Cloning of novel mammary tumor progression and metastasis genes

Crucial to the prognosis of cancer patients is not growth of the primary tumor, but rather dissemination of neoplastic cells to other organs As the process of the activation and inactivation of genes that are involved in tumorigenesis and metastasis is still poorly understood, the aim of this study is to identify novel mammary cancer progression and metastasis genes in vivo. Proviral insertions of mouse mammary tumor virus (MMTV) in mammary epithelial cells are able to activate flanking oncogenes, leading to mammary tumor induction. Classical examples of MMTV-induced oncogenes are Wnt1 and Fgf3. Full neoplastic transformation to an invasive and metastasizing tumor requires activation of collaborating onco/metastasis genes. Thus, additional proviral insertions may lead to metastasis-inducing genes. In this study we used a BALB/c⁺ mouse strain (ie a BALB/c substrain that aquired C3H-MMTV by forster-nursing), and compared extra proviral integrations in a series of sets of independent primary tumors and metastases. As a first step, the isolated tumor sets (primary tumor and metastases) were analyzed by Southern blotting using a MMTV-LTR specific probe. A number of the lung metastases indeed carried additional MMTV integrations, which were not found in the primary tumor. These additional integrations might activate genes being responsible for the lung metastases. To analyze the flanking sequences more efficiently, an adaptor ligation-mediated PCR (Splinkerette-PCR) was modified for the metastasis-related proviral MMTV integrations. To this end, genomic DNA was digested and ligated to a suitable splinkerette linker. The subsequent PCR gets its specificity by using a unique MMTV-LTR-related oligonucleotide and a splinkerette-specific oligonucleotide, which is only able to bind to the DNA if extension of the MMTV oligonucleotide occurs. BLAST/NIX (DNA analysis software) analysis of the derived additional sequences from 23 tumor sets resulted in the discovery of a novel common integration site. The effect of this putative metastasis gene on the metastic potential of mammary tumor cells is presently being investigated.