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PARKZ>FRA6E and Afadin in breast cancer

A Letessia et al

Fijiurc 5 Afadin knockdown in MDCKIl oclls Icads to thc Uestabili/ation of ccii ccii junctions. (al DiITcrcnt miRNA exprcs-sion \cctors wcre tcstcd in Cos cclls. Iwo dilTercnt miRNA cxpression pbsmids (CmCil-P-jniR-neg and I.mCil P-miR-Lac/.l wcrc uscd as irrclcvant Controls to asscss thc spccilicity of thc LinCil P-miR-Afadin plasmid. Cos lysates 10//g wcre separatcd on SDS PAG li thcn immunoblottcd willi Afadin mAb (top) and p85 to control loading (hottom). (I>) MDCKIl cclls stably exprcssing thc I.mCil P-miR-Afadin plasmid. Afadin is localized al ccii celi junctions in conllucnt MIX KII cclls as prcviously dcscribcd (arrowhcads). High limGFP cxprcssion corrclatcs willi a markcd knockdown of Afadin cxprcssion. thc dcstabili/ation of celi ccii junctions and thc cxdnction of Afadin signal at ccii ccii junctions (arrowsi.

the Iretjucncy of PARK2 break and its poicntial impaci on clinical outcoine. Break of one alicie of PARK2 was obsersed in 6% of tumors. Ii correlated willi decreased 5-ycar MFS. Loss of Parkin cxpression was observcd in around 13% of cases. Similarly, Parkin expression is decreased in a large proportion of ovarian tumors (Cesari et al., 2003; Denison et a/.. 2003a). Hosvevcr. we lound lluu loss of Parkin was not correlated with a break of PARK2. Tliis was in sharp contrast lo the good corrclation obscrved between alterations al FRA3E and PRAJ6D and FIUT and WW()X protein levels, respcctisely (Ginestier et al.. 2003: Park et a/.. 2004). The absence of correlalion between FRA6E break and Parkin exprcssion suggests that other mechanisms are responsible lor abnormal expression ol’ Parkin. Abnor-mal methylalion may be one ol’ ihese mechanisms (Agirre et al., 2005).

We hypolhesized that the conscqucnces of the PARK2 break on the clinical outeome may be due to a

subsequcnl eITcct on a 6q gene. lelomeric of PARK2. Genes located lelomeric of PRA6E may be poicntial breast cancer genes. Frecjucnt LOH are obsers ed in ihis region in breast cancer (Orphanos et a!.. 1995; Nosiello et al.. 1996; Cesari et al.. 2003). The tumorigcniciiy of breasl celi lines oan be suppressed by microcell-medialed iratisfer of a part ol’ human chromosome 6 (Negrini et al.. 1994). The 6q26 6q27 region conlains seseral genes including FOP. TTLL2. AF-6 MLLT4. KIF25, T1IBS2. and TBP. FOP encodes a centrosomal protein that is fused lo FGFR1 kinase in myeloproliferative disorders (Popovici et al.. 1999 ; Delasal et al.. 2005). It eould be interesting lo examine if this gene could play a role in oncogenesis om of its fusion willi FGFRF TTLL2 (Tubulin Tyrosine Ligase-likc 2) encodes a member of the TTL homology domain protein family, wliicli catalyses ihe ligalion of glutamie acid to tubulin. In ncuronal systems, tubulin polygliitamination could regulate the organization of microtubule network (Bonnet et al.. 2001) ihus controlling centriole stability and mitosis (Bobinnec et al.. 1998a. b). K/F25 encodes a protein of thc kinesin superfamily, KIF25. which is insolsed in molecular transport away from the ceniro-somc (Miki et al.. 2005). No role in neoplasia has been reported yet for KIF25. The promoter of TIIBS2 (ihrombospondin 2) gene is methylaled (62.5%) in primary endometrial carcinoma (Whitcomb et al.. 2003). High TIIBS2 expression may be associated with an angiogenic phcnotype in endometrial cancer and TI1BS2 expression is a marker of poor prognosis in ihis disease (Sęki et al.. 2001). The TATA-binding protein encoded by TBP is associated with transcriptional celi ul ar systems. Modulation of TBP concentration has an impaci on gene exprcssion that can mediale potential celi iransformalion (Johnson et a/.. 2003a.b).

AF-6 MLLT4 encodes Afadin. which is involvcd in epithelial physiology. li is ubiquitously expressed in normal epithelial cclls. w herc ii localizes al adherens and tight junctions (Mandai et a/.. 1997). Afadin is a scaffold protein that links adhesion proleins, ccllular receptors and signaling effectors to ihe actin cytoskeleton (Mandai et al.. 1997; Buchert et al.. 1999). Mice lacking the Ąf-6 gene die al 10 days post coitum of płacenia failure (Ikeda et a/.. 1999; Zhadanov et al., 1999). AF-6 could play a key role in the dcvclopmcnl of carcinomas. AF-6 is fused lo the MLL gene in the 1(6;I I)(q27:q23) chromosomal iranslocation. which is the most common translocation found in acutc lymphoid leukemia (Prasad et al.. 1993). We ilius chose lo study Afadin bccause of iis role in epithelial physiology and potential insolse-menl in cancer. bul also for tcclmical rcasons. The iwo anti-Afadin antibodics work ssell in IIK on paraflin embedded tissues whereas no appropriate antibody is available for the other proteins encoded by ihe FRA6E-telomeric genes.

The Afadin status in breast tumors was addrcssed by using IIK’ on TMA. Complete loss of Afadin was obsersed in 14.5% of tumors. Loss was correlated with the break of PARK2 and willi a bad outeome for patients without lymph node insasion. We propose that loss of Afadin expression can be due lo a break of

Oncogene