, as analyzed by immunohistochemical staining of F4/80, iNOS and L-arginase. PL treatment also inhibited the expression of vimentin and slug, the markers of epithelial to mesenchymal transition (EMT) in prostate tumors. In summary, the results indicate that dietary PL inhibits growth of both primary and CRPC, which was accompanied inhibition of EMT and modulation of tumor microenvironment. We conclude that PL may be worthy of evaluation for the prevention and treatment of human PCa (Support: CA138761).nnCitation Format: Bilal B. Hafeez, Joseph W. Fischer, Ashok Singh, Ala Mustafa, Louise Meske, Ajit K. Verma. Plumbagin, a medicinal plant-derived 1,4-napthoquinone, inhibits prostate carcinogenesis in intact and castrated Pten knockout mice by modulation of tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2142. doi:10.1158/1538-7445.AM2014-2142

Abstract 4875: Topically applied Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin inhibits ultraviolet radiation-induced development of cutaneous squamous cell carcinomas.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnSkin cancer is the second most common malignancy encountered in the U.S. with an expected diagnosis of 1.3 million new cases of non-melanoma skin cancer each year.nnCutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the most common non-melanoma forms of human skin cancer. SCC, unlike BCC, invades the nearby tissues. SCC is mainly caused by cumulative ultraviolet radiation (UVR) exposure over the course of a lifetime. Certain vulnerable populations, e.g., organ transplant recipients, have extraordinarily high rates of SCC (>50%) with markedly worse morbidity and mortality. To date, there are no accepted criteria for defining or managing these SCC patients. We found that PKCe is an important component of UVR-induced signal transduction pathways to the development of SCC. Also, PKCe is expressed in human and mouse SCC.nnTo find clues about the mechanism by which PKCe may impart susceptibility to UVR-induced development of SCC, we found that PKCe interacts with Hsp90β. In this experiment, PKCe transgenic mice were exposed once to a UVR (4 kJ/m2). PKCe-Hsp90 interaction was analyzed, by co-immunoprecipitation and Western blotting, at 1 and 2hr post UVR treatment.nnPKCe- Hsp90β interaction appears to be enhanced as early as 1hr post UVR treatment. The co-localization of PKCe with Hsp90β was observed in mouse epidermis, UVR-induced SCC in SKH-1 hairless mice and human SCC. Topical application of Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG) to mice inhibited UVR-induced Hsp90β-PKCe interaction, decreased PKCe expression levels and inhibited UVR-induced development of SCC. To determine the effects of topical 17AAG on UVR-induced development of SCC, the SKH1 hairless mice (6-7 week old) were exposed to UVR. The UVR source was Kodacel- filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). Mice were exposed to UVR (1.8 kJ/m2) three times weekly (Monday, Wednesday and Friday). The mice in the vehicle group (n=18) received topical treatment of 200μl vehicle (DMSO: acetone: 1:40 v/v) before and after UVR exposures. The mice in treatment group (n=13) received freshly prepared 500nmol of 17AAG (DMSO: acetone: 1:40 v/v) before and afternnUVR exposure. Topical 17AAG decreased SCC latency by 17 weeks and significantly (P<0.001) inhibited SCC incidence.nn17AAG-caused inhibition of UVR-induced SCC accompanied inhibition ofnnUVR-induced hyperplasia, Hsp90β andnnPKCe expression levels. Interestingly, 17AAG-caused inhibition of UVR-induced Hsp90β accompanied an increase in Hsp70 protein level. These results led us to conclude that disruption of UVR-induced interaction of Hsp90β with PKCe and other oncogenic client proteins bynnHsp90 inhibitor 17AAG may result in prevention and treatment of UVR-inducednnSCC. (Support: RO1CA102431).nnCitation Format: Anupama Singh, Samuel J. Bauer, Ashok Singh, Jordan M. Sand, Bilal B. Hafeez, Louise Meske, Ajit K. Verma. Topically applied Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin inhibits ultraviolet radiation-induced development of cutaneous squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4875. doi:10.1158/1538-7445.AM2013-4875

Abstract 5020: Characterization of epidermal stem cells in SKH1 hairless mice, a widely used mouse model to investigate ultraviolet radiation carcinogenesis.

Human cutaneous squamous cell carcinoma (SCC) is the second most common type of non-melanoma skin cancer (NMSC) after basal cell carcinoma (BCC) in US. Evidence indicates that the precursor cells are the precursor cells for the origin of SCC. The SCCs probably arise from bulge stem cell niche of hair follicle but not from the transit amplifying cells of epidermis. The SKH1 hairless mice is a widely used mouse model for ultraviolet radiation (UV)-induced carcinogenesis. However, SKH1 mice have never been evaluated for status of skin stem cell probably due to absence of hair and other associated hairless phenotype. It has recently become possible to isolate living hair follicle stem and progenitor cells from mouse skin because of the discovery of cell surface marker (CD34) that facilitate enrichment. The cell surface protein CD34, more widely known as a hematopoietic stem and progenitor cell marker, was found to be uniquely expressed in the mouse hair follicle bulge region using immunohistochemistry staining techniques. In addition, CD34 expression was restricted to the hair follicle regardless of hair follicle stage, making this a potentially important tool for selected enrichment of hair follicle bulge region keratinocytes. In combination with alpha 6-integrin staining and fluorescence activated cell sorting (FACS), CD34 is used to successfully isolate a small population of CD34+/alpha 6-integrin+ cells from a single cell preparation of mouse keratinocytes. CD34-expressing keratinocytes were confirmed to have properties consistent with stem and progenitor cells in that they were shown to be slowly cycling and have a high proliferative capacity in culture, growing larger colonies relative to those from CD34− keratinocytes. In the present experiments, the SKH1 mouse skin stem cell population was identified using stem cell markers (CD34+/α6-integrin+, Keratin-15, Gli1, and Sox9). The results of FACS analysis of live cells, isolated from untreated SKH1 mouse skin, indicate that there are 0.23% CD34+ and 1.05% CD34+/α6-integrin+ cells. The FACS sorted double positive (CD34+/α6-integrin+) stem cells of SKH1 are also observed to be positive for K15, Gli1, and Sox9 markers of stem cells in cytospin slides. In summary, the quantitative FACS analysis and qualitative immunofluorescence data in skin tissues confirms the presence of the stem cell populations in SKH1 mice. Support: (RO1CA102431). Citation Format: Ashok Singh, Anupama Singh, Ajit K. Verma. Characterization of epidermal stem cells in SKH1 hairless mice, a widely used mouse model to investigate ultraviolet radiation carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5020. doi:10.1158/1538-7445.AM2013-5020