Prohibitin in breast cancer cell lines: loss of antiproliferative activity is linked to 3' untranslated region mutations

Abstract

The evolutionarily conserved prohibitin gene is located on human chromosome 17q21, and two alleles have been identified. Our previous studies characterizing prohibitin in immortalized cells, classified into four complementation groups (A-D) based on the ability of whole-cell hybrids to become senescent, have suggested that it has tumor suppressor activity in group B cells. Only the cell lines assigned to group B are sensitive to the antiproliferative activity of prohibitin, and all are homozygous for an allele designated B because of its exclusive association with this group. Prohibitin genotyping of 22 breast cancer cell lines identified 17 homozygous for the B allele, 5 homozygous for the non-B allele, and no heterozygotes. Four of these cell lines were chosen for further characterization of prohibitin. In cell proliferation assays, the homozygous B breast cancer cell lines (BT-20, SK-BR-3, and MCF7) are all inhibited from traversing the cell cycle following the introduction of wild-type prohibitin transcripts. The cell line homozygous for the alternative non-B allele (BT-549) is not inhibited by transcripts. All of the breast cancer cell lines overexpress the longer form of the prohibitin mRNA (1.9 kb) and the protein. Mutational analysis of the protein-coding region detected no mutations in any of the lines. However, BT-20, SK-BR-3, and MCF7 cells are all mutated in the final 200 bases of the 3' untranslated region (3'UTR) exclusive to the 1.9-kb transcript, but BT-549 cells had no alterations in this region of the 3'UTR. Functional mapping experiments performed in the mutated SK-BR-3 line showed that the wild-type 3'UTR alone is sufficient to inhibit cell cycle progression, indicating that the antiproliferative activity of the prohibitin transcript is localized to this region. Overall, our results show that most (80%) of the cell lines derived from breast tumors have a common prohibitin genotype, suggesting that they belong to the same group of immortalized cells, group B. The results also show that the prohibitin 3'UTR exhibits the characteristics of a trans-acting regulatory RNA (riboregulator), the tumor suppressor activity of which is inactivated by mutation in group B immortalized cells.