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2012 ?2012 Elsevier Inc.

725 The EMT program is orchestrated through a network of tran- scription factors, including Twist1 (Yang et al., 2004), Snail1/2 (Batlle et al., 2000;

Cano et al., 2000;

Hajra et al., 2002), Zeb1/2 (Comijn et al., 2001;

Eger et al., 2005), and FOXC2 (Mani et al., 2007). Our previous study found that Twist1 is a potent inducer of EMT and invadopodia-mediated ECM degradation (Eckert et al., 2011;

Yang et al., 2004). In mouse and human breast tumor xenograft models, Twist1 expression can promote tumor metas- tasis (Yang et al., 2004). Clinical studies have also associated expression of Twist1 in primary tumors with disease aggressive- ness and poor survival in many types of human cancers, such as squamous cell carcinoma, breast cancer, prostate cancer, and gastric cancer (Eckert et al., 2011;

Kallergi et al., 2011;

Peinado et al., 2007;

Watson et al., 2007). Unlike human carcinoma metastases, most established meta- static tumor cell lines present a permanent mesenchymal pheno- type (Blick et al., 2008) and cannot be used to address the dynamic EMT process during tumor metastasis in vivo. Recent elegant studies using autochthonous mouse tumor models observed the occurrence of EMT in primary carcinoma, but how EMT spatiotemporally regulates metastasis has not been investigated in these models (Hu ¨ semann et al., 2008;

Rhim et al., 2012). The chemical carcinogenesis mouse skin model has been shown to recapitulate the multistep process of human carcinoma progression, including initiation, growth, invasion, and metastasis (Kemp, 2005;

Perez-Losada and Balmain, 2003). At the molecular and genetic levels, the skin carcinogen- esis model shares strong similarities with a number of carcinoma in humans, including activating mutations in Ras family mem- bers, activation of PI3K- and Stat3-mediated signaling path- ways, elevated expression of transforming growth factor b1 (TGFb1), and activation of the TGFb/Smad signaling pathways and, at later stages, Trp53 mutations (DiGiovanni, 1992;

Kemp, 2005). Importantly, like human squamous cell carcinoma, this model develops distant metastases with an epithelial mor- phology in lymph nodes and lungs (Han et al., 2005), making it a suitable model to study the involvement of EMT in vivo. Furthermore, extensive studies have shown that expression of Twist1 in primary human squamous cell carcinoma, including esophageal cancer (Sasaki et al., 2009;

Xie et al., 2009;

Yuen et al., 2007) and head and neck cancer (Ou et al., 2008;

Wushou et al., 2012), correlates with distant metastasis and poor prognosis. In this study, we investigate the importance of the dynamic EMT process in metastasis in vivo using the skin carci- nogenesis model. RESULTS Induction of Twist1 Promotes Invasive Carcinoma Conversion Previous studies have demonstrated the necessary role of Twist1 as an inducer of EMT. To understand the contribution of Twist1 in metastatic carcinoma, we analyzed

99 primary human carcinomas with patient-matched lymph node metastases for Twist1 expression. Of the

20 cases with high Twist1 expression in the primary tumor, we found

16 cases with over 50% drop in Twist1 levels in the lymph node metastases (Figures S1A and S1B available online), suggesting Twist1 is activated in the primary tumor but not distant metastases. To study how dynamic activation of Twist1 directly impacts carcinoma progression, we generated skin-speci?c Twist1 Tet-on inducible mice by crossing transgenic mice carrying a single copy of a TetOP-Twist1 transgene with Keratin