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. 2021 Jul;81(10):667-682.
doi: 10.1002/pros.24144. Epub 2021 May 6.

Inverse agonism at the Na/K-ATPase receptor reverses EMT in prostate cancer cells

Moumita Banerjee  1 , Zhichuan Li  2 , Yingnyu Gao  1   3 , Fangfang Lai  2   4 , Minqi Huang  1 , Zhongbing Zhang  2   5 , Liquan Cai  1 , Juan Sanabria  1   6 , Tianyan Gao  7 , Zijian Xie  1 , Sandrine V Pierre  1
Affiliations

Inverse agonism at the Na/K-ATPase receptor reverses EMT in prostate cancer cells

Moumita Banerjee et al. Prostate. 2021 Jul.

Abstract

The surface expression of Na/K-ATPase α1 (NKA) is significantly reduced in primary prostate tumors and further decreased in bone metastatic lesions. Here, we show that the loss of cell surface expression of NKA induces epithelial-mesenchymal transition (EMT) and promotes metastatic potential and tumor growth of prostate cancer (PCa) by decreasing the expression of E-cadherin and increasing c-Myc expression via the activation of Src/FAK pathways. Mechanistically, reduced surface expression of NKA in PCa is due to increased endocytosis through the activation of NKA/Src receptor complex. Using a high-throughput NKA ligand-screening platform, we have discovered MB5 as an inverse agonist of the NKA/Src receptor complex, capable of blocking the endocytosis of NKA. MB5 treatment increased NKA expression and E-cadherin in PCa cells, which reversed EMT and consequently decreased the invasion and growth of spheroid models and tumor xenografts. Thus, we have identified a hitherto unrecognized mechanism that regulates EMT and invasiveness of PCa and demonstrated for the first time the feasibility of identifying inverse agonists of receptor NKA/Src complex and their potential utility as anticancer drugs. We, therefore, conclude that cell surface expression of α1 NKA can be targeted for the development of new therapeutics against aggressive PCa and that MB5 may serve as a prototype for drug development against EMT in metastatic PCa.

Keywords: EMT; Na/K-ATPase; inverse agonist; metastasis; prostate cancer.

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Conflict of interest statement

CONFLICT OF INTERESTS

The authors Moumita Banerjee, Zhongbing Zhang and Zijian Xie are inventors on patents related to MB5. The remaining authors declare that there are no other conflict of interests.

Figures

Fig. 1
Fig. 1. Loss of α1 NKA in DU145 induces EMT and promotes invasion.
(A) Generation cell subclones from DU145 and α1 NKA knockdown DU145 (KD) cells. Representative immunoblots for α1 and β1 NKA expression are shown for comparison. (B) Quantitative analysis of α1 NKA expression (Western blot analysis), relative to tubulin. **p<0.01, ***p<0.001 as indicated (N=4, one-way ANOVA). (C) Quantitative analysis of β1 NKA expression relative to tubulin. *p<0.05, n.s=not significant, as indicated (N=4, one-way ANOVA). (D) Representative immunoblots for basal phospho and/or total forms of Src, FAK and Myc. Tubulin blot confirms equal loading. *p<0.05, **p<0.01 and ***p<0.001 relative to clone 5 (N=3–4, one-way ANOVA). (E) Representative phase-contrast images of subclones 5, 4 and 2. (N=4, scale bar = 50μm). (F) Representative immunoblots for epithelial (E-cadherin, ZO-1, ZO-2, occludin) and mesenchymal markers (SNAIL and ZEB1) in indicated subclones. (G) Quantitative analyses (Western blot analysis)*p<0.05 and **p<0.01 relative to clone 5 (N=4, one-way ANOVA). (H) qPCR analyses of EMT markers. *p<0.05 and **p<0.01 compared with sub-clone 5 (N=6, one-way ANOVA). (I) Relative cell migration at 16 hours (Boyden chamber assay) **p<0.01 compared with sub-clone 5 (N=6, one-way ANOVA). (J) Spheroid formation assay at day 7 (representative phase contrast images), (N=4, scale bar = 50μm). (K) Spheroid invasion assay. Representative images at indicated timepoints. (N=8, scale bar = 50μm) (L) Quantitative analysis of invasion. ***p<0.0001(N=8, Students t test). (M) Representative immunoblot for MMPs secreted by spheroids. Ponceau stained nitrocellulose membrane is shown as loading control (N=3). ANOVA, analysis of variance; EMT, epithelial-mesenchymal transition; MMP, matrix metalloproteinase; NKA, Na/K-ATPase α1
Fig. 2
Fig. 2. α1 NKA endocytosis and EMT.
(A) α1 NKA endocytosis (internalization) using cell surface biotinylation assay. **p<0.01 compared with DU145 (N=3, one-way ANOVA). (B) Representative confocal images of α1 NKA cellular distribution. DU145 and PC3 imaged at 3X exposure to C4–2(scale bar=50μm). (C, D) Representative immunoblots showing α1 NKA, epithelial marker(left) and mesenchymal marker (right) expression in common PCa cell lines (top). Quantitative analyses (bottom) *p<0.05 and **p<0.01 relative to C4–2 (N=4, two-way ANOVA). ANOVA, analysis of variance; EMT, epithelial-mesenchymal transition; MMP, matrix metalloproteinase; α1 NKA, Na/K-ATPase α1
Fig. 3
Fig. 3. Genetic rescue of α1 NKA counters tumor growth.
(A) Representative immunoblot showing rat α1 NKA expression (anti-NASE antibody recognizes only rat α1 polypeptide) in rescued cells and parental DU145. Bottom panel shows total α1 NKA expression (α6f antibody recognizes both human and rat NKA α1 polypeptide), (N=3). (B) MTT assay showing effect of ouabain on cell viability of DU145, KD and rat α1 rescued cells. (N=5–6). (C) Cell proliferation assay, *p<0.05 as indicated (N=6, one-way ANOVA). (D) Representative immunoblots showing Src activation (phospho-protein vs. total protein), total phosphotyrosine, Myc and tubulin expression (N=4). (E) Quantitative analysis (Western) *p<0.05 and **p<0.01 relative to DU145, (N=3, one-way ANOVA) (F) Tumor weight from α1 KD and α1 rescued cell xenograft. *p<0.05 (N=10, Students t test). ANOVA, analysis of variance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; α1 NKA, Na/K-ATPase α1
Fig. 4
Fig. 4. Schematic diagram of molecular mechanism
(A) Effect of tumor microenvironment on α1 NKA/Src receptor complex activation and its endocytosis leading to EMT (B) MB5 treatment blocks α1 NKA/Src receptor complex in inactive conformation and reverses EMT by stabilizing cell-cell attachment. EMT, epithelial-mesenchymal transition; α1 NKA, Na/K-ATPase α1
Fig 5.
Fig 5.. Validation of MB5 as an inverse agonist of α1 NKA/Src signaling.
(A) Confocal images of ouabain and MB5 on phosphor Src content in LLC-Pk1(N=4). (B) Confocal images of ERK activation/phosphorylation in LLC-PK1 cells, *p<0.05 and **p<0.01, n.s= not statistically significant (N=3, one-way ANOVA). Images are at same scale. (C) Effect of MB5 and ouabain treatment on ERK activation in LLC-PK1 cells (Western blot analysis). **p<0.01 compared with control and ##p<0.01 compared with only ouabain-treated group (N=3, one-way ANOVA). (D) Effect of MB5 on ouabain-induced rat α1 NKA (YFP-tagged) endocytosis(immunostaining). ###p<0.001 compared with control;**p<0.01 and *p<0.05 compared with only ouabain treated cells. (N=3, one-way ANOVA). (E) Effect of MB5 on basal ERK phosphorylation in PY17 cells, *p<0.05 and **p<0.01 compared with control, (N=3, one-way ANOVA). (F) Effect of MB5 treatment on α1 NKA endocytosis ( biotinylation assay) in subclone 2. **p<0.01 and *p<0.05 compared with control (N=3, One-way ANOVA). ANOVA= analysis of variance; α1 NKA, Na/K-ATPase α1
Fig. 6
Fig. 6. MB5 inhibited spheroid growth and invasion by reversing EMT and Src/FAK signaling.
(A) Representative immunoblots showing effect of 100nM MB5 treatment on expression of EMT markers, Src/FAK activation and cell proliferation markers (Myc and PCNA) in DU145-derived subclones. Loading control- tubulin, *p<0.05 and **p<0.01 as indicated, (Students t test, N=3–4). (B) Confocal images showing effect of 1μM MB5 treatment on E-cadherin and Occludin expression in subclone 2 after 16 h (N=3, scale bar=50μm). (C)Representative images (top) and quantitative analyses (bottom) showing effect of MB5 treatment on invasion and growth of subclone 2 spheroids. **p<0.01 compared with 0nM (N=4, one-way ANOVA). (D) Representative immunoblots showing effect of MB5 treatment on MMPs secretion by subclone 2 spheroids. N=3, loading control= Ponceau stained nitrocellulose membrane. (E) Boyden chamber migration assay of subclone 2 cells pretreated with MB5, PP2 (Src inhibitor) or FAK inhibitor. DMSO= vehicle, n.s.= not significant, **p<0.01 compared with –DMSO treated group (N=6, One-way ANOVA). (F) Effect of MB5 treatment on growth of sub-clone 5 spheroids, representative images (top) and quantitative analyses (bottom). *p<0.05 and **p<0.01 compared with 0nM (N=4, One-way ANOVA). ANOVA= analysis of variance; DMSO, dimethyl sulfoxide; EMT, epithelial-mesenchymal transition; MMP, matrix metalloproteinase
Fig. 7
Fig. 7. Effect of MB5 on PC3 and C4–2.
(A) Effect of MB5 treatment on PC3 spheroid invasion and growth. Representative images (top) and quantitative analyses (bottom). *p<0.05 and **p<0.01 compared with 0nM (N=6, one-way ANOVA). Spheroid growth from day 1 to 7, **p<0.01 compared with 0nM (N=5, one-way ANOVA). (B) Representative images of E-cadherin immunostaining in PC3 cells in ultralow attachment plate. (N=4, scale bar =50μm). (C) Representative immunoblots showing effect of MB5 on α1 NKA and Myc expression and Src activation in PC3, *p<0.05 and **p<0.01 as indicated (N=3, Students t test) (D) Representative blot of cytoplasmic and nuclear fractions on EMT markers in PC3 cells with MB5 treatment(N=3). (E) Representative blot showing effect of 1μM MB5 treatment on EMT phenotype in α1 KD cells derived from C4–2, *p<0.05 and **p<0.01 as indicated (N=3, Students t test).; α1 NKA, Na/K-ATPase α1
Fig. 8
Fig. 8. Effect of MB5 treatment on xenografted tumor growth in NOD/SCID mice.
(A) Effect of MB5 treatment (20mg/kg/day) on tumor growth in NOD/SCOD mice xenografted with DU145 or α1 KD cells. Quantitative analysis of tumor weight (bottom) and representative images of tumors with/without MB5 treatment (top). *p<0.05, ***p<0.001 as indicated (Students’ t test) (N=10 mice per group). (B) Tumor volume and weight of xenografted subclone 4 and 2 cells, ***p<0.001 (one-way ANOVA), *p<0.05 (Students’ t test, N=10). (C) Effect of MB5 treatment (10mg/kg/day) on xenografted tumor growth from aggressive sub-clones 4 and 2. ***p<0.001 and *p<0.05 as indicated (Students t test). N=10 tumors per group. Tumor weight (bottom) *p<0.05 (Students t test). (D) Protein expression analyses of tumor lysates (from sub-clone 2) by Western blot analyses. ***p<0.001 and *p<0.05 as indicated (one-way ANOVA), N=4–6 tumors per group. ANOVA= analysis of variance; KD, knockdown
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