Date

July 2008

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Cell and Developmental Biology

Institution

Oregon Health & Science University

Abstract

During my thesis work, I found that TGFβ signaling mediators, Smad2 and Smad4, but not Smad3, were frequently lost in human skin squamous cell carcinomas (SCCs). Previous studies reveal that Smad4-/- mouse epidermis develops spontaneous SCCs whereas Smad3-/- mice are resistant to carcinogen-induced skin cancer. To evaluate the significance of Smad2 loss in skin cancer, we generated mice with keratinocyte-specific Smad2 deletion. These mice exhibited accelerated formation and malignant progression of chemically-induced skin tumors compared to wildtype mice. Consistent with the loss of Smad2 in poorly-differentiated human SCCs, Smad2-/- tumors were poorly differentiated and underwent epithelial-mesenchymal transition (EMT) prior to spontaneous Smad4 loss. Reduced E-cadherin and activation of its transcriptional repressor Snail were also found in Smad2-/- mouse epidermis, and occurred more frequently in Smad2 negative human SCCs than in Smad2 positive SCCs. Knocking down Snail abrogated Smad2 loss-associated EMT, suggesting that Snail upregulation is a major mediator for Smad2 loss-associated EMT. Further, Smad2 loss led to a significant increase in Smad4 binding to the Snail promoter, and knocking down either Smad3 or Smad4 in keratinocytes abrogated Smad2 loss-associated Snail overexpression. Additionally, Smad2-/- skin has increased HMGA2 bound to the Snail promoter and loses transcriptional co-repressor TGIF binding. My data suggest that enhanced Smad3/Smad4-mediated Snail transcription contributed to Smad2 loss-associated EMT during skin carcinogenesis. Squamous cell carcinomas (SCCs) derived from keratinocyte-specific Smad2 or Smad4 knockout mice had three times the blood vessels of SCCs derived from wildtype mice. Smad4, but not Smad2, knockout tumors expressed increased vascular endothelial growth factor (VEGF), TGFβ1, and TGFβ-mediated proangiogenic components. In contrast, Smad2, but not Smad4, knockout tumors had increased hepatocyte growth factor (HGF). HGF and VEGF pathways converged downstream on pAKT. Preneoplastic skin lacking Smad2 or Smad4 had increased blood vessels and molecular alterations consistent with tumor angiogenesis. Consistently, Smad4 knockdown in human keratinocytes induced VEGF, which was abrogated by Smad3 co-knockdown. Smad2 knockdown in human keratinocytes induced HGF, which was abrogated by concomitant Smad3 or Smad4 co-knockdown. HGF promoter activity was increased by Smad2 knockdown, which was abrogated by Smad3 or Smad4 co-knockdown. Comparative chromatin immunoprecipitation showed Smad2-null skin had increased transcriptional activators bound to the HGF promoter, whereas Smad4-null skin had increased transcriptional repressors bound. Coimmunoprecipitation of complexes involving Smad2-HDAC3 or Smad4-CBP/p300 to the HGF promoter indicated that Smad2 acts to repress, while Smad4 activates HGF expression. HGF expression in human skin SCCs correlated with Smad2-negative Smad4-positive tumors. Therefore, I conclude deletion of Smad2 and Smad4 created an angiogenic microenvironment through common and distinct mechanisms.

Identifier

doi:10.6083/M4028PG3

School

School of Medicine

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