HOXB9, a gene overexpressed in breast cancer, promotes tumorigenicity and lung metastasis

Abstract

The mechanisms underlying tumoral secretion of signaling molecules into the microenvironment, which modulates tumor cell fate, angiogenesis, invasion, and metastasis, are not well understood. Aberrant expression of transcription factors, which has been implicated in the tumorigenesis of several types of cancers, may provide a mechanism that induces the expression of growth and angiogenic factors in tumors, leading to their local increase in the tumor microenvironment, favoring tumor progression. In this report, we demonstrate that the transcription factor HOXB9 is overexpressed in breast carcinoma, where elevated expression correlates with high tumor grade. HOXB9 induces the expression of several angiogenic factors (VEGF, bFGF, IL-8, and ANGPTL-2), as well as ErbB (amphiregulin, epiregulin, and neuregulins) and TGF-ss, which activate their respective pathways, leading to increased cell motility and acquisition of mesenchymal phenotypes. In vivo, HOXB9 promotes the formation of large, well-vascularized tumors that metastasize to the lung. Thus, deregulated expression of HOXB9 contributes to breast cancer progression and lung metastasis by inducing several growth factors that alter tumor-specific cell fates and the tumor stromal microenvironment.

Fig. 1.

Expression of HOXB9 in breast cancer. (A) (Left) Normal human breast tissues derived from reduction mammoplasty were hybridized in situ with antisense and sense-HOXB9 probes. (Right) HOXB9 mRNA expression in breast carcinoma and matched normal glands. The normal tissue and tumor are indicated by closed and open arrows, respectively. S, stroma; T, tumor; N normal. (Original magnification 400×) (B) qPCR analysis of HOXB9 expression in microdissected breast carcinoma samples. HOXB9 mRNA was elevated in 17 patients of a 40-breast cancer patient cohort. The table shows the tissues analyzed for each of the 17 patients (1–17) with elevated HOXB9 expression. The level of HOXB9 mRNA in each tissue compartment within a given patient is shown. The HOXB9 expression level in the matched normal gland was set at 1. NA, tissue not available; N, normal.

Fig. 2.

HOXB9 induces EMT, cell motility, and angiogenesis. (A) (Left) Morphology of MCF10A cells expressing HOXB9 protein. (Right) Expression of HOXB9, E-cadherin, β-catenin, and vimentin in vector (V) and HOXB9 (B9)-MCF10A cells. (B) HOXB9 expression increases cell migration and invasion. Chemomigration and chemoinvasion of vector (V) and HOXB9 (B9)-MCF10A cells were assayed in the presence and absence of chemoattractant. The mean was derived from cell counts of nine fields. (C) HOXB9 induces angiogenesis in vivo. The dorsal air sac assay chambers contained PBS alone, vector-MCF10A (V), or HOXB9-MCF10A (B9) cells. Arrowheads highlight newly formed vasculature with a characteristic zigzag pattern in the HOXB9-MCF10A sample. (Original magnification 40×) The numbers under the images specify the mean number of newly formed vessels larger than 3 mm counted in each animal per experimental group (n = 5 mice per group). (D) Knockdown of HOXB9 suppresses angiogenesis in vivo. The dorsal air sac assay chambers contained MDA-MB-231 cells infected with shGFP or shHOXB9 (shB9). Arrowheads highlight newly formed vasculature with the characteristic zigzag pattern in the MDA-MB-231-shGFP samples. (Original magnification 40×) The numbers specify the mean number of newly formed vessels larger than 3 mm counted in each animal per experimental group (n = 5 mice per group). (E) RNA from vector and HOXB9-MCF10A cells and from MDA-MB-231 cells infected with shGFP and shHOXB9 was analyzed by qPCR to determine the expression of angiogenic factors shown in the figure. The fold change in expression relative to GAPDH is shown. The level of expression for each gene in vector-MCF10A and shGFP-MDA-MB-231 cells was set at 1.

Fig. 3.

Suppression of ErbB and Akt activation inhibits HOXB9 enhanced cell motility. (A) Expression of NRGs, ERG, and ARG is increased in HOXB9-MCF10A cells. GAPDH is shown to control for equal loading. (B) ErbB ligands are transcriptional targets of HOXB9. MCF10A cells infected with LacZ and V5-tagged HOXB9 lentiviruses were analyzed by ChIP with anti-V5 and control IgG antibodies. Total lysates were used as controls for input. Precipitated DNA was subjected to PCR using primers spanning the promoter region containing the putative HOX-binding sites. (C) (Left) HOXB9 expression is associated with increased ErbB receptor phosphorylation. Proteins from vector and HOXB9-expressing cells were analyzed for activation of ErbB1, ErbB2, and ErB3, and Akt by Western blot. The lysates were also probed for total protein. (Right) Inhibition of ErbB receptor phosphorylation abolishes Akt phosphorylation. HOXB9-MCF10A cells were treated with 10 μM ErbB receptor tyrosine kinase inhibitor (ErbBR-TKI) for 2 h, and proteins were analyzed by Western blot to monitor ErbB receptor and Akt phosphorylation and total ErbB receptor and Akt levels. (D) Suppression of ErbB receptor and Akt phosphorylation is associated with abrogation of cell migration. Migration of ErbBR-TKI–treated HOXB9-expressing MCF10A cells was assayed. The mean was derived from cell counts of nine fields.

Fig. 4.

TGF-β activation contributes to EMT and increased motility of HOXB9-MCF10A cells. (A) Expression of TGF-β1 and TGF-β2 is increased in HOXB9-MCF10A cells. GAPDH is shown to control for equal loading. (B) (Left) HOXB9 expression is associated with increased Smad2 phosphorylation. Proteins from vector (V) and HOXB9 (B9)-MCF10A cells were analyzed for phospho-Smad2 and total Smad2 protein. (Right) HOXB9-MCF10A cells were treated with 10 μM LY364947 for 24 h. Proteins were analyzed for phospho-Smad2 and total Smad2 levels. (C) HOXB9-MCF10A cells were treated with 10 μM LY364947, and total protein was analyzed for E-cadherin, fibronectin, and vimentin expression. (D) Migration and invasion of LY364947-treated HOXB9-MCF10A cells was assayed.

Fig. 5.

HOXB9 expression promotes tumor growth, angiogenesis, and distal metastasis to the lung. (A) MCF10A cells expressing activated H-Ras alone or activated H-Ras + HOXB9 were inoculated s.c. into mice; the mean change in tumor volume ± SD for each group is shown (n = 8 mice per group). (B) HOXB9-expressing tumors exhibit a higher proliferation index. Tumors from the two groups were stained with antibody to the proliferation marker PCNA. The mean number of PCNA-positive cells ± SD per field is shown (n = 10 fields). (C) HOXB9+activated-Ras tumors exhibit increased vascularization. Vessels are indicted with arrowheads. Quantification of vessels by CD31 staining is shown below. (Original magnification 200×) (D) HOXB9 expression promotes lung metastasis. Lungs of activated H-Ras+HOXB9 tumor-bearing mice (50%) demonstrate micrometastases, whereas none of the animals (0%) bearing activated H-Ras tumors show signs of lung metastasis. The GFP-expressing cell clusters in the lung were visualized under a dissecting microscope. The right panel shows a higher-magnification image of the inset. (Original magnification 40×).