(A) Cluster diagram of Affymetrix microarray data using Rosetta Resolver to compare gene expression profiles of matched normal mammary tissue and adjacent primary breast cancers, revealing significantly lower HOXA9 transcript levels in 4 out of 5 expression sets analyzed (P ≤ 0.01). (B) Levels of HOXA9 mRNA (by quantitative RT-PCR) in primary human mammary tumors (n = 47) compared with normal breast tissue (n = 16). ****P = 0.00035. (C) In situ hybridization using a HOXA9 probe on nonmalignant (n = 4) or malignant (n = 6) mammary epithelial tissue. Scale bar: 100 μm. (D) Immunofluorescent staining for HOXA9 (red) and nuclei (blue) demonstrates robust cytoplasmic and nuclear localized HOXA9 protein in the epithelium of nonmalignant human breast tissue and reduced levels in breast tumors. Top right insets (original magnification, ×20) show a broader view of the breast tissue, with arrows indicating regions blown up in the main images. The arrow in the center main image corresponds with the bottom left inset, which shows a view of HOXA9 staining (original magnification, ×30). Scale bar: 50 mm. The 2^o control shows no nonspecific staining. (E) Breast cancer patients whose tumors expressed the lowest HOXA9 level (lowest expression quartile; red line) experienced significantly reduced disease-free survival compared with all other patients in the study (blue line). An “X” is used to denote each censored sample. P = 0.025. (F) Patients with the lowest HOXA9 levels in their tumors (lowest expression quartile; red line) also had significantly increased metastasis as a first event when compared with all other patients (blue line). An “X” is used to denote each censored sample. P = 0.02. (G) Bar graph demonstrating relative HOXA9 gene expression levels in nonmalignant and malignant MEC lines.

(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.

(A) Immunofluorescence images of β-catenin (red) and nuclei (blue) in T4-2 colonies reexpressing HOXA9 or BRCA1. Arrows indicate cleared lumens. Scale bar: 10 μm. (B) Immunofluorescence images of β-catenin (red) and nuclei (blue) in T4-2 colonies reexpressing HOXA9 alone or with a mutant BRCA1. Scale bar: 10 μm. (C) Quantification of colony size of T4-2 cells reexpressing HOXA9 or BRCA1. ***P = 0.001. (D) Quantification of cross-sectional area of T4-2 colonies formed by cells reexpressing HOXA9 alone or with a mutant BRCA1. *P = 0.05, ***P = 0.001. (E) Quantification of anchorage-independent growth and survival in T4-2 cells reexpressing either HOXA9 or BRCA1. ***P = 0.001. (F) Quantification of the percentage of T4-2 colonies that formed lumens following the reexpression of HOXA9 or BRCA1. *P = 0.0263. (G) Quantification of lumen formation in rBM T4-2 colonies with reexpressed HOXA9, when BRCA1 function has been compromised through coexpression of a mutant BRCA1. (H) Quantification of proliferation in T4-2 cells following HOXA9 reexpression with a mutant BRCA1. **P = 0.0025, ***P = 0.0003. (I) The time course of the progressive increase in xenograft size (5–30 days). Reexpression of HOXA9 in T4-2 tumor cells significantly reduced the rate of lesion expansion (filled circles) compared with the T4-2 vector controls (filled squares), which could be restored to that of T4-2 breast tumor cells if coexpressed with a mutant BRCA1 (filled triangles with red line). ***P = 0.001. (J) Lesion size (28 days) in each experimental group. **P = 0.01, ***P = 0.001.

(A) Semiquantitative PCR gel, indicating HOXA9 mRNA levels expressed in human nonmalignant (MCF10A), metastatic (MDA-231), and transformed (T4-2) MECs. 18S rRNA was used as a control. (B) Semiquantitative PCR gel showing transgenic HOXA9 mRNA levels in MDA-231 and T4-2 cell lines. Immunofluorescence images of nuclei (blue) and FLAG-tagged HOXA9 (red) in MDA-231 and T4-2 cells. Arrows indicate localization of Flag-tagged HOXA9-positive cells. Scale bar: 10 μm. (C) Proliferation in MDA 231 and T4-2 cells following HOXA9 reexpression. **P = 0.0025, ***P = 0.0003. (D) Cross-sectional area of MDA-231 and T4-2 breast tumor colonies in rBM expressing either the vector or HOXA9 transgene. ****P = 0.0001, **P = 0.0068. (E) Immunofluorescence images of β₄ integrin (red), Laminin-5 (red), β-catenin (red), and nuclei (blue) in T4-2 colonies expressing the vector or HOXA9 or phenotypically reverted acini (anti-EGFR) by inhibiting EGFR activity using tyrphostin. Arrows indicate cleared lumen. Scale bar: 10 μm. (F) Lumens observed in rBM-generated T4-2 colonies expressing the vector, HOXA9, or anti-EGFR phenotypically reverted acini. *P = 0.0188, **P = 0.0076. (G) The percentage of tumor colonies greater than 30 μm in diameter, and phase contrast images of tumor colonies embedded within soft agar. Scale bar: 50 μm (top panel); 20 μm (bottom panel). *P = 0.0221, **P = 0.018, ***P = 0.0005. (H) High- and low-magnification phase images of H&E-stained tissue sections of control tumor (T4-2 vector) and HOXA9 reexpressing tumor (T4-2 HOXA9) xenografts. Vascularized regions of T4-2 control tumors are indicated with white arrows, and cystic regions of T4-2 HoxA9 reexpressing tumors are indicate with black arrows. Original magnification, ×40; ×10 (insets). Scale bar: 100 μm.

(A) Immunofluorescence images of β-catenin (red), β₄ integrin (green), Laminin-5 (green), and nuclei (blue) in MCF-10A colonies expressing either luciferase control shRNA or shRNA-HOXA9 clone 3. Arrows indicate cleared lumen. Scale bar: 50 μm. (B) Colony size of MCF-10A cells cultured within a rBM and expressing reduced HOXA9 levels. ****P = 0.0001. (C) Lumens observed in MCF-10A colonies expressing luciferase control shRNA as compared with those with shRNA-mediated HOXA9 knockdown. **P = 0.0010. (D) The response (BRCA1 protein levels) of MCF-10A cells expressing reduced HOXA9 levels to 5 Gray irradiation compared with nonirradiated samples. *P < 0.001. (E and F) Phase images of tumor colonies embedded within soft agar (E) and the percentage of colonies greater than 40 μm in diameter (F). Scale bar: 50 μm. *P = 0.001. (G) Immunofluorescence images of β-catenin (red) and nuclei (blue) in MCF-10A colonies, expressing either luciferase control shRNA or shRNA-BRCA1 clone 5. The arrow indicates cleared lumen. Scale bar: 50 μm. (H) Colony size of MCF-10A cells cultured within a rBM and expressing reduced BRCA1 levels. ***P = 0.0024. (I) Quantification of lumens observed in MCF-10A colonies expressing luciferase control shRNA as compared with those with shRNA-mediated BRCA1 knockdown. ****P = 0.0003. (J) Immunofluorescence images of β-catenin (red) and nuclei (blue) in MCF-10A colonies expressing vector or mutant BRCA1 (BRCA1 MT). The arrow indicates cleared lumen. Scale bar: 10 μm. (K) Quantification of cross-sectional area of MCF-10A colonies in cells cultured within a rBM and coexpressing mutant BRCA1. *P = 0.05. (L) Quantification of lumens observed in MCF-10A colonies expressing vector control as compared with those expressing a mutant BRCA1. *P = 0.05.

(A) Line graph illustrating that significant correlations exist between HOXA9 and BRCA1 mRNA levels expressed in a cohort of normal (shown in blue; P ≤ 0.0001; r² = 0.8534) and tumorigenic (shown in red; P ≤ 0.0001; r² = 0.7160) human breast tissue specimens (n = 53). (B) Graph illustrating the relationship between HOXA9 and BRCA1 protein levels in human ER^–/PR^–/ErbB2^– breast samples. (C) Immunohistochemistry showing colocalized expression of HOXA9 and BRCA1 protein in the epithelium of normal human breast tissue and HOXA9⁺/BRCA1⁺ tumor samples, as compared with HOXA9^–/BRCA1^– tumor samples. (HOXA9 was “stained” with Fast Red, and BRCA1 was “stained” with DAB). Scale bar: 100 μm.