Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.     

Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA

MicroRNAs are an extensive family of approximately 22-nucleotide-long noncoding RNAs expressed in a wide range of eukaryotes, including humans, and they are important in development and disease. We found that microRNA Mir-17-5p has extensive complementarity to the mRNA of AIB1 (named for "amplified in breast cancer 1"). Cell culture experiments showed that AIB1 expression was downregulated by Mir-17-5p, primarily through translational inhibition. Expression of Mir-17-5p was low in breast cancer cell lines. We also found that downregulation of AIB1 by Mir-17-5p resulted in decreased estrogen receptor-mediated, as well as estrogen receptor-independent, gene expression and decreased proliferation of breast cancer cells. Mir-17-5p also completely abrogated the insulin-like growth factor 1-mediated, anchorage-independent growth of breast cancer cells. Our results reveal that Mir-17-5p has a role as a tumor suppressor in breast cancer cells.     

Impaired DNA damage response in cells expressing an exon 11-deleted murine Brca1 variant that localizes to nuclear foci

Both human and mouse cells express an alternatively spliced variant of BRCA1, BRCA1-Delta11, which lacks exon 11 in its entirety, including putative nuclear localization signals. Consistent with this, BRCA1-Delta11 has been reported to reside in the cytoplasm, a localization that would ostensibly preclude it from playing a role in the nuclear processes in which its full-length counterpart has been implicated. Nevertheless, the finding that murine embryos bearing homozygous deletions of exon 11 survive longer than embryos that are homozygous for Brca1 null alleles suggests that exon 11-deleted isoforms may perform at least some of the functions of Brca1. We have analyzed both the full-length and the exon 11-deleted isoforms of the murine Brca1 protein. Our results demonstrate that full-length murine Brca1 is identical to human BRCA1 with respect to its cell cycle regulation, DNA damage-induced phosphorylation, nuclear localization, and association with Rad51. Surprisingly, we show that endogenous Brca1-Delta11 localizes to discrete nuclear foci indistinguishable from those found in wild-type cells, despite the fact that Brca1-Delta11 lacks previously defined nuclear localization signals. However, we further show that DNA damage-induced phosphorylation of Brca1-Delta11 is significantly reduced compared to full-length Brca1, and that gamma irradiation-induced Rad51 focus formation is impaired in cells in which only Brca1-Delta11 is expressed. Our results suggest that the increased viability of embryos bearing homozygous deletions of exon 11 may be due to expression of Brca1-Delta11 and suggest an explanation for the genomic instability that accompanies the loss of full-length Brca1.     

Immunohistochemical distribution of the 52-kDa protein in mammary tumors: a marker associated with cell proliferation rather than with hormone responsiveness

We have previously described a secreted glycoprotein of mol. wt 52,000 (52-kDa protein) which is induced by estrogen in some human breast cancer cell lines. This protein has been identified as the proenzyme of a lysosomal cathepsin-D-like protease which is secreted in large proportions in breast cancer cells. To determine which information may be generated by this marker when detected in mammary tumors, in comparison with hormone receptors, we used monoclonal antibodies interacting specifically with the 52-kDa protein and its related cellular processed products (mols. wts 48 and 34 kDa). A high concentration of this protein has been shown in proliferative ductal mastopathies and cysts, suggesting its value in detecting high-risk mastopathies. We now present the immunoperoxidase distribution of this protein in breast carcinoma compared to the cytosolic hormone receptors assayed in parallel. In 232 breast cancers, no correlation was found between the cellular 52-kDa protein content and cytosolic estrogen or progesterone receptor concentrations. This absence of correlation was also shown by the constitutive production of this protein by estrogen-receptor-negative breast cancer cell lines and confirmed by double immunostaining of breast cancer cell aspirates showing a dissociation between the cytoplasmic staining of this 52-kDa lysosomal protease and the nuclear staining of the estrogen receptor. These clinical results, associated with the in vitro mitogenic and proteolytic activities of this protein, strongly suggest that the 52-kDa protein staining in tissue is associated with tumor proliferation and/or invasion, rather than with hormone responsiveness.