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The Huntington disease protein accelerates breast tumour development and metastasis through ErbB2/HER2 signalling.

Moreira Sousa C, McGuire JR, Thion MS, Gentien D, de la Grange P, Tezenas du Montcel S, Vincent-Salomon A, Durr A, Humbert S - EMBO Mol Med (2013)

Bottom Line: In agreement, mutant huntingtin accelerates epithelial to mesenchymal transition and enhances cell motility and invasion.Finally, we report that in HD, the dynamin dependent endocytosis of the ErbB2/HER2 receptor tyrosine kinase is reduced.This leads to its accumulation and to subsequent increases in cell motility and proliferation.

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Affiliation: Institut Curie, Paris, France; CNRS UMR 3306, Orsay, France.

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PolyQ-huntingtin inhibits ErbB2/HER2 endocytosis through a dynamin dependent mechanismA,B. Human SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23, Htt Q23) and polyQ-huntingtin (pARIS-mCherry-httQ100, Htt Q100) and treated with Geldanamycin (GA) as indicated. n.t.: not transfected. (A) Cells are stained for HER2 and huntingtin (Htt). HER2 intensity at the membrane is quantified before and after treatment (at least n = 24 cells analysed per condition, three independent experiments). Htt Q23 DMSO vs. Htt Q100 DMSO: *p-value = 0.0204; n.t. GA vs. Htt Q100 GA: ***p-value < 0.0001; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0006. (B) Cells are fixed, immunostained with the antibody recognizing the extracellular part of HER2 and analysed by flow cytometry. A representative flow cytometry profile is shown (left). The graphs in B, C and D represent mean HER2-APC fluorescence level (surface HER2; three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 DMSO versus Htt Q100 DMSO: *p-value = 0.0141; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0009.C. SKBr3 cells are transfected with a construct expressing a GTPase-defective mutant K44A dynamin (Dyn2 K44A), treated with Geldanamycin as indicated and analysed by flow cytometry. (Three independent experiments, 10,000 cells analysed per condition and experiment). DMSO versus GA, ***p-value < 0.0001; DMSO versus Dyn2 K44A DMSO, p-value > 0.9999; GA versus Dyn2 K44A GA, ***p-value < 0.0001; Dyn2 K44A DMSO versus Dyn2 K44A GA, ***p-value = 0.0002. n.s., not significant.D. SKBr3 cells are transfected with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100 and a construct encoding wild-type dynamin (Dyn2 WT) and analysed by flow cytometry (three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 versus Htt Q100, *p-value = 0.0491; Htt Q100 versus Htt Q100 + Dyn2 WT, **p-value = 0.0016; Htt Q23 + Dyn2 WT versus Htt Q100 + Dyn2 WT, *p-value = 0.0136.E. Huntingtin and dynamin interact in a polyQ-dependent manner. Huntingtin immunoprecipitation experiments were performed on cellular extracts from MMTV-PyVT/HdhQ7/Q7 (HdhQ7/Q7) and MMTV-PyVT/HdhQ111/Q111 (HdhQ111/Q111) tumours. Immunoprecipitation with mouse IgG (IgG m) is used as a control.F. SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23) and polyQ-huntingtin (pARIS-mCherry-httQ100) and stained for huntingtin (Htt) and dynamin.
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fig06: PolyQ-huntingtin inhibits ErbB2/HER2 endocytosis through a dynamin dependent mechanismA,B. Human SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23, Htt Q23) and polyQ-huntingtin (pARIS-mCherry-httQ100, Htt Q100) and treated with Geldanamycin (GA) as indicated. n.t.: not transfected. (A) Cells are stained for HER2 and huntingtin (Htt). HER2 intensity at the membrane is quantified before and after treatment (at least n = 24 cells analysed per condition, three independent experiments). Htt Q23 DMSO vs. Htt Q100 DMSO: *p-value = 0.0204; n.t. GA vs. Htt Q100 GA: ***p-value < 0.0001; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0006. (B) Cells are fixed, immunostained with the antibody recognizing the extracellular part of HER2 and analysed by flow cytometry. A representative flow cytometry profile is shown (left). The graphs in B, C and D represent mean HER2-APC fluorescence level (surface HER2; three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 DMSO versus Htt Q100 DMSO: *p-value = 0.0141; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0009.C. SKBr3 cells are transfected with a construct expressing a GTPase-defective mutant K44A dynamin (Dyn2 K44A), treated with Geldanamycin as indicated and analysed by flow cytometry. (Three independent experiments, 10,000 cells analysed per condition and experiment). DMSO versus GA, ***p-value < 0.0001; DMSO versus Dyn2 K44A DMSO, p-value > 0.9999; GA versus Dyn2 K44A GA, ***p-value < 0.0001; Dyn2 K44A DMSO versus Dyn2 K44A GA, ***p-value = 0.0002. n.s., not significant.D. SKBr3 cells are transfected with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100 and a construct encoding wild-type dynamin (Dyn2 WT) and analysed by flow cytometry (three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 versus Htt Q100, *p-value = 0.0491; Htt Q100 versus Htt Q100 + Dyn2 WT, **p-value = 0.0016; Htt Q23 + Dyn2 WT versus Htt Q100 + Dyn2 WT, *p-value = 0.0136.E. Huntingtin and dynamin interact in a polyQ-dependent manner. Huntingtin immunoprecipitation experiments were performed on cellular extracts from MMTV-PyVT/HdhQ7/Q7 (HdhQ7/Q7) and MMTV-PyVT/HdhQ111/Q111 (HdhQ111/Q111) tumours. Immunoprecipitation with mouse IgG (IgG m) is used as a control.F. SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23) and polyQ-huntingtin (pARIS-mCherry-httQ100) and stained for huntingtin (Htt) and dynamin.

Mentions: Huntingtin is involved in intracellular trafficking and endocytosis, and polyQ-huntingtin impairs these functions (Caviston et al, 2007; Gauthier et al, 2004; Velier et al, 1998). Given the increase in ErbB2 at the plasma membrane in the presence of polyQ-huntingtin, we wondered whether polyQ-huntingtin would influence ErbB2 internalization. We induced ErbB2 internalization by inhibiting the specific regulator of ErbB2 stability Hsp90, using Geldanamycin (Citri et al, 2004). To address this question, we used as a model system the human SKBr3 breast cancer cell line, which as it was derived from a HER2-positive tumour, expresses high levels of HER2 (Fig 6A). In these cells, HER2 is mainly localized at the plasma membrane. We transfected SKBr3 cells with full-length wild-type (pARIS-mCherry-httQ23, Htt Q23) and polyQ-huntingtin (pARIS-mCherry-httQ100, Htt Q100; Pardo et al, 2010). Upon Geldanamycin treatment of SKBr3 cells, the staining of HER2 at the plasma membrane was markedly lower in cells expressing exogenous huntingtin of normal CAG length (Fig 6A; upper panel, star) and in non-transfected cells (arrowhead). In contrast, the decrease of HER2 staining at the membrane triggered by Geldanamycin treatment was much less efficient in the presence of exogenously expressed polyQ-huntingtin (compare transfected cell – bottom panel, star – with non polyQ-huntingtin transfected cell – arrowhead). We then specifically addressed the effect of mutant huntingtin on internalization by examining levels of HER2 at the cell surface by flow cytometry analysis (Fig 6B). SKBr3 cells expressing wild-type and mutant huntingtin were treated with Geldanamycin and immunostained with an antibody recognizing the extracellular part of HER2 prior to flow cytometry. As expected (Figs 5 and 6A), in the absence of Geldanamycin treatment, HER2 accumulated at the SKBr3 cell surface when mutant huntingtin was expressed as compared with control cells. Upon Geldanamycin treatment, the internalization of HER2 in cells expressing Htt Q23 was greater than in cells expressing Htt Q100 (Fig 6B). These results indicate that polyQ-huntingtin interferes with HER2 internalization.


The Huntington disease protein accelerates breast tumour development and metastasis through ErbB2/HER2 signalling.

Moreira Sousa C, McGuire JR, Thion MS, Gentien D, de la Grange P, Tezenas du Montcel S, Vincent-Salomon A, Durr A, Humbert S - EMBO Mol Med (2013)

PolyQ-huntingtin inhibits ErbB2/HER2 endocytosis through a dynamin dependent mechanismA,B. Human SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23, Htt Q23) and polyQ-huntingtin (pARIS-mCherry-httQ100, Htt Q100) and treated with Geldanamycin (GA) as indicated. n.t.: not transfected. (A) Cells are stained for HER2 and huntingtin (Htt). HER2 intensity at the membrane is quantified before and after treatment (at least n = 24 cells analysed per condition, three independent experiments). Htt Q23 DMSO vs. Htt Q100 DMSO: *p-value = 0.0204; n.t. GA vs. Htt Q100 GA: ***p-value < 0.0001; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0006. (B) Cells are fixed, immunostained with the antibody recognizing the extracellular part of HER2 and analysed by flow cytometry. A representative flow cytometry profile is shown (left). The graphs in B, C and D represent mean HER2-APC fluorescence level (surface HER2; three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 DMSO versus Htt Q100 DMSO: *p-value = 0.0141; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0009.C. SKBr3 cells are transfected with a construct expressing a GTPase-defective mutant K44A dynamin (Dyn2 K44A), treated with Geldanamycin as indicated and analysed by flow cytometry. (Three independent experiments, 10,000 cells analysed per condition and experiment). DMSO versus GA, ***p-value < 0.0001; DMSO versus Dyn2 K44A DMSO, p-value > 0.9999; GA versus Dyn2 K44A GA, ***p-value < 0.0001; Dyn2 K44A DMSO versus Dyn2 K44A GA, ***p-value = 0.0002. n.s., not significant.D. SKBr3 cells are transfected with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100 and a construct encoding wild-type dynamin (Dyn2 WT) and analysed by flow cytometry (three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 versus Htt Q100, *p-value = 0.0491; Htt Q100 versus Htt Q100 + Dyn2 WT, **p-value = 0.0016; Htt Q23 + Dyn2 WT versus Htt Q100 + Dyn2 WT, *p-value = 0.0136.E. Huntingtin and dynamin interact in a polyQ-dependent manner. Huntingtin immunoprecipitation experiments were performed on cellular extracts from MMTV-PyVT/HdhQ7/Q7 (HdhQ7/Q7) and MMTV-PyVT/HdhQ111/Q111 (HdhQ111/Q111) tumours. Immunoprecipitation with mouse IgG (IgG m) is used as a control.F. SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23) and polyQ-huntingtin (pARIS-mCherry-httQ100) and stained for huntingtin (Htt) and dynamin.
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fig06: PolyQ-huntingtin inhibits ErbB2/HER2 endocytosis through a dynamin dependent mechanismA,B. Human SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23, Htt Q23) and polyQ-huntingtin (pARIS-mCherry-httQ100, Htt Q100) and treated with Geldanamycin (GA) as indicated. n.t.: not transfected. (A) Cells are stained for HER2 and huntingtin (Htt). HER2 intensity at the membrane is quantified before and after treatment (at least n = 24 cells analysed per condition, three independent experiments). Htt Q23 DMSO vs. Htt Q100 DMSO: *p-value = 0.0204; n.t. GA vs. Htt Q100 GA: ***p-value < 0.0001; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0006. (B) Cells are fixed, immunostained with the antibody recognizing the extracellular part of HER2 and analysed by flow cytometry. A representative flow cytometry profile is shown (left). The graphs in B, C and D represent mean HER2-APC fluorescence level (surface HER2; three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 DMSO versus Htt Q100 DMSO: *p-value = 0.0141; Htt Q23 GA versus Htt Q100 GA: ***p-value = 0.0009.C. SKBr3 cells are transfected with a construct expressing a GTPase-defective mutant K44A dynamin (Dyn2 K44A), treated with Geldanamycin as indicated and analysed by flow cytometry. (Three independent experiments, 10,000 cells analysed per condition and experiment). DMSO versus GA, ***p-value < 0.0001; DMSO versus Dyn2 K44A DMSO, p-value > 0.9999; GA versus Dyn2 K44A GA, ***p-value < 0.0001; Dyn2 K44A DMSO versus Dyn2 K44A GA, ***p-value = 0.0002. n.s., not significant.D. SKBr3 cells are transfected with pARIS-mCherry-httQ23, pARIS-mCherry-httQ100 and a construct encoding wild-type dynamin (Dyn2 WT) and analysed by flow cytometry (three independent experiments, 10,000 cells analysed per condition and experiment). Htt Q23 versus Htt Q100, *p-value = 0.0491; Htt Q100 versus Htt Q100 + Dyn2 WT, **p-value = 0.0016; Htt Q23 + Dyn2 WT versus Htt Q100 + Dyn2 WT, *p-value = 0.0136.E. Huntingtin and dynamin interact in a polyQ-dependent manner. Huntingtin immunoprecipitation experiments were performed on cellular extracts from MMTV-PyVT/HdhQ7/Q7 (HdhQ7/Q7) and MMTV-PyVT/HdhQ111/Q111 (HdhQ111/Q111) tumours. Immunoprecipitation with mouse IgG (IgG m) is used as a control.F. SKBr3 cells are transfected with plasmids encoding full-length wild-type (pARIS-mCherry-httQ23) and polyQ-huntingtin (pARIS-mCherry-httQ100) and stained for huntingtin (Htt) and dynamin.
Mentions: Huntingtin is involved in intracellular trafficking and endocytosis, and polyQ-huntingtin impairs these functions (Caviston et al, 2007; Gauthier et al, 2004; Velier et al, 1998). Given the increase in ErbB2 at the plasma membrane in the presence of polyQ-huntingtin, we wondered whether polyQ-huntingtin would influence ErbB2 internalization. We induced ErbB2 internalization by inhibiting the specific regulator of ErbB2 stability Hsp90, using Geldanamycin (Citri et al, 2004). To address this question, we used as a model system the human SKBr3 breast cancer cell line, which as it was derived from a HER2-positive tumour, expresses high levels of HER2 (Fig 6A). In these cells, HER2 is mainly localized at the plasma membrane. We transfected SKBr3 cells with full-length wild-type (pARIS-mCherry-httQ23, Htt Q23) and polyQ-huntingtin (pARIS-mCherry-httQ100, Htt Q100; Pardo et al, 2010). Upon Geldanamycin treatment of SKBr3 cells, the staining of HER2 at the plasma membrane was markedly lower in cells expressing exogenous huntingtin of normal CAG length (Fig 6A; upper panel, star) and in non-transfected cells (arrowhead). In contrast, the decrease of HER2 staining at the membrane triggered by Geldanamycin treatment was much less efficient in the presence of exogenously expressed polyQ-huntingtin (compare transfected cell – bottom panel, star – with non polyQ-huntingtin transfected cell – arrowhead). We then specifically addressed the effect of mutant huntingtin on internalization by examining levels of HER2 at the cell surface by flow cytometry analysis (Fig 6B). SKBr3 cells expressing wild-type and mutant huntingtin were treated with Geldanamycin and immunostained with an antibody recognizing the extracellular part of HER2 prior to flow cytometry. As expected (Figs 5 and 6A), in the absence of Geldanamycin treatment, HER2 accumulated at the SKBr3 cell surface when mutant huntingtin was expressed as compared with control cells. Upon Geldanamycin treatment, the internalization of HER2 in cells expressing Htt Q23 was greater than in cells expressing Htt Q100 (Fig 6B). These results indicate that polyQ-huntingtin interferes with HER2 internalization.

Bottom Line: In agreement, mutant huntingtin accelerates epithelial to mesenchymal transition and enhances cell motility and invasion.Finally, we report that in HD, the dynamin dependent endocytosis of the ErbB2/HER2 receptor tyrosine kinase is reduced.This leads to its accumulation and to subsequent increases in cell motility and proliferation.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Paris, France; CNRS UMR 3306, Orsay, France.

Show MeSH
Related in: MedlinePlus