Snail induction is an early response to Gli1 that determines the efficiency of epithelial transformation

Abstract

Gli family members mediate constitutive Hedgehog signaling in the common skin cancer, basal cell carcinoma (BCC). Snail/Snai1 is rapidly induced by Gli1 in vitro, and is coexpressed with Gli1 in human hair follicles and skin tumors. In the current study, we generated a dominant-negative allele of Snail, SnaZFD, composed of the zinc-finger domain and flanking sequence. In promoter-reporter assays, SnaZFD blocked the activity of wild-type Snail on the E-cadherin promoter. Snail loss-of-function mediated by SnaZFD or by one of several short hairpin RNAs inhibited transformation of RK3E epithelial cells by Gli1. Conversely, enforced expression of Snail promoted transformation in vitro by Gli1, but not by other genes that were tested, including Notch1, ErbB2, and N-Ras. As observed for Gli1, wild-type Snail repressed E-cadherin in RK3E cells and induced blebbing of the cytoplasmic membrane. Induction of a conditional Gli1 transgene in the basal keratinocytes of mouse skin led to rapid upregulation of Snail transcripts and to cell proliferation in the interfollicular epidermis. Established Gli1-induced skin lesions exhibited molecular similarities to BCC, including loss of E-cadherin. The results identify Snail as a Gli1-inducible effector of transformation in vitro, and an early Gli1-responsive gene in the skin.

Gli1 acts through Snail and E-cadherin to promote nuclear signaling by β-catenin

“…To temporally correlate Gli1 activity with Wnt transcript levels, we used RTOG10 cells, a clone of RK3E that contains a tetracycline (tet)-inducible Gli1 allele (Li et al, 2006). Microarray analysis of these cells identified Wnt4 and Wnt7b as early responses to Gli1 (3 h, data not shown).…”

“…Microarray analysis of these cells identified Wnt4 and Wnt7b as early responses to Gli1 (3 h, data not shown). By semi- Li et al, 2006). Induction of Wnt4 was slight at 3 h and prominent at 6 h, whereas Wnt2b was induced between 3 and 6 h (Louro et al, 2002).…”

“…We stained sections of BCC-like lesions induced by tet on Gli1 (Li et al, 2006) using an antibody to the active, unphosphorylated form of b-catenin. The superficial epithelium stained weakly, whereas the more infiltrative basaloid cells stained prominently (Figure 2e).…”

“…Key effectors of Shh signaling include zinc-finger proteins of the Gli family. In mouse epidermal cells or in rat kidney epithelial cells immortalized with adenovirus E1A (i.e., RK3E cells), Gli1 rapidly induces transcription of Snail, a zinc-finger protein that represses transcription of E-cadherin and promotes the epithelial-mesenchymal transition (EMT) (Louro et al, 2002;Barrallo-Gimeno and Nieto, 2005;Li et al, 2006). Snail short-hairpin RNAs (shRNAs) or a dominant-negative (DN) allele blocked transformation of RK3E cells by Gli1, whereas exogenous Snail cooperated with Gli1 to induce transformation (Li et al, 2006).…”

“…In mouse epidermal cells or in rat kidney epithelial cells immortalized with adenovirus E1A (i.e., RK3E cells), Gli1 rapidly induces transcription of Snail, a zinc-finger protein that represses transcription of E-cadherin and promotes the epithelial-mesenchymal transition (EMT) (Louro et al, 2002;Barrallo-Gimeno and Nieto, 2005;Li et al, 2006). Snail short-hairpin RNAs (shRNAs) or a dominant-negative (DN) allele blocked transformation of RK3E cells by Gli1, whereas exogenous Snail cooperated with Gli1 to induce transformation (Li et al, 2006). E-cadherin (E-cad) is a type I integral membrane protein that localizes to adherens junctions and mediates Ca 2 þ -dependent, cell-cell adhesion (Cavallaro and Christofori, 2004;Nelson and Nusse, 2004;Gumbiner, 2005;Brembeck et al, 2006).…”