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Regulation of Msx2 Gene Expression by Steroid Hormones in Human Nonmalignant and Malignant Breast Cancer Explants Cultured in Vitro

Muscle segment homeobox genes, which regulate developmental programs and are expressed in embryonic and adult tissue, play a role in development of some malignancies. There are no reports on the expression of these families of genes in breast cancer tissue. The aim of this study was to compare expression of Msx2 gene in breast cancer of different genotypes as well as in surrounding nonmalignant tissues. Explants obtained during surgery were divided according to their sex steroid receptor status determined by immunocytochemistry. Four explants obtained from malignant and nonmalignant tissue of each individual patient were incubated in a control medium or with the addition of progesterone (10− 7 M) alone, estradiol 17 β (10− 5 M) or both. The relative level of Msx2 transcripts was evaluated by a semiquantitative RT-PCR and cell proliferation by Alamar Blue test. Results of RT-PCR analysis showed that the relative expression of Msx2 gene depended on the presence of ER/PR receptors both in nonmalignant and malignant tissues Relative amount of Msx2 mRNA was the highest in surrounding nonmalignant ER + /PR − and ER − /PR + tissue, whereas in ER − /PR − and ER + /PR + tissue it was 1.4–1.6-fold lower. Tumorigenesis led to about a twofold decrease in the relative amount of Msx2 mRNA except for ER + /PR + immunophenotype, where no changes were observed. Addition of estradiol or progesterone to the culture of ER − /PR − type tissue explants did not change significantly the relative amount of Msx2 gene mRNA. An opposite effect was observed in ER + /PR − type of tissue. Addition of estradiol alone, or estradiol and progesterone together to tissue culture explants decreased two to three fold the relative amount of Msx2 gene mRNA in both, malignant and surrounding tissues. Progesterone alone had no effect on Msx2 gene expression in this type of tissue. The most complicated regulation was observed in ER + /PR + type of tissue. Culture of tissue explants supplemented with estradiol significantly increased the relative amount of Msx2 gene mRNA in the surrounding tissue. Progesterone enhanced the stimulatory effect of estradiol in surrounding tissues but not in the malignant tissue. Increased expression of Msx2 correlated with an increased proliferation in ER − /PR − and ER + /PR + types, but not in ER + /PR − type of tissues. In conclusion, obtained results provide evidence that estrogen affects Msx2 gene expression. Significant changes in the relative amount of Msx2 gene mRNA and lack of canonical ERE element in 5′-upstream sequence of this gene suggest that regulation takes place indirectly probably by protein-protein interaction. The decrease in the relative amount of Msx2 gene mRNA in ER + /PR − type tumor suggests that progesterone also affects Msx2 gene expression by an indirect mechanism(s).