Detection of novel genes that are up-regulated (Di12) or down-regulated (T1A12) with disease progression in breast cancer.

Abstract

Carcinogenesis is a multistep process and it is believed that seven to ten alterations are needed to convert a normal cell into a maligant one. Thus, identification of these specific genetic lesions and their role in the progression of cancer will lead to more effective methods for early diagnosis, as well as provide targets for treatment and therapeutic intervention. To isolate a number of genes that are differentially expressed in human breast carcinomas we used subtractive hybridization and differential display cloning techniques. By using these methods we obtained 952 clones, characterized 288 clones by restriction mapping, and 105 by Northern blotting and DNA sequencing. Twenty-four clones were found to be previously unidentified, unique genes. Full-length complementary (c)DNA was isolated from clone T1A12 and Di12, and further characterized by computer data search. Polyclonal antibodies were generated against N- and/or C-terminal peptides of the predicted T1A12 and Di12 amino acid sequences and immunohistochemistry was used to delineate gene function in breast cancers. We found that T1A12 is a novel member of the insulin-like growth factor binding protein family and that its expression decreases with disease progression. In 60 primary breast tissues examined, strong T1A12-specific immunoperoxidase staining was observed in luminal epithelial cells of normal lobules or ducts, and in blood vessels. Moderate protein expression was detected in hyperplastic and ductal carcinoma in situ (DCIS) cells, but no staining was found in invasive ductal carcinoma (IDC) cells. On the contrary, by using the same primary breast specimen Di12, a gene which shares no sequence homology with any protein, was found to be overexpressed in IDC cells but was not detectable in normal breast tissues. Specific strong Di12 staining was seen in the cytoplasm, with a slight increase in perinuclear regions of less well differentiated cells similar to those present in ductal carcinomas with poor prognosis. Both the T1A12 and Di12 genes, studied alone or in combination, could prove valuable for understanding the biology of breast cancer, detecting early and late stage disease, and for selecting appropriate treatments.