The Vacuolar ATPase a2-subunit regulates Notch signaling in triple-negative breast cancer cells

Abstract

Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer with poor prognosis for which no targeted therapies are currently available. Notch signaling has been implicated in breast cancer but the factors that control Notch in TNBC are unknown. Because the Vacuolar ATPase has been shown to be important in breast cancer invasiveness, we investigated the role of a2-subunit isoform of Vacuolar ATPase (a2V) in regulating Notch signaling in TNBC. Confocal microscopy revealed that among all the 'a' subunit isoforms, a2V was uniquely expressed on the plasma membrane of breast cancer cells. Both a2V and NOTCH1 were elevated in TNBC tumors tissues and cell lines. a2V knockdown by siRNA as well as V-ATPase inhibition by Bafilomycin A1 (Baf A1) in TNBC cell lines enhanced Notch signaling by increasing the expression of Notch1 intracellular Domain (N1ICD). V-ATPase inhibition blocked NICD degradation by disrupting autophagy and lysosomal acidification as demonstrated by accumulation of LC3B and diminished expression of LAMP1 respectively. Importantly, treatment with Baf A1 or anti-a2V, a novel-neutralizing antibody against a2V hindered cell migration of TNBC cells. Our findings indicate that a2V regulates Notch signaling through its role in endolysosomal acidification and emerges as a potential target for TNBC.

Keywords: Notch signaling; a2V-ATPase; autophagy; bafilomycin; triple negative breast cancer.

Figure 1. a2V is abundantly expressed on the surface of TNBC cells

ER/PR positive MCF7, Triple–negative MDA-MB-231 and MDA-MB-468 cells were grown on chamber slides. Cells were fixed, permeabilized and processed for immunofluorescence microscopy. A. Representative images taken with a confocal laser-scanning microscope show expression pattern of V-ATPase ‘a’ subunit isoforms a1, a2, and a3 (green). Cultured cells were stained with antibodies specific for a2V-ATPase (red) along with B. plasma membrane marker, pan-cadherin (green) and C. early endosome marker, Rab5 (green). Colocalization was examined by confocal microscopy. Representative z-stack images are shown. Nucleus was stained with DAPI (blue). Scale bars: 10 μm. D. Non-permeabilized Breast cancer cells were cultured, fixed and subjected to surface staining of a2V. Stacked offset histograms are shown. Matched Isotype control from MDA-MB-468 is shown. E. Whole cell lysates from MCF7, SkBR3, MDA-MB-231 and MDA-MB-468 were harvested and subjected to Western blot analysis with specific antibody for a2V. β-actin was used as a loading control. DAPI: diamidino-2-phenylindole.

Figure 2. Expressions of Notch1 pathway genes are elevated in TNBC

Total RNA extracted from MCF7, SKBR3, MDA-MB-231 and MDA-MD-468 cell lines was subjected to qRT-PCR analysis for mRNA levels of A. Notch receptors NOTCH1, NOTCH2 B. Notch ligands JAG1, JAG2 and C. Notch Target Genes HES1 and HEY1. Data represent mean ± standard error, n = 4. D. Protein level expression of Notch1 and Jagged 1 in these cell lines was examined by Western blot analysis. β-actin was used as a loading control. E. Surface staining of Notch1 in non-permeabilized cells is shown as stacked offset histograms. Matched Isotype control from MDA-MB-468 is shown.

Figure 3. a2V and Notch1 are activated in human breast tumors

Tissue microarray containing human breast tumors and normal breast tissues (control) were used to immunolocalize A. a2V and B. Notch1. Tumors were grouped by receptor-defined subtype. 12 sections per subtype were analyzed. Brown staining - DAB, counterstain - hematoxylin. Original magnification: 400X. Corresponding scatter dot plots show immunostaining index score ((ISIS) = Stained area score (SAS) × Immunostaining intensity score (IIS)) for C. a2V and D. Notch1. Data represent mean ± standard error, n = 12. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 between a pair of subtypes. DAB: 3,3′ Diaminobenzidine.

Figure 4. a2V inhibition increases Notch signaling in TNBC

A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after transfection. Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.

Figure 5. V-ATPase inhibition blocks Notch receptor degradation by inhibiting lysosomal acidification and autophagy

TNBC cells were grown on chamber slides and treated with vehicle control or 0.1 μM Baf A1 for 4 hrs. Cells were fixed, permeabilized and processed for immunofluorescence microscopy. Localization of N1ICD (green) and Lamp1 (red) in A. control or B. Baf A1 treated MDA-MB-231 and MDA-MB-468 cells by immunofluorescence. Nucleus was stained with DAPI (blue). Scale bars: 10 μm. C. Lysosensor Green DND 153 staining of acidic intracellular compartments in MDA-MB-231 and MDA-MB-468 cells treated with control or Baf A1. Representative images for each cell line are shown. D. MDA-MB-231 and MBA-MB-468 cells were treated with vehicle Control, 2 μM GSI or 0.1 μM Baf A1 for 24 hrs and immunoblotted for LC3B. β actin was used as loading control.

Figure 6. a2V-ATPase inhibition enhances Wnt signaling in TNBC

A. MDA-MB-231 cells were transfected with scrambled control or a2V siRNA and harvested after 48 hrs of transfection. Fold change in mRNA expression levels of Wnt signaling genes WNT4, β-catenin (CTNNB1), C-MYC and Cyclin D1 (CYCD1) was assessed by qRT PCR. Prior to fold--change calculation, the values were normalized to signal generated from endogenous control 18srRNA. Data represent mean ± standard error, n = 4. *P ≤ 0.05, **P ≤ 0.01 compared to control siRNA. (B and C) TNBC cells were grown on chamber slides and treated with B. vehicle control or C. 0.1 μM Baf A1 for 4 hours. Cells were fixed, permeabilized and processed for immunofluorescence microscopy. Localization of β-catenin (green) is shown. Nucleus was stained with DAPI (blue). Scale bars: 10 μm.

Figure 7. Effect of V-ATPase inhibition on cell viability and migration of TNBC cells

MDA-MB-231 and MDA-MB-468 cells were seeded (1 × 10⁴ cells/well) in 96 well plates and treated with vehicle control, Baf A1 or GSI as indicated at various concentrations for 48 hrs. A. Cell viability measured by ApoTox-Glo™ triplex assay is shown as percent control. (B, C) Effect of V-ATPase inhibition on cell migration was demonstrated by using Oris™ Cell Migration Assay. 5 × 10⁴ cells were plated in 96 well plates with stoppers. When adherent, stoppers were removed and cells were treated with 1 μM Baf A1, 10 μg/mL anti-a2V or their respective controls DMSO or IgG and incubated for 48 hrs to permit cell migration. Quantified fluorescence of Cell Tracker Green CMFDA in the detection zone of B. MDA-MB-231 and C. MDA-MB-468 is shown. Data represent mean ± standard error, n = 3. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared to vehicle control. D. Summary: V-ATPase regulates Notch signaling in TNBC through its role in endolysosomal acidification and autophagy. Following interaction with ligands on signal sending cell, the Notch receptor on signal receiving cell is endocytosed and cleaved resulting in the release of NICD, which translocates to the nucleus and activates Notch target genes HES1 and HEY1. V-ATPase inhibition by Baf A1 or a2V knockdown halts the recycling (early endosomes) and degradative (autophagosome-lysosomes) routes of Notch receptor, thereby accumulating NICD and resulting in enhanced signaling. Furthermore, V-ATPase inhibition induces apoptosis and hinders cell migration of TNBC cells.