DMBT1 expression is down-regulated in breast cancer

Abstract

Background: We studied the expression of DMBT1 (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle.

Methods: Sections from 17 benign lesions and 55 carcinomas were immunostained with anti DMBT1 antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions. DMBT1 gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients.

Results: Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments. DMBT1 protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive.

Conclusions: The redistribution and up-regulation of DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation.

Figure 1

Immunoreactivity of mammary epithelium with anti DMBTh12 Ab: a, supranuclear dot positivity in morphologically normal breast sample; b, strong, abundant surface immunoreactivity in a case of fibrocystic disease; c, intraluminal immunoreactive material in a case of gynecomastia; d, numerous supranuclear and randomly distributed positive dots in intraductal papillomatosis near an infiltrating ductal carcinoma; e, strong supranuclear and baso-lateral cytoplasmic immunoreactivity of hyperplastic ductal cells in a case of mucinous infiltrating carcinoma; f, granular diffuse intracytoplasmic positivity of hyperplastic intraductal cells in a case of carcinoma in situ; g, numerous positive malignant tumour cells of an infiltrating ductal carcinoma; h, few positive cells in an infiltrating ductal carcinoma; i, completely negative neoplastic cells of an invasive carcinoma; the arrow points to positive cells with large elongate nuclei in a non neoplastic duct facing normal negative cells.

Figure 2

a, double staining of morphologically normal mammary epithelium: with anti DMBTh12 Ab (red stain in the cytoplasm) and with anti MCM5 Ab (Brown stain in the nucleus); b, lower magnification of double labelled mammary tissue; arrow points to a structure negative for both antigens.

Figure 3

Immuno-reactivity of normal mammary epithelium in three serial sections incubated with: a, anti DMBTh12 Ab; b, anti MCM5 Ab and c, anti Ki 67 antibody where only few cells are labelled. Inset: higher magnification of the same ductal structure.

Figure 4

DMBT1 expression assessed by RT-PCR. Both DMBT1 (435 bp) and β-2microglobulin (602 bp) products were loaded in the same well. Lane M: size marker (50 bp ladder), lane 1: cell line Hs 578T, lanes 2, 3, 4, 5: breast carcinomas, lane 6: negative control without template.