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Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth.

Ren XR, Wang J, Osada T, Mook RA, Morse MA, Barak LS, Lyerly HK, Chen W - Breast Cancer Res. (2015)

Bottom Line: Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment.In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform.We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment.

Methods: We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform.

Results: We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo.

Conclusions: This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.

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Perhexiline promotes cell surface-expressed Flag-HER3ΔNLS2 receptor internalization and the ubiquitination-mediated degradation of HER3 receptors through lysosome and proteasome. (A) Cell surface-expressed Flag-HER3ΔNLS2 receptors are internalized upon perhexiline treatment. HEK293 cells transfected with Flag-HER3ΔNLS2 were incubated with the anti-FLAG antibody on ice for 30 minutes (i) and subsequently incubated at 37°C for 1 hour untreated (ii) or in the presence of DMSO or perhexiline (10 μM) (iii, iv). Cells were fixed and localization patterns of Flag-HER3ΔNLS2 were visualized using confocal microscopy. Internalized Flag-HER3ΔNLS2 vesicles were shown as indicated by arrowheads in DMSO-treated cells whereas arrows indicate large puncta of internalized receptors in perhexiline-treated cells. (B) Perhexiline induces HER3 ubiquitination. MDA-MB-468 cells were treated with perhexiline (10 μM) for the indicated time. The endogenous HER3 was immunoprecipitated and the amount of ubiquitination and total HER3 were detected using the anti-ubiquitin and HER3 antibodies, respectively. (C) Perhexiline-induced downregulation of HER3 is blocked by proteasome and lysosome inhibitors. MDA-MB-468 cells were treated with DMSO, MG132 (10 μM), or BFA1 (30 nM) in the absence or presence of perhexiline (10 μM) for 8 hours. The endogenous HER3 was detected by the anti-HER3 antibody. (D) Perhexiline induces the redistribution of endogenous HER3 to the lysosome. MDA-MB-468 cells treated with DMSO (i to iii) or 10 μM perhexiline (iv to vi) for 2 hours were fixed and analyzed using confocal microscopy. The localization of HER3 and the lysosome was visualized using the anti-HER3 antibody (green) and LysoTracker (red), respectively. Co-localization of HER3 (green) with lysosome (visualized by LysoTracker Red) was observed in perhexiline-treated cells as yellow puncta marked by arrows. DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; HER3, human epidermal growth factor receptor 3.
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Fig2: Perhexiline promotes cell surface-expressed Flag-HER3ΔNLS2 receptor internalization and the ubiquitination-mediated degradation of HER3 receptors through lysosome and proteasome. (A) Cell surface-expressed Flag-HER3ΔNLS2 receptors are internalized upon perhexiline treatment. HEK293 cells transfected with Flag-HER3ΔNLS2 were incubated with the anti-FLAG antibody on ice for 30 minutes (i) and subsequently incubated at 37°C for 1 hour untreated (ii) or in the presence of DMSO or perhexiline (10 μM) (iii, iv). Cells were fixed and localization patterns of Flag-HER3ΔNLS2 were visualized using confocal microscopy. Internalized Flag-HER3ΔNLS2 vesicles were shown as indicated by arrowheads in DMSO-treated cells whereas arrows indicate large puncta of internalized receptors in perhexiline-treated cells. (B) Perhexiline induces HER3 ubiquitination. MDA-MB-468 cells were treated with perhexiline (10 μM) for the indicated time. The endogenous HER3 was immunoprecipitated and the amount of ubiquitination and total HER3 were detected using the anti-ubiquitin and HER3 antibodies, respectively. (C) Perhexiline-induced downregulation of HER3 is blocked by proteasome and lysosome inhibitors. MDA-MB-468 cells were treated with DMSO, MG132 (10 μM), or BFA1 (30 nM) in the absence or presence of perhexiline (10 μM) for 8 hours. The endogenous HER3 was detected by the anti-HER3 antibody. (D) Perhexiline induces the redistribution of endogenous HER3 to the lysosome. MDA-MB-468 cells treated with DMSO (i to iii) or 10 μM perhexiline (iv to vi) for 2 hours were fixed and analyzed using confocal microscopy. The localization of HER3 and the lysosome was visualized using the anti-HER3 antibody (green) and LysoTracker (red), respectively. Co-localization of HER3 (green) with lysosome (visualized by LysoTracker Red) was observed in perhexiline-treated cells as yellow puncta marked by arrows. DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; HER3, human epidermal growth factor receptor 3.

Mentions: Our data showed that perhexiline stimulates HER3 internalization and degradation. To further prove that the internalized receptor was from the cell surface, we generated an N-terminal Flag-tagged HER3ΔNLS2 construct. Live cells expressing surface-localized Flag-HER3ΔNLS2 receptors were labeled on ice with fluorescence-conjugated Flag antibody to prevent internalization of the receptor (Figure 2A, panel i). Raising the temperature to 37°C caused a small amount of Flag-HER3ΔNLS2 receptors to internalize as indicated by the redistribution of labeled receptors to the cytoplasm in untreated or DMSO-treated cells (Figure 2A, panels ii and iii). However, more internalized receptors were observed in cells treated with perhexiline for 1 hour, and these internalized receptors formed larger puncta inside the cells (Figure 2A, panel iv).Figure 2


Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth.

Ren XR, Wang J, Osada T, Mook RA, Morse MA, Barak LS, Lyerly HK, Chen W - Breast Cancer Res. (2015)

Perhexiline promotes cell surface-expressed Flag-HER3ΔNLS2 receptor internalization and the ubiquitination-mediated degradation of HER3 receptors through lysosome and proteasome. (A) Cell surface-expressed Flag-HER3ΔNLS2 receptors are internalized upon perhexiline treatment. HEK293 cells transfected with Flag-HER3ΔNLS2 were incubated with the anti-FLAG antibody on ice for 30 minutes (i) and subsequently incubated at 37°C for 1 hour untreated (ii) or in the presence of DMSO or perhexiline (10 μM) (iii, iv). Cells were fixed and localization patterns of Flag-HER3ΔNLS2 were visualized using confocal microscopy. Internalized Flag-HER3ΔNLS2 vesicles were shown as indicated by arrowheads in DMSO-treated cells whereas arrows indicate large puncta of internalized receptors in perhexiline-treated cells. (B) Perhexiline induces HER3 ubiquitination. MDA-MB-468 cells were treated with perhexiline (10 μM) for the indicated time. The endogenous HER3 was immunoprecipitated and the amount of ubiquitination and total HER3 were detected using the anti-ubiquitin and HER3 antibodies, respectively. (C) Perhexiline-induced downregulation of HER3 is blocked by proteasome and lysosome inhibitors. MDA-MB-468 cells were treated with DMSO, MG132 (10 μM), or BFA1 (30 nM) in the absence or presence of perhexiline (10 μM) for 8 hours. The endogenous HER3 was detected by the anti-HER3 antibody. (D) Perhexiline induces the redistribution of endogenous HER3 to the lysosome. MDA-MB-468 cells treated with DMSO (i to iii) or 10 μM perhexiline (iv to vi) for 2 hours were fixed and analyzed using confocal microscopy. The localization of HER3 and the lysosome was visualized using the anti-HER3 antibody (green) and LysoTracker (red), respectively. Co-localization of HER3 (green) with lysosome (visualized by LysoTracker Red) was observed in perhexiline-treated cells as yellow puncta marked by arrows. DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; HER3, human epidermal growth factor receptor 3.
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Related In: Results  -  Collection

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Fig2: Perhexiline promotes cell surface-expressed Flag-HER3ΔNLS2 receptor internalization and the ubiquitination-mediated degradation of HER3 receptors through lysosome and proteasome. (A) Cell surface-expressed Flag-HER3ΔNLS2 receptors are internalized upon perhexiline treatment. HEK293 cells transfected with Flag-HER3ΔNLS2 were incubated with the anti-FLAG antibody on ice for 30 minutes (i) and subsequently incubated at 37°C for 1 hour untreated (ii) or in the presence of DMSO or perhexiline (10 μM) (iii, iv). Cells were fixed and localization patterns of Flag-HER3ΔNLS2 were visualized using confocal microscopy. Internalized Flag-HER3ΔNLS2 vesicles were shown as indicated by arrowheads in DMSO-treated cells whereas arrows indicate large puncta of internalized receptors in perhexiline-treated cells. (B) Perhexiline induces HER3 ubiquitination. MDA-MB-468 cells were treated with perhexiline (10 μM) for the indicated time. The endogenous HER3 was immunoprecipitated and the amount of ubiquitination and total HER3 were detected using the anti-ubiquitin and HER3 antibodies, respectively. (C) Perhexiline-induced downregulation of HER3 is blocked by proteasome and lysosome inhibitors. MDA-MB-468 cells were treated with DMSO, MG132 (10 μM), or BFA1 (30 nM) in the absence or presence of perhexiline (10 μM) for 8 hours. The endogenous HER3 was detected by the anti-HER3 antibody. (D) Perhexiline induces the redistribution of endogenous HER3 to the lysosome. MDA-MB-468 cells treated with DMSO (i to iii) or 10 μM perhexiline (iv to vi) for 2 hours were fixed and analyzed using confocal microscopy. The localization of HER3 and the lysosome was visualized using the anti-HER3 antibody (green) and LysoTracker (red), respectively. Co-localization of HER3 (green) with lysosome (visualized by LysoTracker Red) was observed in perhexiline-treated cells as yellow puncta marked by arrows. DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; HER3, human epidermal growth factor receptor 3.
Mentions: Our data showed that perhexiline stimulates HER3 internalization and degradation. To further prove that the internalized receptor was from the cell surface, we generated an N-terminal Flag-tagged HER3ΔNLS2 construct. Live cells expressing surface-localized Flag-HER3ΔNLS2 receptors were labeled on ice with fluorescence-conjugated Flag antibody to prevent internalization of the receptor (Figure 2A, panel i). Raising the temperature to 37°C caused a small amount of Flag-HER3ΔNLS2 receptors to internalize as indicated by the redistribution of labeled receptors to the cytoplasm in untreated or DMSO-treated cells (Figure 2A, panels ii and iii). However, more internalized receptors were observed in cells treated with perhexiline for 1 hour, and these internalized receptors formed larger puncta inside the cells (Figure 2A, panel iv).Figure 2

Bottom Line: Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment.In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform.We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment.

Methods: We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform.

Results: We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo.

Conclusions: This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.

Show MeSH
Related in: MedlinePlus