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Loss of AF6/Afadin, a Marker of Poor Outcome in Breast Cancer, Induces Cell Migration, Invasiveness and Tumor Growth

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Loss of AF6/Afadin, a Marker of Poor Outcome in Breast Cancer, Induces Cell Migration, Invasiveness and Tumor Growth Oncogene (2011) 30, 3862–3874 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Loss of AF6/afadin, a marker of poor outcome in breast cancer, induces cell migration, invasiveness and tumor growth G Fournier1,4,5, O Cabaud1,4,5, E Josselin1,4,5, A Chaix2,4,5, J Ade´laı¨de4,5, D Isnardon4,5, A Restouin3,4,5, R Castellano3,4,5, P Dubreuil2,4,5, M Chaffanet1,4,5, D Birnbaum1 and M Lopez1,4,5 1Inserm, UMR 891, Laboratoire d’oncologie mole´culaire, CRCM, Marseille, France; 2Inserm, UMR 891, Laboratoire d’he´matopoı¨e`se fonctionnelle et mole´culaire, CRCM, Marseille, France; 3Inserm, UMR 891, Plateforme TrGET essais pre´-cliniques, CRCM, Marseille, France; 4Institut Paoli-Calmettes, Marseille, France and 5Univ. Me´diterrane´e, Marseille, France Afadin/AF6, an F-actin-binding protein, is ubiquitously leading to improvement of classification and prognosis expressed in epithelia and has a key role during develop- (Sorlie et al., 2001). Despite this, adverse evolution can ment, through its regulatory role in cell–cell junction still be observed for patients with apparent good organization. Afadin loss of expression in 15% of breast prognosis at diagnosis and vice versa. New molecular carcinoma is associated with adverse prognosis and markers that help define prognosis are needed. We increased risk of metastatic relapse. To determine the previously showed that afadin expression is lost in 15% role of afadin in breast cancer, we studied the functional of breast cancer irrespective of the molecular subtype consequences of afadin protein extinction using in vitro (Letessier et al., 2007). We further found that afadin is and in vivo models. Three different breast cancer cell lines an independent adverse prognosis factor for patients representative of the major molecular subtypes were without axillary lymph node invasion. Absence of afadin stably repressed for afadin expression (knockdown of expression in tumors correlates with reduced 5-year afadin (afadin KD)) using RNA interference. Collective metastasis-free survival (MFS). and individual migrations as well as Matrigel invasion The MLLT4 gene encoding afadin is located on were markedly increased in afadin KD cells. Heregulin-b1 chromosome 6 band q27 (Saha et al., 1995). No paralog (HRG-b1)-induced migration and invasion were increased gene exists in mammals. Loss of afadin expression in by twofold in afadin KD cells. Conversely, ectopic breast tumors correlates with break at the FRA6E expression of afadin in the afadin-negative T47D cell line common fragile site located at 6q26 (Letessier et al., inhibited spontaneous and HRG-b1-induced migrations. 2007). Afadin is a multidomain F-actin-binding protein RAS/MAPK and SRC kinase pathways were activated in expressed in almost all epithelial tissues, and in a variety afadin KD cells. Activation levels positively correlated of other cell types including neurons, fibroblasts, with migration and invasion strength. Use of MEK1/2 endothelial and epithelial cells (Mandai et al., 1997; (U0126) and SRC kinases (SU6656) inhibitors reduced Takai et al., 2008). From the N- to C-terminus, the long afadin-dependent migration and invasion. Afadin extinc- isoform of afadin comprises two RAS/RAP1 binding, a tion in the SK-BR-3 cell line markedly accelerated tumor Forkhead, a Dilute, a PDZ and an actin-binding growth development in mouse mammary gland and lung domains (Hofmann and Bucher, 1995; Ponting, 1995; metastasis formation. These results may explain why the Mandai et al., 1997; Linnemann et al., 1999; Yamamoto loss of afadin expression in tumors correlates with high et al., 1999). The short isoform lacks the actin-binding tumor size and poor metastasis-free survival in patients. domain (Saito et al., 1998). Afadin role is complex, and Oncogene (2011) 30, 3862–3874; doi:10.1038/onc.2011.106; still not precisely known. Afadin interacts with various published online 11 April 2011 proteins (receptor tyrosine kinases, cell adhesion mole- cules, cytoskeletal components, regulatory and signaling Keywords: afadin; breast cancer; migration; invasion; molecules). Studies of afadin have been carried out in tumorigenicity; SRC and MAPK different species, at different developmental stages and in different cellular models in vitro. In mouse, afadin knockout results in embryonic lethality with defects starting at gastrulation. This includes disorganization of Introduction the ectoderm, impaired migration of the mesoderm and loss of somites (Ikeda et al., 1999; Zhadanov et al., Breast cancer is a heterogeneous disease. Different 1999). In Drosophila, afadin homolog Canoe (Cno) molecular subtypes of breast tumors have been defined, (Miyamoto et al., 1995) is essential during morphogen- esis to maintain adherens junctions by connecting adherens junctions to actin (Sawyer et al., 2009). Correspondence: Dr M Lopez, Laboratoire d’oncologie mole´culaire, However, in contrast to mammals, Cno is dispen- UMR891 Inserm, 27 Bd Leı¨Roure, 13009 Marseille, France. E-mail: [email protected] sable for cadherin–catenin-based adherens junctions Received 3 September 2010; revised and accepted 15 February 2011; assembly and establishment of cell polarity in the fly published online 11 April 2011 (Sawyer et al., 2009). Cno contributes to regulate signal Afadin loss in breast cancer and tumorigenicity G Fournier et al 3863 transduction during morphogenesis and genetically interacts with RAS, JNK, NOTCH and WNT pathways (Miyamoto et al., 1995; Takahashi et al., 1998; Matsuo et al., 1999; Boettner et al., 2003; Carmena et al., 2006). Cno regulates cell shape changes during dorsal closure, asymmetric divisions and cell fate choice (Takahashi et al., 1998; Carmena et al., 2006; Speicher et al., 2008). The precise mechanisms that support afadin and Cno function are still obscure, but the data have assigned a role for afadin in cell–cell adhesion and/or in the regulation of signal transduction. Afadin has also been described as a regulator of integrin-mediated cell adhe- sion (Su et al., 2003). More recently, afadin has been described as a regulator of cell migration. However, afadin contribution as positive or negative regulator of migration is still largely controversial (Lorger and Figure 1 (a) Afadin knockdown in breast tumor cell lines was Moelling, 2006; Miyata et al., 2009; Severson et al., obtained by stable RNAi strategy. SK-BR-3, MCF7 and MDA- 2009). The role of afadin in oncogenesis has been docu- MB-231 cell lines were tested by western blot using two different monoclonal antibodies. Top: the anti-AF6 (BD) directed against mented in some acute lymphoid and myeloid leukemias. the PDZ domain of afadin. Bottom: the anti-AF6 (HBT) directed The MLLT4 gene is a fusion partner of the mixed against the last C-terminus amino-acid residues present in the long lineage leukemia gene MLL (Prasad et al., 1993). Self- isoform. Both antibodies were used at a concentration of 1 mg/ml, association of afadin activates the oncogenic potential gave similar pattern and revealed the 220-kDa long isoform of afadin. (À): miRNA LacZ control vector; ( þ ): miRNA afadin of MLL–afadin fusion protein (Liedtke et al., 2010). vector. The P85 regulatory subunit of phosphoinositide-3 kinase To determine the functional consequences of afadin was used as loading charge control. (b) Afadin knockdown using loss of expression in breast cancer physiopathology, we three different miRNA afadin vectors (1, 2 and 3) gave variable studied in vitro and in vivo models. We inhibited afadin levels of inhibition compared with the miRNA LacZ control vector expression in breast tumor cells expressing afadin using (4). Afadin extinction level in cell population was controlled to be homogenous based on GFP signal, and immunofluorescence RNA interference (RNAi). We demonstrated that stable studies using anti-afadin antibody (data not shown). knockdown of afadin (afadin KD) expression led to a marked increase in collective and individual cell migra- tions, as well as invasion. In afadin KD cells, increased the F-actin-binding domain and a 190-kDa isoform migration and invasion were concomitant and depen- missing the F-actin-binding domain (Mandai et al., dent of the activation of the RAS/MAPK and SRC 1997). The latter is usually less frequently expressed than kinase pathways. In vivo, afadin extinction led to the former. Because functional differences have been accelerated tumor growth development in mouse mam- reported between these two isoforms (Buchert et al., mary gland and lung metastasis formation. Our results 2007) we first characterized the isoforms of our cellular assigned afadin as a new breast cancer biomarker models. We used two different antibodies. One was involved in tumor progression. directed against the C-terminus F-actin-binding domain and thus specific for the 220-kDa isoform. The other, directed against the PDZ domain, recognized both Results isoforms. Both antibodies revealed a major band at 220 kDa, suggesting that the 220-kDa isoform is Afadin knockdown using miRNA sequences prominently expressed in the three cell lines To analyze the functional consequences of afadin loss of (Figure 1a). The knockdown expression of afadin in expression, we used an RNAi strategy. This was done the three cell lines was equally detected by both using an expression vector that enables the expression of antibodies. This ascertained the specificity of the engineered microRNA (miRNA) sequences and Emer- engineered miRNA vectors and the two antibodies ald GFP (EmGFP) as reporter (Letessier et al., 2007). (Figure 1a). Afadin knockdown efficiency was quanti- Three different afadin miRNA sequences were designed fied for the three different miRNAs in the three cell and cloned in this vector. These miRNA sequences were lines. Residual expression of afadin after knockdown located at positions 2515 (AF6-1), 3481 (AF6-2) and ranged from 12 to 38% for MCF7, from 0.5 to 37% for 4088 (AF6-3) in the afadin cDNA sequences. Valida- SK-BR-3 and from 33 to 67% for MDA-MB-231 tions of the respective vectors were done in Cos cells (Figure 1b).
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