HOXB7 Overexpression Leads Triple-Negative Breast Cancer Cells to a Less Aggressive Phenotype

Abstract

HOX genes appear to play a role in breast cancer progression in a molecular subtype-dependent way. The altered expression of HOXB7, for example, was reported to promote breast cancer progression in specific subtypes. Here we induced HOXB7 overexpression in MDA-MB-231 cells, a cellular model of the Triple-Negative breast cancer molecular subtype, and evaluated the phenotypic changes in cell viability, morphogenesis, migration, invasion, and colony formation. During the phenotypic characterization of the HOXB7-overexpressing cells, we consistently found less aggressive behavior represented by lower cell viability, inhibition of cell migration, invasion, and attachment-independent colony formation capacities added to the more compact and organized spheroid growth in 3D cultures. We then evaluated the expression of putative downstream targets and their direct binding to HOXB7 comparing ChIP-qPCR data generated from HOXB7-overexpressing cells and controls. In the manipulated cells, we found enriched biding of HOXB7 to CTNNB1, EGFR, FGF2, CDH1, DNMT3B, TGFB2, and COMMD7. Taken together, these results highlight the plasticity of the HOXB7 function in breast cancer, according to the cellular genetic background and expression levels, and provide evidence that in Triple-Negative breast cancer cells, HOXB7 overexpression has the potential to promote less aggressive phenotypes.

Keywords: HOXB7; MDA-MB-231; breast cancer; cell phenotype.

Figure 1

HOXB7 expression in MDA231 cells after transfection. (A) HOXB7 mRNA expression in wild-type cells (WT), empty-vector transfected cells (EV), B7 (pool of cells transfected with HOXB7-expression vector), and D3 (clone of cells transfected with HOXB7-expression vector). p-Values for each comparison were: EV×B7 (p = 0.0199), EV×D3 (p = 0.0127), and B7×D3 (p = 0.0146). (B) HOXB7 cytoplasmic protein expression in WT, EV, B7, and D3 cells. (C) HOXB7 nuclear protein expression in WT, EV, B7, and D3 cells. p-Values for each comparison were: EVxD3 (p = 0.0263) and B7×D3 (p = 0.00486). The bars represent the mean ± SD obtained in three independent experiments. *, Statistically significant differences, p ≤ 0.05, obtained by Brown–Forsythe and Welch ANOVA tests (Multiple comparisons) with Games–Howell’s correction. n-fold represents the fold change in the indicated comparison taking EV or B7 values as reference.

Figure 2

HOXB7 overexpression effect on cell spheroids in 3D cultures and on viability and Docetaxel sensitivity in MDA231 cells. (A) Cell spheroids in 3D cultures from WT, EV, and D3 cells. WT and EV cells have similar patterns of 3D-growth with spread spheroids with protrusions (arrows). D3 cells grow in smaller and more compact spheroids without protrusion formations. (B) MTT cell-viability measure in EV and D3 cells at 24 h, 48 h, 72 h, and 96 h post-plating. p-values for 48 h and 96 h were p = 0.0140 and p = 0.0017, respectively. (C) EV and D3 cell sensitivity to 5 nM and 50 nM Docetaxel treatment at 24 h, 48 h, 72 h, and 96 h post-plating. p-values for ETOH-96 h was p = 0.0102; for Docetaxel 5 nM-24 h was p = 0.0003 and for Docetaxel 50 nM-24 h was p < 0.0001. Cells were kept in ETOH as control of vehicle action. The graphs represent the mean ± SD obtained in four independent experiments. *, Statistically significant differences (*, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001 and ****, p ≤ 0.0001) obtained through 2way ANOVA test (Multiple comparisons) with Sidak’s correction. The % values refer to the difference in D3 cell behavior in comparison to EV cells.

Figure 3

HOXB7 overexpression effect on MDA231 cells migration capacity. (A) Representative images of the wound-healing assay in EV and D3 cells at 0 h, 9 h, and 15 h after the scratch wound. Images captured using InCell Analyser 2000, 10x objective. (B) Wound areas of the EV and D3 cells along the time. The graph represents the mean ± SD obtained in four independent experiments. ***, Statistically significant differences, p ≤ 0.001, obtained through 2way ANOVA test (Multiple comparisons) with Sidak’s correction. The p-values for the D3 × EV comparisons are p = 0.0001 at 6 h and p = 0.0003 at 9 h, 12 h, and 15 h. The percentage values refer to the difference in D3 cells wound areas in comparison to EV cells.

Figure 4

HOXB7 overexpression effect on MDA231 cell invasion capacity. (A) Representative images of the nuclei of the EV and D3 cells that invaded the Matrigel^® layer of the invasion chamber and migrate through the control chambers. (B) Invasion percentage of EV and D3 cells with the respective invasion index. The graph represents the mean ± SD obtained in three independent experiments. * Statistically significant differences, p ≤ 0.05, obtained by unpaired T-test with Welch’s correction (p = 0.0395).

Figure 5

HOXB7 overexpression effect on anchorage-independent colony formation and on CTNNB1 expression in MDA231 cells. (A) Number of colonies formed by MCF7 and MDA231 (WT, EV, and D3). The graph represents the mean ± SD obtained in three independent experiments. *, Statistically significant differences, p ≤ 0.05, obtained by Brown–Forsythe and Welch ANOVA tests with Games–Howell’s correction. p-values were: MCF7xWT (p = 0.0093), MCF7xEV (p = 0.0071), WTxD3 (p = 0.0408), and EVxD3 (p = 0.0071). n_fold represents the fold change in the indicated comparison and taking MCF7 or EV values as reference. (B,C) CTNNB1 mRNA and total protein relative expression in EV and D3 cells. (D) Western blot representative of the CTNNB1 total protein analyses in EV and D3 cells with the expression of Tubulin as the loading control. The bars represent the mean ± SD obtained in three independent experiments. *, Statistically significant differences (*, p ≤ 0.05 and **, p ≤ 0.01), obtained by unpaired T-test with Welch’s correction. p-values for mRNA and protein expression analyses were p = 0.0207 and p = 0.0066, respectively. n_fold represents the fold change in the expression of D3 cells in comparison to EV cells.

Figure 6

HOXB7 protein direct interaction with the promoter regions of EGFR (A), FGF2 (A), CTNNB1 (B), CDH1 (C), DNMT3B (D), TGFB2 (E), and COMMD7 (F). IgG, Immunoprecipitations with anti-IgG antibody (interaction negative control). HOXB7, Immunoprecipitations with anti-HOXB7 antibody. The bars represent the mean ± SD obtained in three independent experiments. *, Statistically significant differences, (*, p ≤ 0.05, **, p ≤ 0.01 and *** p ≤ 0.001) obtained by 2way ANOVA test (Multiple comparisons) with Bonferroni’s correction (EGFR, FGF2, CTNNB1 DISTAL, CTNNB1 PROX, CDH1 CpG, and CDH1 PROM) or by Brown–Forsythe and Welch ANOVA tests (Multiple comparisons) with Games–Howell’s correction (DNMT3B, TGFB2, and COMMD7). The calculated p-values were: EGFR (IgG_D3 x D3 = 0.01), FGF2 (IgG_EV x EV = 0.035, IgG_D3 × D3 = 0.036), CTNNB1 (DISTAL: IgG_EV × EV = 0.032, IgG_D3 × D3 = 0.011. PROX: IgG_EV × EV = 0.022, IgG_D3 × D3 = 0.017), CDH1 (CpG: IgG_EV× EV = 0.018, IgG_D3 × D3 = 0.002. PROM: IgG_EV × EV = 0.034, IgG_D3 × D3 = 0.0001, EV × D3 = 0.031), DNMT3B (IgG_D3 × D3 = 0.033), and COMMD7 (IgG_D3 × D3 = 0.013). n_fold represents the fold change in the indicated comparison and taking IgG or EV values as reference.

Figure 7

Summary of the HOXB7 nuclear overexpression effects in the TNBC cell line MDA-MB-231. The HOXB7 nuclear overexpression leads the MDA-MB-231 cells to a less aggressive phenotype represented by a more organized spheroid formation in 3D culture and reduced cell viability, migration, invasion, and anchorage-independent colony formation. The observed phenotypes could be related to changes in CTNNB1 expression and to HOXB7 binding to genes with important roles in breast cancer progression. An exception was made for COMMD7 whose function in breast cancer is still understudied. HOXB7 tridimensional structure within the nucleus is by Emw—Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=8820026 (accessed on 29 April 2021).