The Na⁺/H⁺ exchanger (NHE1) as a novel co-adjuvant target in paclitaxel therapy of triple-negative breast cancer cells

Abstract

Dysregulation of Na⁺/H⁺ exchanger isoform one (NHE1) activity is a hallmark of cells undergoing tumorigenesis and metastasis, the leading cause of patient mortality. The acidic tumor microenvironment is thought to facilitate the development of resistance to chemotherapy drugs and to promote extracellular matrix remodeling leading to metastasis. Here, we investigated NHE1 as a co-adjuvant target in paclitaxel chemotherapy of metastatic breast cancer. We generated a stable NHE1-knockout of the highly invasive, triple-negative, MDA-MB-231 breast cancer cells. The NHE1-knockout cells proliferated comparably to parental cells, but had markedly lower rates of migration and invasion in vitro. In vivo xenograft tumor growth in athymic nude mice was also dramatically decreased compared to parental MDA-MB-231 cells. Loss of NHE1 expression also increased the susceptibility of knockout cells to paclitaxel-mediated cell death. NHE1 inhibition, in combination with paclitaxel, resulted in a dramatic decrease in viability, and migratory and invasive potential of triple-negative breast cancer cells, but not in hormone receptor-positive, luminal MCF7 cells. Our data suggest that NHE1 is critical in triple-negative breast cancer metastasis, and its chemical inhibition boosts the efficacy of paclitaxel in vitro, highlighting NHE1 as a novel, potential co-adjuvant target in breast cancer chemotherapy.

Figure 1. Na/H exchanger expression and activity in parental MDA-MB-231 (231-WT) and NHE1-knockout cells (231-KO)

A, Western blot analysis of NHE1 expression in 231-WT, 231-KO, MDA-MB-468 and MCF7 cells probed with anti-NHE1 antibody. B, Changes in intracellular pH (pH_i) in response to acid loading. Examples of traces illustrating the recovery from an acute acid load. Intracellular pH was examined in cells that were transiently acidified using ammonium chloride. Periods of NH₄Cl, NaCl and Na-free solution are indicated. An entire example of the recovery is indicated for stimulated 231-WT cells (231s). An example of recovery is indicated for unstimulated 231-WT cells (231u) and MDA-MB-231 NHE1-knockout cells (231-KO). Fluorescence of BCECF, a pH-sensitive indicator dye, was used to record and quantify changes in pH_i post-acute acid load induced by ammonium chloride. NHE1 activity was calculated from the slope of the first 20 sec of recovery from acidification and was expressed as ΔpH/sec. C, Relative rate of Na⁺/H⁺ exchange activity of 231-KO cells relative to 231-WT cells, and in MDA-MB-468 and MCF7 cells. Values of ΔpH/sec from 231-WT and 231-KO cells were normalized to the unstimulated control values for 231-WT cells. Data for MCF7 and MDA-MB-468 were normalized to their control unstimulated values respectively. No discernible exchanger activity was detected in the knockout cells. Background drift and buffering capacity were not significantly different between cell types. In stimulated conditions (0.2% serum), NHE1 activity is significantly increased in 231-WT, MDA-MB-468 and MCF7 cells [*P<0.001, ⁺ P<0.01, N=8].

Figure 2. Characterization of NHE1-knockout (231-KO) MDA-MB-231 cells

A, B, Cell proliferation and viability in response to increasing paclitaxel doses was assessed by spectrophotometric analysis of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) absorbance at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of mean OD values at indicated time points for each cell type relative to 0 hr. A, Proliferation of 231-WT and 231-KO cells. In stimulated (0.2% serum) conditions, proliferation of knockout cells is not significantly different from parental MDA-MB-231 cells. However, both 231-WT and 231-KO cells are more proliferative over 48 hr. compared to MDA-MB-468 (468) and MCF7 cells. B, Effect of paclitaxel on cell viability of 231-WT and 231-KO cells. Paclitaxel was significantly more cytotoxic to NHE1-knockout cells compared to parental MDA-MB-231 cells at concentrations of 0.1 nM and higher in stimulated conditions over 24 hours [*P<0.001, ⁺ P<0.01, ^# P<0.05, N=3]. In contrast, stimulated MDA-MB-468 and MCF7 cells did not show any changes in viability dependent on paclitaxel concentration.

Figure 3. Effect of knocking out NHE1 on MDA-MB-231 xenograft tumor growth in female athymic nude mice

A, Average tumor volume over time in mice subcutaneously injected with either 231-WT or 231-KO cells. Suspensions of 231-WT and 231-KO cells in Matrigel were subcutaneously injected into the right and left dorsal flanks of female athymic nude mice to determine their tumor-promoting potential. Tumor growth was monitored weekly and mice were euthanized at Day 60. Tumor volume was calculated based on the volume of a sphere (V = (4/3)πr³), or the length and width of the tumor mass (V = 0.5x X 2y, where x is the length and y is the width of the tumor mass). There were 4 mice per group: 7 of 8, and 3 of 8, tumor xenografts developed in the wild type and knockout group respectively. B, Excised tumors from all mice. 231-WT xenografted cells developed into significantly larger, heterogeneous tumors than the 231-KO cells starting at Day 28 [^# P<0.05, N=8], and showed a marked increase tumor growth after Day 35 [*P<0.01, N=8]. No significant tumor growth was observed in the knockout group.

Figure 4. Effect of paclitaxel in combination with NHE1 inhibitors on cell viability of wild type (231-WT) and NHE1-knockout (231-KO) MDA-MB-231 cells, and MDA-MB-468 (468) and MCF7 cells

Cells were treated for 24 hours in reduced (0.2%) serum media (stimulated conditions), with 1 nM paclitaxel (TAX), 10 μM EMD87580 (EMD) (A), or 10 nM HMA (HMA) (B), or with both paclitaxel and EMD87580 (ET, A), or paclitaxel and HMA (HT, B) [*P<0.001, ⁺ P<0.01, N=5]. Media only control cells (CTRL) were left untreated. Viability was assessed by the catalytic conversion of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan in live cells. Spectrophotometric quantitation of this colorimetric change was recorded as the optical density of the sample at 570 nm, with background subtraction at 630 nm. All data are presented as a ratio of sample means over mean control values for each treatment for each cell type. In 231-KO cells, a significant loss of viability is observed when cells are treated with paclitaxel alone [⁺ P<0.01, N=5]. In stimulated conditions, both triple-negative MDA-MB-231 and MDA-MB-468 cells showed significantly reduced viability when treated with paclitaxel in the presence of NHE1 inhibitors EMD87580 or HMA [*P<0.001, N=5]. MCF7 cell viability, however, was unaffected by drug treatments.

Figure 5. Effect of paclitaxel in combination with NHE1 inhibitors on cell migration of wild type (WT) and NHE1-knockout (KO) MDA-MB-231, MDA-MB-468 (468) and MCF7 cells

The rate of closure of an induced gap was evaluated using a qualitative wound-healing assay as described in the Materials and Methods. A, Rate of gap closure in stimulated (STIM) (0.2% serum) and unstimulated (UNSTIM) (10% serum) 231-KO cells relative to 231-WT cells, and in comparison to MDA-MB-468 and MCF7 cells. Stimulated 231-WT and MDA-MB-468 cells migrate faster than unstimulated cells [*P<0.001, ^# P<0.05, N=10]. In contrast, in 231-KO cells, faster migration is observed in unstimulated conditions [*P<0.001, N=10]. B, Pictorial representation of gap closure (at 10X magnification) in serum-deprived 231-WT and 231-KO cells over time. C, Combined effect of paclitaxel and NHE1 inhibitors on the rate of migration. 1 nM paclitaxel (TAX) was evaluated in combination with either HMA (10 nM) or EMD87580 (10 μM). Arbitrary measurements of gap closure (normalized to the untreated controls) were pooled over multiple independent experiments and quantified with Image Pro Plus software [*P<0.001, ⁺ P<0.01, ^# P<0.05, N=10]. D, Pictorial representation of gap closure (at 10X magnification) in serum-deprived 231-WT cells treated with paclitaxel, either alone or in combination with EMD87580 or HMA at 18 hr.

Figure 6. Invasiveness of stimulated (0.2% serum) or unstimulated (10% serum) 231-WT, 231-KO, MDA-MB-468 or MCF7 cells treated with paclitaxel +/− NHE1 inhibitors

The rate of cell invasion was determined using a Matrigel-coated Boyden chamber porous insert as described in the Materials and Methods. A, Invasiveness of 231-KO cells, +/− stimulation, relative to 231-WT cells, and in comparison to MDA-MB-468 (468) and MCF7 cells [*P<0.001, N=4]. Side panel (B) illustrates representative images. C, Effect of paclitaxel (1 nM, TAX) in combination with NHE1 inhibitors (10 μM EMD87580 or 10 nM HMA) on cell invasion in wild type and NHE1-knockout MDA-MB-231, and MDA-MB-468 cells [*P<0.001, N=4]. D, Illustration of invasion of stimulated 231-WT cells.

Figure 7. Long-term invasion is dependent on the expression of NHE1 in MDA-MB-231 breast cancer cells

A, Representative image of 231-WT and 231-KO cells in 3-dimensional scaffold over 7 days in 10% serum; arrow shows the direction of invasion from top to bottom of the scaffold. B, Summary of results showing the number of invading cells [*P<0.001, N=3].