Abstract A52: Overexpression of Sp2 increases susceptibility to wound- and carcinogen-induced papillomas in vivo and inhibits cell cycle progression and induces apoptosis of epidermal stem cells in vitro

Abstract

Abstract Transcription factors regulate gene expression and de-regulation of their activities can transform normal cells into tumor cells. The Sp-family of transcription factors consists of nine distinct genes (Sp1–9) that are evolutionarily-conserved and share a common DNA-binding domain. Sp2 is one of several poorly-characterized members of this transcription factor family. Inactivation of Sp2 function in zebrafish and mice indicates that Sp2 is required for the completion of gastrulation and embryogenesis, respectively, and thus Sp2 is an essential gene. A causal relationship between Sp2 and tumorigenesis is also suggested by the observation that Sp2 expression is correlated directly with the progression of human prostate cancers and murine cutaneous squamous cell carcinomas. To determine if this correlation is functionally meaningful we created transgenic mice that over-express mouse Sp2 in basal cells of stratified epithelia. We reported recently that these animals (Sp2-C) exhibit increased susceptibility to wound- and carcinogen (DMBA/TPA)-induced tumorigenesis, and we concluded that Sp2 over-expression is oncogenic. To determine whether wound-induced papillomas depend on infiltrating T-cells for growth we treated transgenic animals with Tacrolimus, a well-characterized T-cell inhibitor, before and after surgical wounding. We find that Tacrolimus treatment in Sp2-C, but not wild-type animals, increased wound-induced papillomagenesis dramatically indicating that a T-cell mediated immune response limits the outgrowth of these lesions. To determine if Sp2 DNA-binding activity is required for the increased susceptibility to wound- and carcinogen-induced tumorigenesis, we generated a transgenic mouse line (Sp2-Not1) that over-expresses a mutated Sp2 cDNA lacking a functional DNA binding domain. Interestingly, the incidence of papillomagenesis in Sp2-Not1 mice is half that of wild-type littermates when treated with DMBA/TPA. Moreover, Sp2-Not1 mice do not develop wound-induced neoplasms. Instead Sp2-Not1 mice exhibit a decreased capacity to heal surgical wounds. We interpret these results to indicate that the transgene carried by Sp2-Not1 mice negatively regulates keratinocyte proliferation and/or viability in vivo. To determine the molecular mechanisms underlying the phenotypes presented by our transgenic strains we have prepared primary keratinocyte cultures and analyzed rates of proliferation, expression of differentiation-specific markers, cell-cycle progression, and the incidence of apoptosis. CyQUANT® cell proliferation and tritiated-thymidine incorporation assays showed that keratinocytes obtained from Sp2-C and Sp2-Not1 animals proliferate poorly in vitro compared to wild-type keratinocytes. Flow cytometric analyses indicated that Sp2-C and Sp2-Not1 keratinocytes progress through the cell cycle inefficiently and accumulate in all cell-cycle compartments. Flow cytometry following annexin V/PI staining, immunohistochemistry for cleaved caspases-3, and genomic DNA laddering assays indicate increased numbers of apoptotic cells in Sp2-C and Sp2-Not1 cultures relative to cultures prepared from wild-type littermates. In summary, (1) Sp2 over-expression in epidermal progenitor cells is oncogenic and requires Sp2 DNA-binding activity. (2) Wound-induced papillomagenesis in Sp2 transgenic mice is inhibited by infiltrating T-cells. (3) Sp2 over-expression inhibits progression through all cell-cycle compartments in vitro and induces programmed cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A52.