HOXA9 regulates BRCA1 expression to modulate human breast tumor phenotype
Penney M. Gilbert, … , Barbara L. Weber, Valerie M. Weaver
Penney M. Gilbert, … , Barbara L. Weber, Valerie M. Weaver
Published April 12, 2010
Citation Information: J Clin Invest. 2010;120(5):1535-1550. https://doi.org/10.1172/JCI39534.
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Research Article Oncology

HOXA9 regulates BRCA1 expression to modulate human breast tumor phenotype

Abstract

Breast cancer 1, early onset (BRCA1) expression is often reduced in sporadic breast tumors, even in the absence of BRCA1 genetic modifications, but the molecular basis for this is unknown. In this study, we identified homeobox A9 (HOXA9) as a gene frequently downregulated in human breast cancers and tumor cell lines and noted that reduced HOXA9 transcript levels associated with tumor aggression, metastasis, and patient mortality. Experiments revealed that loss of HOXA9 promoted mammary epithelial cell growth and survival and perturbed tissue morphogenesis. Restoring HOXA9 expression repressed growth and survival and inhibited the malignant phenotype of breast cancer cells in culture and in a xenograft mouse model. Molecular studies showed that HOXA9 restricted breast tumor behavior by directly modulating the expression of BRCA1. Indeed, ectopic expression of wild-type BRCA1 phenocopied the tumor suppressor function of HOXA9, and reducing BRCA1 levels or function inhibited the antitumor activity of HOXA9. Consistently, HOXA9 expression correlated with BRCA1 in clinical specimens and with tumor aggression in patients lacking estrogen receptor/progesterone receptor expression in their breast tissue. These findings indicate that HOXA9 restricts breast tumor aggression by modulating expression of the tumor suppressor gene BRCA1, which we believe provides an explanation for the loss of BRCA1 expression in sporadic breast tumors in the absence of BRCA1 genetic modifications.

Authors

Penney M. Gilbert, Janna K. Mouw, Meredith A. Unger, Johnathon N. Lakins, Mawuse K. Gbegnon, Virginia B. Clemmer, Miriam Benezra, Jonathan D. Licht, Nancy J. Boudreau, Kelvin K.C. Tsai, Alana L. Welm, Michael D. Feldman, Barbara L. Weber, Valerie M. Weaver

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Figure 3

HOXA9 regulates BRCA1 expression.

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HOXA9 regulates BRCA1 expression. (A) Semiquantitative PCR gel, indicati...
(A) Semiquantitative PCR gel, indicating increased BRCA1 expression with the reexpression of HOXA9 in MDA-231 cells. (B) Bar graph quantifying immunoblot data from multiple experiments, showing increased BRCA1 protein in MDA-231 or T4-2 breast tumor cells reexpressing HOXA9. *P = 0.0457, **P = 0.0028. (C) Representative gel of ChIP studies in breast cancer cells, revealing coprecipitation of HOXA9 with the BRCA1 promoter and acetylated acetyl-H3-histone with the β-globin promoter. (D) Bar graphs quantifying ChIP experiments in MDA-231 (n = 2) and T4-2 cells (*P = 0.0178; n = 4). (E) Luciferase reporter analysis, showing a dose-dependent increase in BRCA1 promoter activity in response to addition of wild-type HOXA9. **P = 0.001. (F) Luciferase reporter analysis, displaying loss of BRCA1 promoter activity upon addition of HOXA9 containing an N255T (DNA BM) mutation in the conserved DNA binding domain. *P = 0.03 (G) Luciferase reporter analysis, indicating enhanced HOXA9-mediated BRCA1 promoter activity upon addition of PBX1 cofactor (2 μg), compared with Pbx1 alone or Pbx1 cotransfected with a shRNA reducing HOXA9 expression. *P = 0.0259. (H) Luciferase reporter analysis, showing a diminished responsiveness of a BRCA1 promoter construct containing a deletion in residues –223 to +44, which contains 3 putative Hox binding sites (gray bar). Data are normalized to matched vector control (black bars). Negative numbers refer to basepairs upstream of the BRCA1 transcription start site. **P = 0.02.