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. 2022 Sep 27;11(19):3007.
doi: 10.3390/cells11193007.

Hypoxia-Inducible Expression of Annexin A6 Enhances the Resistance of Triple-Negative Breast Cancer Cells to EGFR and AR Antagonists

Stephen D Williams  1 , Tunde M Smith  1 , LaMonica V Stewart  1   2 , Amos M Sakwe  1
Affiliations

Hypoxia-Inducible Expression of Annexin A6 Enhances the Resistance of Triple-Negative Breast Cancer Cells to EGFR and AR Antagonists

Stephen D Williams et al. Cells. .

Abstract

Physiological changes such as hypoxia in the tumor microenvironment (TME) endow cancer cells with malignant properties, leading to tumor recurrence and rapid progression. Here, we assessed the effect of hypoxia (1% Oxygen) on the tumor suppressor Annexin A6 (AnxA6) and the response of triple-negative breast cancer (TNBC) cells to epidermal growth factor receptor (EGFR) and androgen receptor (AR) targeted therapies. We demonstrate that brief exposure of TNBC cells to hypoxia (within 24 h) is associated with down regulation of AnxA6 while > 24 h exposure cell type dependently stimulated the expression of AnxA6. Hypoxia depicted by the expression and stability of HIF-1/2α led to up regulation of the HIF target genes SLC2A1, PGK1 as well as AR and the AR target genes FABP-4 and PPAR-γ, but the cellular levels of AnxA6 protein decreased under prolonged hypoxia. Down regulation of AnxA6 in TNBC cells inhibited, while AnxA6 over expression enhanced the expression and cellular levels of HIF-1/2α, SLC2A1 and PGK1. RNAi mediated inhibition of hypoxia induced AnxA6 expression also strongly inhibited glucose uptake and ROS production in AnxA6 expressing TNBC cells. Using a luciferase reporter assay, we confirm that short-term exposure of cells to hypoxia inhibits while prolonged exposure of cells to hypoxia enhances AnxA6 promoter activity in HEK293T cells. Compared to cells cultured under normoxia, TNBC cells were more resistant to lapatinib under hypoxic conditions, and the downregulation of AnxA6 sensitized the cells to EGFR as well as AR antagonists. These data suggest that AnxA6 is a hypoxia inducible gene and that targeting AnxA6 upregulation may be beneficial in overcoming TNBC resistance to EGFR and/or AR targeted therapies.

Keywords: EGFR; Lapatinib; androgen receptor; annexin A6; hypoxia; therapy resistance; triple-negative breast cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Differential Response of TNBC cells to hypoxic conditions. (A,B) Representative TNBC cell models depicting AnxA6-high mesenchymal-like (BT-549) and AnxA6-low basal-like (MDA-468) TNBC cell lines were incubated under hypoxic conditions (37 °C; 1% O2; 5% CO2; 94% N2) for the indicated times. Whole cell lysates were prepared and assessed by Western blotting using antibodies against the indicated proteins. Detection of β-actin was used as a loading control. (C,D) TNBC cells were cultured under hypoxic conditions for 96 h and the expression of AnxA6, HIF-1/2A, and its downstream target genes SLCA21- and PGK-1 genes were assessed by RT-PCR. Statistical significance was calculated using the Mann–Whitney U-test in GraphPad. In parts C,D, each bar represents the mean ± SEM of duplicate analysis from three independent experiments. Asterisks (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001) compared to normoxic values.
Figure 2
Figure 2
Altered Expression of AnxA6 influences HIF-1α and/or HIF-2α. (A) AnxA6 downregulation in the representative mesenchymal-like BT-549 TNBC cells. (B) Control and AnxA6 downregulation as well as empty vector and AnxA6 overexpression in the representative basal-like (MDA-468) TNBC cell line. Cells were incubated under hypoxic conditions for 96 h, and whole cell lysates were assessed by Western blotting using antibodies against HIF-1/2α and AnxA6. Detection of β-actin was used as a loading control. (C,D) Effect of AnxA6 expression status, and hypoxia on the mRNA expression of HIF-1/2A, and downstream target genes. In parts C,D, each bar represents the mean ± SEM of duplicate analysis from three independent experiments. Asterisks (ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). p-values were analyzed by Two-way ANOVA.
Figure 3
Figure 3
The Proximal AnxA6 promoter is responsive to Hypoxia. (A) The proximal promoter of AnxA6 was truncated as indicated and cloned into pGL4 basic, then used to transfect HEK293T cells and the luciferase activity was assayed following treatment of the cells with 0.3 mM Ni2+ for 24 h using the dual luciferase assay kit. Renilla luciferase expressing vector was used as the transfection control. (B) HEK293T cells transfected with pGL4-A6-4 promoter were treated with the indicated concentrations of Ni2+ for 24 h followed by luciferase assay. (C) HEK293T cells transfected with EV and pGL4-A6-4 promoter were cultured under normoxia, hypoxia, with or without lapatinib (2 µM) treatment and luciferase activity was assays as in (A) above. (D) HEK293T cells transfected with pGL4-A6-4 promoter were cultured in hypoxic conditions for up to 96 h followed by luciferase assays. All data are presented as mean ± SEM, n = 3. Asterisks (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). p-values were analyzed by One-way ANOVA.
Figure 4
Figure 4
Effects of AnxA6 Downregulation on Glucose uptake, ATP production, and ROS levels during hypoxia. (A-C) Control AnxA6 expressing (NSC) and AnxA6 down regulated (A6sh5) MDA-468 and BT-549 TNBC cell lines were seeded in 96-well plates overnight and incubated with or without hypoxia for 48 h. Glucose uptake (A), ATP production (B), and ROS generation (C) were measured as described in materials and methods and normalized to normoxia values. All data are presented as mean ± SEM, n = 3. All data is normalized to control NSC-normoxia treatment groups for respective cell lines. Asterisks (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). p-values were analyzed by Two-way ANOVA.
Figure 5
Figure 5
Hypoxia induced AnxA6 expression is associated with lapatinib resistance in TNBC cells Parental TNBC cells (A,D), and control AnxA6 expressing NSC and AnxA6 down regulated (A6sh5) MDA-468 and BT-549 cells (E,H), were seeded at a density of 1.0 x 104 cells/well in quadruplicate in 96-well plates and incubated overnight. Cells were then treated with serial dilutions of the drug ± hypoxia for a further 72 h and cell proliferation was measured using the Prestoblue cell viability reagent. Cell viability was calculated relative to untreated DMSO controls, and the IC50 values were calculated by non-linear regression using GraphPad prism, v.9.3. All data are presented as mean ± SEM, n = 4.
Figure 6
Figure 6
Reduced AnxA6 expression is associated with increased AR expression and sensitivity of TNBC cells to AR antagonist. (A) Representative Western blot showing AR expression in MDA-468, BT-549 as well as in control AnxA6 expressing and (NSC), and AnxA6 down regulated (A6sh5) BT-549 cell lines. LNCaP cells were used as the AR-positive control. Detection of β-actin was used as a loading control. (B) The effect of AnxA6 expression with or without hypoxia on the expression of AR, PPARγ, and FABP-4 genes by qRT-PCR. (C, D) Effect of AR antagonists bicalutamide (Bic) and enzalutamide (Enz) on the viability of TNBC cells cultured under hypoxia. Cells were seeded in quadruplicate in 96 well plates, then treated with the indicated concentrations of the drugs under normoxia or hypoxia for 72 h. The viability of the cells was assessed by using the PrestoBlue cell viability assay reagent. All data are presented as mean ± SEM, n = 2. All data are normalized to NSC-DMSO control treatment groups for each cell line. Asterisks (* p ≤ 0.05; ** ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001) indicate significance relative to NSC-DMSO treated cells under normoxia. p-values were analyzed by Two-way ANOVA.
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