WHSC1L1, a new candidate oncogene within the 8p11-12 amplicon induces a proliferative advantage in breast cancer Free

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Amplification of chromosomal regions leads to increases in DNA copy number and expression of oncogenes in human breast cancer (HBC). The identification and functional characterization of tumor-specific oncogene targets has potential diagnostic and therapeutic implications. The 8p11-12 genomic region is amplified in 15% of human breast cancers and harbors several candidate oncogenes. Using Agilent oligonucleotide array-based comparative genomic hybridization (CGH), we have recently mapped the 8p11-p12 amplicon in three human breast cancer cell lines developed in our lab, and in 22 primary human breast cancers. Quantitative RT-PCR was used to determine the expression level of 53 genes from this region in breast cancer specimens. From these studies, we identified 21 candidate genes based on statistical association between copy number and expression. To elucidate the individual and cooperating oncogenic properties of these genes in human breast cancer, we established a cDNA expression library containing candidate genes using a lentiviral expression system. We transduced the lentiviral library with individual and different combinations of candidate genes into MCF-10A human mammary epithelial cells and selected recipient cells in serum-free medium lacking insulin-like growth factor and/or epidermal growth factor. In previous work, we demonstrated that three genes from the 8p11 region (LSM1, BAG4 and C8orf4) induce transformed phenotypes when over expressed in MCF10A cells. In the present study we established additional lentiviral gene constructs for SPFH2, PROSC, BRF2, DDHD2, WHSC1L1, TM2D2, AP3M2 and VDAC3. MCF10A cells were infected with these lentiviral vectors and tested for expression of growth factor-independence phenotypes. 5 of the 8 genes tested including BRF2, DDHD2 and WHSC1L1 could induce insulin-like growth factor-independent growth but not independence of EGF individually. However, growth curves and colony formation assays in MCF10A cells with equalized viral titer of the tested genes indicated that over expression of WHSC1L1 enhanced cell proliferation most dramatically in insulin deficient media. Furthermore, three-dimensional morphogenesis assays in Matrigel showed that MCF10A cells over expressing WHSC1L1 formed large, highly proliferative and abnormal structures compared with control cells. Thus, our data indicates that the 8p11-12 amplicon harbors multiple oncogenes includingWHSC1L1, a new candidate gene with transforming potential in human mammary epithelial cells.