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. 2020 Jun 10:11:848.
doi: 10.3389/fphar.2020.00848. eCollection 2020.

KV11.1 Potassium Channel and the Na+/H+ Antiporter NHE1 Modulate Adhesion-Dependent Intracellular pH in Colorectal Cancer Cells

Jessica Iorio  1 , Claudia Duranti  1 , Tiziano Lottini  1 , Elena Lastraioli  1 , Giacomo Bagni  1 , Andrea Becchetti  2 , Annarosa Arcangeli  1
Affiliations

KV11.1 Potassium Channel and the Na+/H+ Antiporter NHE1 Modulate Adhesion-Dependent Intracellular pH in Colorectal Cancer Cells

Jessica Iorio et al. Front Pharmacol. .

Abstract

Increasing evidence indicates that ion channels and transporters cooperate in regulating different aspects of tumor pathophysiology. In cancer cells, H+/HCO3 - transporters usually invert the transmembrane pH gradient typically observed in non-neoplastic cells, which is thought to contribute to cancer malignancy. To what extent the pH-regulating transporters are functionally linked to K+ channels, which are central regulators of cell membrane potential (Vm), is unclear. We thus investigated in colorectal cancer cells the implication of the pH-regulating transporters and KV11.1 (also known as hERG1) in the pH modifications stimulated by integrin-dependent cell adhesion. Colorectal cancer cell lines (HCT 116 and HT 29) were seeded onto β1 integrin-dependent substrates, collagen I and fibronectin. This led to a transient cytoplasmic alkalinization, which peaked at 90 min of incubation, lasted approximately 180 min, and was inhibited by antibodies blocking the β1 integrin. The effect was sensitive to amiloride (10 µM) and cariporide (5 µM), suggesting that it was mainly caused by the activity of the Na+/H+ antiporter NHE1. Blocking KV11.1 with E4031 shows that channel activity contributed to modulate the β1 integrin-dependent pHi increase. Interestingly, both NHE1 and KV11.1 modulated the colorectal cancer cell motility triggered by β1 integrin-dependent adhesion. Finally, the β1 integrin subunit, KV11.1 and NHE1 co-immunoprecipitated in colorectal cancer cells seeded onto Collagen I, suggesting the formation of a macromolecular complex following integrin-mediated adhesion. We conclude that the interaction between KV11.1, NHE1, and β1 integrin contributes to regulate colorectal cancer intracellular pH in relation to the tumor microenvironment, suggesting novel pharmacological targets to counteract pro-invasive and, hence, pro-metastatic behavior in colorectal cancer.

Keywords: Collagen I; beta 1 integrin subunit; cariporide; hERG1; integrins; lateral motility.

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Figures

Figure 1
Figure 1
Effect of Collagen I, Fibronectin, and Vitronectin on pHi in HCT 116 and HT 29 cells. The time course of pHi is reported in panel (A) (HCT 116) and in panel (B) (HT 29). Simbol ▪: No coating surface, •: PL coating, ▲: Col-I coating, ▼: FN coating, ♦: VN coating, ○: Col-I anti-β1 Ab. On the right of the panel, 90 min pHi values are reported. Light grey bar: No coating surface, dark grey bar: Col-I, black bar: FN and striped bar: Col-I anti-β1 Ab. Number represent mean ± s.e.m (of three different experiments). *, P < 0.05; **, P < 0.01 and ***, P < 0.001. p value after 30 min of seeding, panel (A): ***P < 0.001: Col-I vs Control: 0.0005, FN vs Control: 0.0005; p value after 90 min of seeding, panel (A): ***P < 0.001: Col-I vs Control: 0.0001, FN vs Control: 0.0001; *P < 0.05: No coat vs Control: 0.01, PL vs Control: 0.01 and VN vs Control: 0.02. p value after 180 min of seeding, panel (A): *P < 0.05: No coat vs Control: 0.01, PL vs Control: 0.01 and VN vs Control: 0.01, Col-I vs Control: 0.01, FN vs Control: 0.01. p value after 30 min of seeding, panel (B) ***P < 0.001: Col-I vs Control: 0.0001 and FN vs Control: 0.0001; p value after 90 min of seeding, panel (B) ***P < 0.001: Col-I vs Control: 0.0001 and FN vs Control: 0.0001; *P < 0.05: No coat vs Control: 0.01, PL vs Control: 0.01 and VN vs Control: 0.01. p value after 180 min of seeding, panel (B) *P < 0.05: No coat vs Control: 0.01, PL vs Control: 0.01, VN vs Control: 0.01, Col-I vs Control: 0.01 and FN vs Control: 0.01. p value after 30 min of seeding, panel (A): ***P < 0.001: Col-I vs No coat: 0.001 and FN vs No coat: 0.001. p value after 90 min of seeding, panel (A): Col-I vs No coat: 0.0001, FN vs No coat: 0.0001; Col-I anti-β1 Ab vs Col-I: 0.0007; Col-I anti-β1 Ab vs FN: 0.0006. p value after 30 min of seeding, panel (B) ***P < 0.001: Col-I vs No coat: 0.001 and FN vs No coat: 0.001. p value after 90 min of seeding, panel (B) Col-I vs No coat: 0.001 and FN vs No coat: 0.001. Col-I anti-β1 Ab vs Col-I: 0.0006; Col-I anti-β1 Ab vs FN: 0.0005. Representative images of cells seeded for 90 min on Col-I, no treated and treated with anti-β1 Ab are reported in panels AI (HCT 116) and BI (HT 29), 100 µm scale bar. The conditions are shown on the top of each picture. Cells seeded on Col-I are elongated and attached, cells seeded on Col-I and treated with anti-β1 Ab are round and detached.
Figure 2
Figure 2
Effect of Acetazolamide, Amiloride, S0859, Cariporide, E4031, and E4031 plus Cariporide on pHi in cells seeded on collagen I, 90 min treatment, in HCT 116 and HT 29 cells. pHi values of HCT 116 are reported in panel (A) and for HT 29 in panel (B). Red line: pHi value at time zero. Number represent mean ± s.e.m (of three different experiments). *, P < 0.05 and ***, P < 0.001. p value panel (A): ***P < 0.001: Control vs Amil: 1.9e^-05, Control vs Carip: 1.9e^-05, Control vs E4031: 0.0001, Control vs E4031+Carip: 1.8e^-05Acet vs Carip: 1.7e^-05; *P < 0.05: Control vs S0859: 0.03, Acet vs S0859: 0.03, Amil vs S0859: 0.02; Carip vs S0859: 0.02; S0859 vs E4031+ Carip: 0.02. p value panel (B): ***P < 0.001: Control vs Amil: 1.5e^-05, Control vs Carip: 1.4e^-05, Acet vs Carip: 1.3e^-05; Control vs E4031: 0.0001 and Control vs E4031+Carip: 1.9e^-05; *P < 0.05: Control vs S0859: 0.04, Acet vs S0859: 0.03; S0859 vs E4031+ Carip: 0.02.
Figure 3
Figure 3
Effect of E4031 and E4031 plus Cariporide on motility index in cells seeded on col-I, 90 min treatment. Motility index values are reported for HCT 116 in panel (A) and for HT 29 in panel C. Number represent mean ± s.e.m (of three different experiments). Representative images are reported in panel (B) for HCT 116 and in panel (D) for HT 29, 200 µm scale bar. In figure the statistically significant differences between control and treatments are reported. **, P < 0.01. p value panel (A): **, P < 0.01: Control vs E4031: 0.002 and Control vs E4031+Carip: 0.001. p value panel (C): **, P < 0.01: Control vs E4031: 0.003 and Control vs E4031+Carip: 0.001.
Figure 4
Figure 4
β1-integrin, KV11.1, and NHE1 protein complex. (A) Co-immunoprecipitation of β1 Integrin, Kv 11.1 and NHE1 in HCT 116 cells, seeded on no coating surface and Col-I for 90 min. Densitometric analysis is reported in panel (A). In panel A with “WB” is indicated the protein signal in the co-ip and with “INPUT” the protein signal in the total lysate. Pre-seeding condition is reported as pre, No coating as No coat and Collagen I as Col-I; The immunoprecipitation with anti β1 integrin antibody is indicated as IP β1 and with anti KV11.1 antibody is reported as IP KV11.1. Complex quantification is reported in panel B, black bar: β1-integrin, KV11.1 and NHE1 protein complex and white bar: β1-integrin and KV11.1 protein complex. Number represent mean ± s.e.m (of three different experiments). **, P < 0.01 and ***, P < 0.001. p value panel (B), ***, P < 0.001: β1-integrin/KV11.1/NHE1 complex, Pre vs Col-I IP β1, p: 0.0008; β1-integrin/KV11.1 complex, Pre vs Col-I IP β1, p: 0.00075; β1-integrin/KV11.1/NHE1 complex, Pre vs Col-I IP KV11.1, p: 0.00076; β1-integrin/KV11.1 complex, Pre vs Col-I IP KV11.1 p: 0.00074. β1-integrin/KV11.1/NHE1 complex, No coat vs Col-I IP β1, p: 1.7e^-05; β1-integrin/KV11.1 complex No coat vs Col-I IP β1, p: 0.00072; β1-integrin/KV11.1/NHE1 complex, No coat vs Col-I IP KV11.1, p: 1.9e^-05; β1-integrin/KV11.1 complex, No coat vs Col-I IP KV11.1, p: 0.00073. **, P < 0.01: β1-integrin/KV11.1/NHE1 complex, Pre vs No coat: 0.002; β1-integrin/KV11.1/NHE1 complex vs β1-integrin/KV11.1 complex, No coat IP β1: 0.002. Cropped images of blots are reported.
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