Gene BRCC-2 and diagnostic and therapeutic uses thereof

#The present invention relates to a gene that encodes a polypeptide that modulates apoptosis. This polypeptide is a useful target for identifying compounds that inhibit cancer progression by modulating apoptosis. Also, this polypeptide is useful as a diagnostic target for detecting cancers wherein this polypeptide is overexpressed, e.g., breast cancer and lung cancer.

#Alterations in the cellular genes which directly or indirectly control cell growth and differentiation are considered to be the main cause of cancer. The raf gene family includes three highly conserved genes termed A-, B- and c-raf (also called raf-1). Raf genes encode protein kinases that are thought to play important regulatory roles in signal transduction processes that regulate cell proliferation. Expression of the c-raf protein is believed to play a role in abnormal cell proliferation since it has been reported that 60% of all lung carcinoma cell lines express unusually high levels of c-raf mRNA and protein. Rapp et al., The Oncogene Handbook, E. P. Reddy, A. M Skalka and T. Curran, eds., Elsevier Science Publishers, New York, 1988, pp. 213-253.

#Malignant tumors develop through a series of stepwise, progressive changes that lead to the loss of growth control characteristic of cancer cells, i.e., continuous unregulated proliferation, the ability to invade surrounding tissues, and the ability to metastasize to different organ sites. Carefully controlled in vitro studies have helped define the factors that characterize the growth of normal and neoplastic cells and have led to the identification of specific proteins that control cell growth and differentiation.

#FIG. 1. cDNA sequence of BRCC2, SEQ ID NO:1. The figure shows SEQ ID NO:1, the nucleotide sequence of a BRCC2 cDNA clone (862 bp), isolated from human breast cancer cells (MDA-MB 231) by the differential display of mRNA approach (GenBank Accession nos. AF220061 and AF303179). Nucleotide positions are indicated by numbers on the right. Shaded area represents the coding region (294-620 bp). Based on the genomic database search, BRCC2 is an intronless gene located on chromosome 11q (GenBank accession no. AP001359, clone RP11-820L6).

#FIG. 2. cDNA, SEQ ID NO:1, and predicted amino acid sequences of BRCC2, SEQ ID NO:2. The figure shows the nucleotide positions are indicated by numbers on the left. The predicted longest open reading frame (ORF) (108 amino acids) is shown. Motifs and other signature sequences were detected by domain database search (PROSITE). Underlined amino acids, N- glycosylation site; shaded residues, Protein Kinase C phosphorylation site; bolded residues, Casein Kinase II phosphorylation site. The nuclear localization of BRCC2 protein was predicted by Reinhardt's method.

#FIG. 3. Normal tissue distribution of BRCC2 gene expression. Human adult tissue mRNA blots (Clontech) were probed with 32P-labeled BRCC2 cDNA fragment. The blots were reprobed with β-Actin cDNA. As shown, BRCC mRNA (˜1.2 kb) is expressed in most normal tissues.

#The molecular genetic factors that negate cell death and contribute to tumor growth and metastasis can be attractive targets for therapeutic intervention. In a search for such genes, the present inventors have identified a full-length cDNA encoding a gene which is hereby named as BRCC2 that is a modulator of apoptosis.

#The expression of the gene BRCC-2 (FIG. 1 SEQ ID NO:1) is decreased in human breast cancer cells (BRCC) treated with a novel antisense raf oligonucleotide (AS-raf-ODN) (FIG. 5). AS-raf-ODN causes programmed cell death in cancer cells by decreasing the amount of a proliferation and survival—promoting protein Raf-1. Thus, the present inventors have shown that BRCC-2 is a component of growth and cell survival/death pathway in cancer cells and is regulated by Raf-1 protein. In linking the expression of BRCC2 to the modulation of Raf-1, the present inventors have shown that BRCC2 is up regulated by Raf-1 and therefore this newly discovered gene is a component of the cell survival/cell death pathway. More particularly, the present invention is based, at least in part, on the discovery that the BRCC2 gene plays a role down stream of raf-1.

#Other aspects of the present invention are based on the discovery that the manipulation of the level of BRCC-2 in cancer cells provides therapeutic advantages. For example, decreasing the amount of BRCC-2 induces many of the in vivo effects of antisense raf oligonucleotide such as tumor growth arrest, tumor regression, tumor cell death and/or potentiate radiation/drug-induced cytotoxicity. In addition, being a target potentially downstream of Raf-1, it is anticipated that greater specificity is obtained by targeting the action of BRCC2 as compared with Raf-1.