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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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Related in: MedlinePlus

Identification of perhexiline as a novel agent that promotes HER3 internalization and degradation. (A) Perhexiline promotes HER3ΔNLS2-YFP internalization. As a control to HER3ΔNLS2-YFP cellular distribution, endogenous HER3 expression in MDA-MB-468 cells was detected by immunofluorescence staining (i). U2OS cells stably expressing HER3ΔNLS2-YFP were used to screen for compounds that promote HER3 internalization. Representative confocal images shown in panels (ii to v) were taken from cells expressing HER3ΔNLS2-YFP that were treated with DMSO, 10 μM perhexiline for 30 minutes and 4 hours, and 20 μM CPT-1 inhibitor etomoxir for 4 hours, respectively. Arrows indicate intracellularly localized puncta of internalized HER3ΔNLS2-YFP receptors. (B) Structure of perhexiline maleate. (C) Perhexiline induces dose-dependent downregulation of endogenous HER3 receptors. Cell lysates prepared from SK-BR-3 cells treated with different concentrations of perhexiline for 8 hours were analyzed for the expression of HER3, HER2, and EGFR. β-actin was used as a loading control. (D) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (C) were quantified by normalizing to β-actin. (E) Time course of perhexiline-induced downregulation of endogenous HER3 in SK-BR-3 cells. Cells treated with 10 μM perhexiline for the indicated time were analyzed for endogenous HER3, HER2, and EGFR expression. (F) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (E) were quantified by normalizing to β-actin. CPT-1, mitochondrial carnitine palmitoyltransferase-1; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; HER3, human epidermal growth factor receptor 3.
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Fig1: Identification of perhexiline as a novel agent that promotes HER3 internalization and degradation. (A) Perhexiline promotes HER3ΔNLS2-YFP internalization. As a control to HER3ΔNLS2-YFP cellular distribution, endogenous HER3 expression in MDA-MB-468 cells was detected by immunofluorescence staining (i). U2OS cells stably expressing HER3ΔNLS2-YFP were used to screen for compounds that promote HER3 internalization. Representative confocal images shown in panels (ii to v) were taken from cells expressing HER3ΔNLS2-YFP that were treated with DMSO, 10 μM perhexiline for 30 minutes and 4 hours, and 20 μM CPT-1 inhibitor etomoxir for 4 hours, respectively. Arrows indicate intracellularly localized puncta of internalized HER3ΔNLS2-YFP receptors. (B) Structure of perhexiline maleate. (C) Perhexiline induces dose-dependent downregulation of endogenous HER3 receptors. Cell lysates prepared from SK-BR-3 cells treated with different concentrations of perhexiline for 8 hours were analyzed for the expression of HER3, HER2, and EGFR. β-actin was used as a loading control. (D) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (C) were quantified by normalizing to β-actin. (E) Time course of perhexiline-induced downregulation of endogenous HER3 in SK-BR-3 cells. Cells treated with 10 μM perhexiline for the indicated time were analyzed for endogenous HER3, HER2, and EGFR expression. (F) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (E) were quantified by normalizing to β-actin. CPT-1, mitochondrial carnitine palmitoyltransferase-1; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; HER3, human epidermal growth factor receptor 3.

Mentions: In contrast to the other members of the HER family of receptor kinases, the HER3 receptor is not an active kinase [1]. As a result, strategies targeting the kinase activity to develop small molecule therapeutics against HER3 similar to those employed to develop small molecules against EGFR and HER2, are not applicable. To overcome this hurdle, we developed an imaging-based high-throughput screening strategy to search for small molecules that promote HER3 internalization based on the notion that receptor internalization often results in loss of receptor function. Screening compound libraries for their effects on membrane trafficking has proven to be a highly rewarding strategy for identifying inhibitors and activators of other enigmatic membrane receptors such as Frizzled and Smoothened [20,21]. Since endogenous HER3 receptors were expressed at the plasma membrane, the cytosol, and the nucleus in MDA-MB-468 cells (Figure 1A, panel i), therefore in this report, we utilized an YFP-tagged HER3ΔNLS2 (HER3ΔNLS2-YFP) expression construct, with the C-terminal nuclear localization sequence (NLS2, a four Arg-repeat) [22] removed in order to enhance membrane localization. We found that this mutant receptor functions similarly as wild-type (WT) HER3 in promoting downstream signaling upon stimulation with neuregulin β1 (NRGβ1) (Figure S1 in Additional file 1). Stable U2OS cells were generated to express HER3ΔNLS2-YFP and the receptors were predominantly expressed at the cell surface basally (Figure 1A, panel ii), which is different from endogenous HER3 expression throughout the cell (Figure 1A, panel i). Using this cell line, we screened the Prestwick Chemicals Library containing FDA and foreign regulatory agency-approved drugs, and discovered perhexiline maleate (Figure 1B) induced HER3 receptor internalization (Figure 1A, panels iii and iv). Perhexiline is a drug once used to treat angina (Figure 1B) [19]. The mechanism responsible for its anti-angina activity is believed to result from inhibition of mitochondrial CPT-1. To test whether inhibition of CPT-1 induced HER3 receptor internalization, we tested the effect of another CPT-1 inhibitor, etomoxir, on HER3 internalization. Our result showed that etomoxir had no effect on HER3 localization (Figure 1A, panel v) suggesting perhexiline operates through a mechanism independent of its ability to inhibit CPT-1 [23].Figure 1


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)

Identification of perhexiline as a novel agent that promotes HER3 internalization and degradation. (A) Perhexiline promotes HER3ΔNLS2-YFP internalization. As a control to HER3ΔNLS2-YFP cellular distribution, endogenous HER3 expression in MDA-MB-468 cells was detected by immunofluorescence staining (i). U2OS cells stably expressing HER3ΔNLS2-YFP were used to screen for compounds that promote HER3 internalization. Representative confocal images shown in panels (ii to v) were taken from cells expressing HER3ΔNLS2-YFP that were treated with DMSO, 10 μM perhexiline for 30 minutes and 4 hours, and 20 μM CPT-1 inhibitor etomoxir for 4 hours, respectively. Arrows indicate intracellularly localized puncta of internalized HER3ΔNLS2-YFP receptors. (B) Structure of perhexiline maleate. (C) Perhexiline induces dose-dependent downregulation of endogenous HER3 receptors. Cell lysates prepared from SK-BR-3 cells treated with different concentrations of perhexiline for 8 hours were analyzed for the expression of HER3, HER2, and EGFR. β-actin was used as a loading control. (D) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (C) were quantified by normalizing to β-actin. (E) Time course of perhexiline-induced downregulation of endogenous HER3 in SK-BR-3 cells. Cells treated with 10 μM perhexiline for the indicated time were analyzed for endogenous HER3, HER2, and EGFR expression. (F) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (E) were quantified by normalizing to β-actin. CPT-1, mitochondrial carnitine palmitoyltransferase-1; 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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Fig1: Identification of perhexiline as a novel agent that promotes HER3 internalization and degradation. (A) Perhexiline promotes HER3ΔNLS2-YFP internalization. As a control to HER3ΔNLS2-YFP cellular distribution, endogenous HER3 expression in MDA-MB-468 cells was detected by immunofluorescence staining (i). U2OS cells stably expressing HER3ΔNLS2-YFP were used to screen for compounds that promote HER3 internalization. Representative confocal images shown in panels (ii to v) were taken from cells expressing HER3ΔNLS2-YFP that were treated with DMSO, 10 μM perhexiline for 30 minutes and 4 hours, and 20 μM CPT-1 inhibitor etomoxir for 4 hours, respectively. Arrows indicate intracellularly localized puncta of internalized HER3ΔNLS2-YFP receptors. (B) Structure of perhexiline maleate. (C) Perhexiline induces dose-dependent downregulation of endogenous HER3 receptors. Cell lysates prepared from SK-BR-3 cells treated with different concentrations of perhexiline for 8 hours were analyzed for the expression of HER3, HER2, and EGFR. β-actin was used as a loading control. (D) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (C) were quantified by normalizing to β-actin. (E) Time course of perhexiline-induced downregulation of endogenous HER3 in SK-BR-3 cells. Cells treated with 10 μM perhexiline for the indicated time were analyzed for endogenous HER3, HER2, and EGFR expression. (F) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (E) were quantified by normalizing to β-actin. CPT-1, mitochondrial carnitine palmitoyltransferase-1; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; HER3, human epidermal growth factor receptor 3.
Mentions: In contrast to the other members of the HER family of receptor kinases, the HER3 receptor is not an active kinase [1]. As a result, strategies targeting the kinase activity to develop small molecule therapeutics against HER3 similar to those employed to develop small molecules against EGFR and HER2, are not applicable. To overcome this hurdle, we developed an imaging-based high-throughput screening strategy to search for small molecules that promote HER3 internalization based on the notion that receptor internalization often results in loss of receptor function. Screening compound libraries for their effects on membrane trafficking has proven to be a highly rewarding strategy for identifying inhibitors and activators of other enigmatic membrane receptors such as Frizzled and Smoothened [20,21]. Since endogenous HER3 receptors were expressed at the plasma membrane, the cytosol, and the nucleus in MDA-MB-468 cells (Figure 1A, panel i), therefore in this report, we utilized an YFP-tagged HER3ΔNLS2 (HER3ΔNLS2-YFP) expression construct, with the C-terminal nuclear localization sequence (NLS2, a four Arg-repeat) [22] removed in order to enhance membrane localization. We found that this mutant receptor functions similarly as wild-type (WT) HER3 in promoting downstream signaling upon stimulation with neuregulin β1 (NRGβ1) (Figure S1 in Additional file 1). Stable U2OS cells were generated to express HER3ΔNLS2-YFP and the receptors were predominantly expressed at the cell surface basally (Figure 1A, panel ii), which is different from endogenous HER3 expression throughout the cell (Figure 1A, panel i). Using this cell line, we screened the Prestwick Chemicals Library containing FDA and foreign regulatory agency-approved drugs, and discovered perhexiline maleate (Figure 1B) induced HER3 receptor internalization (Figure 1A, panels iii and iv). Perhexiline is a drug once used to treat angina (Figure 1B) [19]. The mechanism responsible for its anti-angina activity is believed to result from inhibition of mitochondrial CPT-1. To test whether inhibition of CPT-1 induced HER3 receptor internalization, we tested the effect of another CPT-1 inhibitor, etomoxir, on HER3 internalization. Our result showed that etomoxir had no effect on HER3 localization (Figure 1A, panel v) suggesting perhexiline operates through a mechanism independent of its ability to inhibit CPT-1 [23].Figure 1

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