Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression

Abstract

TGF-β and its signaling mediators, Smad2, -3, and -4, are involved with tumor suppression and promotion functions. Smad4 -/-mouse epidermis develops spontaneous skin squamous cell carcinomas (SCCs), and Smad3 -/-mice are resistant to carcinogen-induced skin cancer; however, the role of Smad2 in skin carcinogenesis has not been explored. In the present study, we found that Smad2 and Smad4, but not Smad3, were frequently lost in human SCCs. Mice with keratinocyte-specific Smad2 deletion exhibited accelerated formation and malignant progression of chemically induced skin tumors compared with WT 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 of Smad2 loss-associated EMT. Furthermore, 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. Our data suggest that enhanced Smad3/Smad4-mediated Snail transcription contributed to Smad2 loss-associated EMT during skin carcinogenesis.

Role of SARA (SMAD Anchor for Receptor Activation) in Maintenance of Epithelial Cell Phenotype

“…In kidney proximal tubular epithelial cells, basal expression of ␣SMA increased as Smad2 was depleted via siRNA (27), and in studies using Cre/LoxP-mediated gene targeting, Smad2 knock-out hepatocytes spontaneously acquired mesenchymal cell features characteristic of EMT (28). How Smad2 ablation promotes increased EMT was investigated in a recent study using keratinocytes (29). In that study, it was shown that mice with keratinocyte-specific deletion of Smad2 had accelerated formation and malignant progression of skin tumors when compared with wild-type mice.…”

Smad3 Is a Key Nonredundant Mediator of Transforming Growth Factor β Signaling in Nme Mouse Mammary Epithelial Cells

“…Another study showed that Smad2 knockout hepatocytes acquire mesenchymal features and increased cell proliferation even in the absence of exogenous TGFβ stimulation (29), suggesting that Smad2 inhibits epithelial characteristics and proliferation, at least of hepatocytes. Likewise, Smad2 knockout keratinocytes have been reported to exhibit reduced levels of E-cadherin protein and increased levels of nuclear Snail protein in vivo, thus contributing to the accelerated formation and malignant progression of chemically induced skin tumors in mice (30). Moreover, depletion of Smad2 in human epithelial cells using siRNA was reported to enhance the Smad3-dependent cytostatic response to TGFβ (26).…”

Targeting TGF-β Signaling for Therapeutic Gain

“…A study of 34 matched primary and recurrent breast tumors shows that, despite apparent lack of TGFBR2 mutations in primary tumors, 12% of recurrent breast tumors contain receptor kinase-attenuating point mutations, suggesting that TGFBR2 mutation in a minority of breast tumors is a late event rather than a driver (Lucke et al 2001). In human cutaneous squamous cell carcinoma (cSCC), attenuated expression of Smad proteins has been reported (Hoot et al 2008), but whether this is owing to mutation or transcriptional or epigenetic dysregulation was not investigated. In head and neck squamous cell carcinoma (HNSCC), mutations in TGFBR2 and SMAD4 occur but at low frequency (1% or less) (TCGA Network 2015).…”

“…Examination of Smad activation in archival human tumor samples, probed by phosphoSmad ( pSmad) immunoreactivity, may be misleading (Xie et al 2002;Ogino et al 2007;Hoot et al 2008;Harradine et al 2009), not only because of the common limitations of immunohistochemistry (IHC), such as specificity and nonlinear sensitivity, but also because pSmad2 activation is heterogeneous and highly dynamic within the tumor. IHC analysis has shown increased levels of activated nuclear pSmad2 at the invasive front of human breast tumors (Kang et al 2005), and intravital microscopy using a fluorescent reporter to track Smad activation in live mouse tumors reveals the dynamic nature of this activity within breast cancer cells in vivo (Giampieri et al 2010).…”

“…Expression of E-cadherin, which can suppress tumor progression, is commonly down-regulated within many carcinomas during EMT (Caulin et al 1995;Portella et al 1998;Lacher et al 2006;Fransvea et al 2008;Hoot et al 2008). The TGF-b-Smad pathway mediates expression of high-mobility group A2 (HMGA2), which contributes to the induction of the expression of Snail and Slug, two zinc-finger transcription factors that repress the E-cadherin gene (Padua and Massagué 2009).…”