Regulation of voltage-gated sodium channel β1 subunit function and localisation by γ-secretase in metastatic breast cancer cells

TY - CONF

T1 - Regulation of voltage-gated sodium channel β1 subunit function and localisation by γ-secretase in metastatic breast cancer cells

AU - Haworth, Alexander

AU - Baumann, Christoph George

AU - Brackenbury, William John

PY - 2018/1/15

Y1 - 2018/1/15

N2 - Voltage-gated Na+ channels (VGSCs), composed of a pore-forming α-subunit and auxiliary β-subunits (β1-β4), are responsible for the inward Na+ current underlying neuronal action potentials. VGSC expression is deregulated in cancer cells. β1 is upregulated in breast cancer specimens relative to control tissue and β1 overexpression in MDA-MB-231 breast cancer cells promotes tumour growth and metastasis and induces neurite-like outgrowths. β1-mediated neurite outgrowth in immature neurons is dependent on γ-secretase cleavage, which releases a β1 intracellular domain (ICD). Our study investigates the role of γ-secretase cleavage in β1 localisation and function in MDA-MB-231 cells. We hypothesised that β1-ICD, the γ-secretase product, translocates to the nucleus, as seen with β2-ICD, and that γ-secretase cleavage regulates β1 function. Imaged using confocal microscopy, β1-ICD-GFP showed significant nuclear enrichment in MDA-MB-231 cells (MDA-MB-231-β1-ICD-GFP), compared to full-length β1-GFP (P<0.0001), but with similar enrichment to GFP (P=0.98; n=14; one-way ANOVA). Other γ-secretase cleavage products like APP-ICD translocate as multimeric complexes. To determine whether β1-ICD-GFP nuclear localisation was distinct from GFP nucleocytoplasmic diffusion, nuclear import was assessed using fluorescence recovery after photobleaching. The half-maximal recovery time for β1-ICD-GFP fluorescence in the nucleus was two-fold greater than that observed for GFP (P<0.001; n=10; Mann-Whitney test), suggesting a distinct nuclear import mechanism for β1-ICD-GFP. Morphology and transcellular adhesion assays were used to assess the functional impact of γ-secretase cleavage. MDA-MB-231-β1-ICD-GFP cells showed similar morphology in monoculture, evaluated by measuring process length, to MDA-MB-231-β1-GFP cells (P=0.09), but almost double the process length of MDA-MB-231-GFP cells (P<0.01; n=32; one-way ANOVA), suggesting β1-ICD recapitulates aspects of β1-mediated cell morphology. Inhibiting γ-secretase in MDA-MB-231-β1-GFP cells significantly increased transcellular adhesion, but had no effect on control MDA-MB-231 cells (P<0.01; n=3; t-test), implying γ-secretase modulates β1-mediated cell-cell adhesion. Further work will focus on elucidating the role of nuclear β1-ICD in MDA-MB-231 breast cancer cells.

AB - Voltage-gated Na+ channels (VGSCs), composed of a pore-forming α-subunit and auxiliary β-subunits (β1-β4), are responsible for the inward Na+ current underlying neuronal action potentials. VGSC expression is deregulated in cancer cells. β1 is upregulated in breast cancer specimens relative to control tissue and β1 overexpression in MDA-MB-231 breast cancer cells promotes tumour growth and metastasis and induces neurite-like outgrowths. β1-mediated neurite outgrowth in immature neurons is dependent on γ-secretase cleavage, which releases a β1 intracellular domain (ICD). Our study investigates the role of γ-secretase cleavage in β1 localisation and function in MDA-MB-231 cells. We hypothesised that β1-ICD, the γ-secretase product, translocates to the nucleus, as seen with β2-ICD, and that γ-secretase cleavage regulates β1 function. Imaged using confocal microscopy, β1-ICD-GFP showed significant nuclear enrichment in MDA-MB-231 cells (MDA-MB-231-β1-ICD-GFP), compared to full-length β1-GFP (P<0.0001), but with similar enrichment to GFP (P=0.98; n=14; one-way ANOVA). Other γ-secretase cleavage products like APP-ICD translocate as multimeric complexes. To determine whether β1-ICD-GFP nuclear localisation was distinct from GFP nucleocytoplasmic diffusion, nuclear import was assessed using fluorescence recovery after photobleaching. The half-maximal recovery time for β1-ICD-GFP fluorescence in the nucleus was two-fold greater than that observed for GFP (P<0.001; n=10; Mann-Whitney test), suggesting a distinct nuclear import mechanism for β1-ICD-GFP. Morphology and transcellular adhesion assays were used to assess the functional impact of γ-secretase cleavage. MDA-MB-231-β1-ICD-GFP cells showed similar morphology in monoculture, evaluated by measuring process length, to MDA-MB-231-β1-GFP cells (P=0.09), but almost double the process length of MDA-MB-231-GFP cells (P<0.01; n=32; one-way ANOVA), suggesting β1-ICD recapitulates aspects of β1-mediated cell morphology. Inhibiting γ-secretase in MDA-MB-231-β1-GFP cells significantly increased transcellular adhesion, but had no effect on control MDA-MB-231 cells (P<0.01; n=3; t-test), implying γ-secretase modulates β1-mediated cell-cell adhesion. Further work will focus on elucidating the role of nuclear β1-ICD in MDA-MB-231 breast cancer cells.

M3 - Abstract

SP - 394

EP - 395

T2 - UK Interdisciplinary Breast Cancer Symposium

Y2 - 15 January 2018 through 16 January 2018

ER -