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Snai1 and Snai2 Collaborate on Tumor Growth and Metastasis Properties of Mouse Skin Carcinoma Cell Lines

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Snai1 and Snai2 Collaborate on Tumor Growth and Metastasis Properties of Mouse Skin Carcinoma Cell Lines Oncogene (2008) 27, 4690–4701 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Snai1 and Snai2 collaborate on tumor growth and metastasis properties ofmouse skin carcinoma cell lines D Olmeda1,3, A Montes1, G Moreno-Bueno1, JM Flores2, F Portillo1 and A Cano1 1Departamento de Bioquı´mica, UAM, Instituto de Investigaciones Biome´dicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain and 2Departamento de Cirugı´a y Medicina Animal, Facultad de Veterinaria, UCM, Madrid, Spain Snai1 (Snail) and Snai2 (Slug), the two main members of 2006). Local invasion is presently considered as the Snail family factors, are important mediators of epithe- leading event for carcinoma metastasis (Christofori, lial-mesenchymal transitions and involved in tumor 2006; Gupta and Massague, 2006). During the invasive progression. We recently reported that Snai1 plays a process, tumor cells lose their cell–cell adhesion proper- major role in tumor growth, invasion and metastasis, but ties and undergo profound changes in their phenotype the contribution ofSnai2 to tumorigenesis is not yet well known as epithelial-mesenchymal transition (EMT), a understood. To approach this question we have silenced process reminiscent of developmental EMT (Nieto, Snai2 and/or Snai1 by stable RNA interference in two 2002; Thiery, 2002). Downregulation of E-cadherin is independent mouse skin carcinoma (HaCa4 and CarB) a hallmark of EMT and occurs frequently during cell lines. We demonstrate that Snai2 knockdown has a carcinoma progression (Birchmeier and Behrens, 1994; milder effect, but collaborates with Snai1 silencing in Christofori and Semb, 1999; Thiery, 2002). Transcrip- reduction oftumor growth potential ofeither carcinoma tional repression mechanisms of E-cadherin have been cell line when injected into nude mice. Importantly, Snai1 intensively investigated in the last years and several or Snai2 silencing dramatically influences the metastatic transcription factors characterized as E-cadherin repres- ability ofsquamous carcinoma HaCa4 cells, inducing a sors (reviewed in Peinado et al., 2007). Among them, strong reduction in liver and lung distant metastasis. two members of the Snail superfamily, Snai1 (Snail; However, only Snai1 knockdown has an effective action Batlle et al., 2000; Cano et al., 2000) and Snai2 (Slug; on invasiveness and fully abolishes tumor cell dissemina- Hajra et al., 2002; Bolos et al., 2003) are presently tion into the spleen. These results demonstrate that Snai1 considered key regulators of EMT (Nieto, 2002). and Snai2 collaborate on primary tumor growth and Although the function of Snai1 and Snai2 can be specifically contribute to site-specific metastasis ofHaCa4 interchangeable in different species (Barrallo-Gimeno cells. These data also indicate that Snai1 is the major and Nieto, 2005), a distinct role for each factor is regulator oflocal invasion, supporting a hierarchical supported from analysis of knockout mice. While Snai1 participation ofboth factorsin the metastatic process. null mice present early embryonic lethality (Carver Oncogene (2008) 27, 4690–4701; doi:10.1038/onc.2008.118; et al., 2001), Snai2 null mice are viable, undergoing a published online 14April 2008 normal program of development (Jiang et al., 1998). The specific contribution of Snai1 and Snai2 to tumor Keywords: Snai1; Snai2; E-cadherin; EMT; invasion; progression is still poorly defined. Recent studies on metastasis Snail factors indicate that either Snai1 and/or Snai2 can be expressed in different carcinomas, including breast, ovarian, colon and squamous cell carcinomas, some- times associated to invasion, metastasis and/or poor prognosis (Peinado et al., 2007). Previous studies on Introduction human breast carcinoma cells also suggested a differ- ential involvement of Snai1 and Snai2 in E-cadherin Despite the enormous knowledge accumulated in the repression or specific invasion patterns (Hajra et al., past decades on the mechanisms of tumor generation, 2002; Come et al., 2006). We recently described an very little is still known about the molecular mechanisms important role for Snai1 in tumor growth, differentia- governing metastatic dissemination (Mehlen and Puisieux, tion and invasion of mouse skin (Olmeda et al., 2007a), and in lymph node metastasis of human breast Correspondence: Dr A Cano, Department of Biochemistry, UAM, carcinoma cells (Olmeda et al, 2007b). Now, we have Instituto de Investigaciones Biome´ dicas ‘Alberto Sols’ CSIC-UAM, investigated the interplay between Snai1 and Snai2 in c/Arturo Duperier 4, Madrid 28029, Spain. tumorigenesis targeting each factor by stable RNA E-mail: [email protected] interference in two mouse skin carcinoma cell lines, 3Current address: Centro Nacional de Investigaciones Oncolo´ gicas, Melchor Ferna´ ndez Almagro, Madrid, Spain. analysing their effect in in vitro invasion, and tumori- Received 5 September 2007; revised 6 February 2008; accepted 7 March genic and metastatic behaviour into nude mice. Our 2008; published online 14April 2008 results indicate that Snai1 and Snai2 collaborate on Snai1 and Snai2 silencing and metastasis inhibition D Olmeda et al 4691 in vivo tumor growth and induction of lung and liver or Snai1 expression. The specificity of the individual distant metastasis. Importantly, Snai1 is determinant for interference was also evidenced; no significant changes local invasion and tumor cell dissemination into spleen. in endogenous Snai1 or Snai2 levels were detected in the Together, these data support an interplay and hierarch- shSnai2- and shSnai1-derived clones (Figures 1a and b). ical participation of Snai1 and Snai2 during tumor Expression of EMT markers was then analysed. Re- progression. expression of E-cadherin transcripts was detected in HaCa4cells after double interference (HSS-C1/C7) to similar levels obtained after Snai1 silencing, but not in HaCa4-shSnai2 cells (Figure 1a, left), indicating that Results Snai1 expression is sufficient to repress E-cadherin in HaCa4cells. No expression of E-cadherin protein could shSnai2 inhibits Snai2 expression without affecting Snai1 be detected in cultures of HaCa4-derived clones We have previously reported that Snai2 represses the (Olmeda et al., 2007a; data not shown). Induction of E-cadherin proximal promoter inducing a full EMT E-cadherin expression could not be detected in CarB- process (Bolos et al., 2003). We first used MDCK-Snai2 derived clones (Figure 1a, right). The absence of genetic cells to prove that siRNA against Snai2 abrogates alterations in the E-cadherin locus in CarB cells its transcriptional repression, by choosing a 19-mer (Navarro et al., 1991; Rodrigo et al., 1999), suggests C-terminal Snai2 sequence not present in Snai1 mRNA that additional repressors or epigenetic mechanisms of any species (Manzanares et al., 2001), cloned into affect E-cadherin expression in this cell line; indeed, pTER-Zeo vector. The shSnai2 derepressed E-cadherin hypermethylation of E-cadherin promoter was detected promoter in MDCK-Snai2 cells in a dose-dependent in CarB cells (Fraga et al., 2004). Expression of the manner (Supplementary Figure S1A). It also blocked mesenchymal markers vimentin and fibronectin, was not the repressive action of Snai2-EGFP in MDCK cells. affected by Snai2 silencing but was significantly Importantly, the effect of shSnai2 is specific as it did not decreased (50–60%) in double interfered CarB cells influence Snai1-EGFP mediated repression of E-cadh- (CSS-D5/D10; Figure 1c, right), similar to CarB- erin promoter (Supplementary Figure S1B). As pre- shSnai1 cells (Olmeda et al., 2007a). Vimentin and viously reported, shSnai1 is specific for Snail1 repression fibronectin expression was moderately reduced in (Supplementary Figure S1B; Olmeda et al., 2007a). double interfered HaCa4(HSS-C1/C7; 30–40%), but not affected in HaCa4-shSnai2 (Figure 1c, left) or Interference of endogenous Snai2 in carcinoma cells HaCa4-shSnai1 cells (Olmeda et al., 2007a). These affects EMT markers and cooperates with Snai1 silencing results, suggest that Snai1 and Snai2 can cooperate in in decreased invasiveness the expression of mesenchymal markers in a cell context The effectiveness of shSnai2 to block endogenous Snai2 dependent manner. Silencing of Snai1 and/or Snai2 expression was analysed in two mouse epidermal induces subtle changes in the epithelioid phenotype of carcinoma cell lines, derived from mouse squamous cell HaCa4cells that grow in compact epithelial-like islands carcinoma (HaCa4) and spindle cell carcinoma (CarB), at low cell density; however, at confluence the phenotype extensively characterized previously as E-cadherin defi- was not influenced (Supplementary Figure S2A, left cient, expressing high levels of Snai1 and Snai2, and panels). No changes in the spindle phenotype of CarB showing highly invasive, tumorigenic and metastatic cells was detected after Snai1 and/or Snai2 knockdown behaviour (Navarro et al., 1991; Llorens et al., 1998; at any density (Supplementary Figure S2B). In agree- Cano et al., 2000). We have recently reported that ment with the morphological phenotype, Snai1 and/or endogenous Snai1 silencing in HaCa4and CarB cells Snai2 silencing did not modify the organization of the leads to decreased expression of mesenchymal markers F-actin and microtubule cytoskeleton of either cell type and invasiveness (Olmeda et al., 2007a). To answer (Supplementary Figures S2A and B). whether Snai2 could collaborate with Snai1 in the We next analysed the effect of Snai2 and/or Snai1 phenotype and
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