Metastatic lymph node 64 (MLN64), a gene overexpressed in breast cancers, is regulated by Sp/KLF transcription factors Fabien Alpy 1 , Anne Boulay 1,2 , Christel Moog-Lutz 1,3 , Kumari L Andarawewa 1 , Se´bastien Degot 1 , Isabelle Stoll 1 , Marie-Christine Rio 1 and Catherine Tomasetto n,1 1 Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (IGBMC), CNRS/INSERM/Universite´Louis Pasteur, BP 10142, 67404 Illkirch, C.U. de Strasbourg, France MLN64, is invariably coamplified and coexpressed with erbB-2 in breast cancers. The human MLN64 and ERBB2 genes are positioned at less than 50 kb from each other, on chromosome 17q12. To understand the molecular basis of MLN64 overexpression in cancer, the genomic region containing the MLN64 and ERBB2 genes was isolated and mapped. The two genes, DARPP32 and Telethonin, flanking MLN64 respectively on its centromeric and telomeric sides, although coamplified, are not over- expressed in breast cancer cells, indicating that gene amplification is not sufficient to allow overexpression. The MLN64 minimal promoter was isolated and found to be a housekeeping gene promoter containing four potential Sp1 binding elements. Using Sp1-deficient Drosophila SL2 cells, MLN64 promoter activity was induced in a dose- dependent manner by exogenous Sp1 addition. Further- more, mutation of each individual Sp1 element resulted in a significant decrease in reporter gene activity, indicating that all the Sp1 binding elements are functional and act together to promote gene expression. Since the ERBB2 promoter is also positively regulated by Sp1, this study indicates that MLN64 and ERBB2 genes share common transcriptional controls together with a physical link on chromosome 17q. We speculate that, in addition to the oncogenic potential of erbB-2 overexpression, the un- balanced action of MLN64 contributes to the poor clinical outcome of breast tumors bearing this amplified region. Oncogene (2003) 22, 3770–3780. doi:10.1038/sj.onc.1206500 Keywords: erbB-2; MLN64; MENTHO; StAR; Sp1 Introduction Human metastatic lymph node (MLN) 64 cDNA was identified from a breast cancer-derived metastatic lymph node cDNA library by differential hybridization using malignant (MLN) versus nonmalignant (breast fibroa- denoma and normal lymph node) tissues (Tomasetto et al. , 1995a). MLN64 cDNA encodes a protein of 445 residues that can be divided into two domains. The amino-terminal half of the protein contains four potential transmembrane regions and targets the protein to the membrane of late endosomes (Alpy et al., 2001). Recently, we have isolated a novel late-endosomal protein, MENTHO (MLN64 N-terminal domain homo- logue), structurally related to the amino-terminal half of MLN64. MENTHO and MLN64 share 70% identity and 83% similarity in an original protein domain that we designated as the MENTAL (MLN64 N-TerminAL) domain (Alpy et al., 2002). The carboxy-terminal half of the protein is structurally related to the steroidogenic acute regulatory (StAR) protein (Moog-Lutz et al., 1997). StAR is a mitochondrial protein that regulates the acute production of steroids in the adrenal and gonads in response to corticotropin and luteinizing hormone, respectively (Stocco, 2001). The functional relation between MLN64 and StAR was previously addressed. It was shown that, like StAR, MLN64 can enhance steroidogenesis in vitro (Watari et al., 1997). Moreover, the region conserved between StAR and MLN64, the StAR-related transfer (START) domain was shown to be a cholesterol-binding domain for both proteins (Tsujishita and Hurley, 2000). While StAR acts as a sterol-carrier directly on the mitochondria (King et al., 1995; Arakane et al., 1998), MLN64 is involved in the cholesterol transport from endosome to cytoplasmic acceptor(s) (Alpy et al., 2001). Chromosomal mapping showed that the MLN64 gene is located in the q12–q21 region of long arm of the chromosome 17 (Tomasetto et al., 1995a). This region is altered in 20–30% of breast cancers, the most common modification being the amplification of proto-oncogene ERBB2 (Slamon et al., 1987). Amplification of ERBB2 is a marker of poor prognosis and tumor aggressiveness in breast cancers and its overexpression in tumors has been correlated to therapy failure (Pegram et al., 1998; Ross and Fletcher, 1999). Although amplification involves a significant chromosomal region, bearing several genes, it is commonly believed that a single gene is the driving force for this mechanism. Nevertheless, recent evidences suggest that ERBB2 is not the unique Received 3 January 2003; revised 17 February 2003; accepted 17 February 2003 *Correspondence: IGBMC, De´partement de Pathologie Mole´culaire, 1 rue Laurent Fries, BP10142, 67404 Illkirch Cedex, France; E-mail: cat@igbmc.u-strasbg.fr 2 Current address: Novartis Pharma AG, Klibeckstrasse VKL 125.3.16, 4002 Basel, Switzerland 3 Current address: U440 INSERM/ IFM; 17, rue du Fer a` Moulin; 75005 Paris, France Oncogene (2003) 22, 3770–3780 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc