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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

Perhexiline treatment inhibits neuregulin-mediated activation of downstream signaling and cell proliferation. (A) MDA-MB-468 cells were cultured in the absence or presence of perhexiline (10 μM) for 6 hours, and subsequently stimulated with DMSO or NRGβ1 (10 ng/ml) for 10 minutes. Cell lysates were analyzed for the phosphorylation of HER3 (Tyr1289), Akt and ERK1/2 as well as total HER3 expression. (B) Perhexiline inhibits NRGβ1-induced cell growth. MB-MDA-468 cells were treated for 72 hours with vehicle (0.2% BSA in PBS) or NRGβ1 (10 ng/ml) in the presence of DMSO or perhexiline (2 μm). Cell growth was measured using the MTS assay. NRGβ1 treatment resulted in 33.5% increase of cell proliferation. Perhexiline inhibits 11.5% cells in vehicle media and 36.3% cell growth in NRGβ1 media. Data are presented as the mean ± SEM (n = 3). The statistical significance between DMSO control and perhexiline treatment in vehicle media or NRGβ1 media was analyzed by a two-tailed Student’s t test, (with *P <0.05 defined as significant compared to the control group with only NRGβ1 treatment). BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated protein kinase; HER3, human epidermal growth factor receptor 3; NRGβ1, neuregulin β1; PBS, phosphate-buffered saline; SEM, standard error of the mean.
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Fig3: Perhexiline treatment inhibits neuregulin-mediated activation of downstream signaling and cell proliferation. (A) MDA-MB-468 cells were cultured in the absence or presence of perhexiline (10 μM) for 6 hours, and subsequently stimulated with DMSO or NRGβ1 (10 ng/ml) for 10 minutes. Cell lysates were analyzed for the phosphorylation of HER3 (Tyr1289), Akt and ERK1/2 as well as total HER3 expression. (B) Perhexiline inhibits NRGβ1-induced cell growth. MB-MDA-468 cells were treated for 72 hours with vehicle (0.2% BSA in PBS) or NRGβ1 (10 ng/ml) in the presence of DMSO or perhexiline (2 μm). Cell growth was measured using the MTS assay. NRGβ1 treatment resulted in 33.5% increase of cell proliferation. Perhexiline inhibits 11.5% cells in vehicle media and 36.3% cell growth in NRGβ1 media. Data are presented as the mean ± SEM (n = 3). The statistical significance between DMSO control and perhexiline treatment in vehicle media or NRGβ1 media was analyzed by a two-tailed Student’s t test, (with *P <0.05 defined as significant compared to the control group with only NRGβ1 treatment). BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated protein kinase; HER3, human epidermal growth factor receptor 3; NRGβ1, neuregulin β1; PBS, phosphate-buffered saline; SEM, standard error of the mean.

Mentions: To determine the effect of perhexiline on HER3-mediated oncogenic signaling in breast cancer cells, serum-starved MDA-MB-468 cells were pretreated with perhexiline and subsequently stimulated with NRGβ1. As shown in Figure 3A, perhexiline treatment significantly decreased NRGβ1-induced phosphorylation of Akt and ERK. This inhibition of signaling coincided with perhexiline-mediated downregulation of HER3 (Figure 3A). In addition, MTS assays were performed to assess the effect of perhexiline on cell proliferation. Results showed that NRGβ1 treatment resulted in a 33.5% increase in cell proliferation consistent with HER3’s role in promoting tumor growth. Treating cells with perhexiline decreased cell proliferation by 11.5% and 36.3% in serum-free and NRGβ1-containing medium, respectively (Figure 3B).Figure 3


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 treatment inhibits neuregulin-mediated activation of downstream signaling and cell proliferation. (A) MDA-MB-468 cells were cultured in the absence or presence of perhexiline (10 μM) for 6 hours, and subsequently stimulated with DMSO or NRGβ1 (10 ng/ml) for 10 minutes. Cell lysates were analyzed for the phosphorylation of HER3 (Tyr1289), Akt and ERK1/2 as well as total HER3 expression. (B) Perhexiline inhibits NRGβ1-induced cell growth. MB-MDA-468 cells were treated for 72 hours with vehicle (0.2% BSA in PBS) or NRGβ1 (10 ng/ml) in the presence of DMSO or perhexiline (2 μm). Cell growth was measured using the MTS assay. NRGβ1 treatment resulted in 33.5% increase of cell proliferation. Perhexiline inhibits 11.5% cells in vehicle media and 36.3% cell growth in NRGβ1 media. Data are presented as the mean ± SEM (n = 3). The statistical significance between DMSO control and perhexiline treatment in vehicle media or NRGβ1 media was analyzed by a two-tailed Student’s t test, (with *P <0.05 defined as significant compared to the control group with only NRGβ1 treatment). BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated protein kinase; HER3, human epidermal growth factor receptor 3; NRGβ1, neuregulin β1; PBS, phosphate-buffered saline; SEM, standard error of the mean.
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Related In: Results  -  Collection

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Fig3: Perhexiline treatment inhibits neuregulin-mediated activation of downstream signaling and cell proliferation. (A) MDA-MB-468 cells were cultured in the absence or presence of perhexiline (10 μM) for 6 hours, and subsequently stimulated with DMSO or NRGβ1 (10 ng/ml) for 10 minutes. Cell lysates were analyzed for the phosphorylation of HER3 (Tyr1289), Akt and ERK1/2 as well as total HER3 expression. (B) Perhexiline inhibits NRGβ1-induced cell growth. MB-MDA-468 cells were treated for 72 hours with vehicle (0.2% BSA in PBS) or NRGβ1 (10 ng/ml) in the presence of DMSO or perhexiline (2 μm). Cell growth was measured using the MTS assay. NRGβ1 treatment resulted in 33.5% increase of cell proliferation. Perhexiline inhibits 11.5% cells in vehicle media and 36.3% cell growth in NRGβ1 media. Data are presented as the mean ± SEM (n = 3). The statistical significance between DMSO control and perhexiline treatment in vehicle media or NRGβ1 media was analyzed by a two-tailed Student’s t test, (with *P <0.05 defined as significant compared to the control group with only NRGβ1 treatment). BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated protein kinase; HER3, human epidermal growth factor receptor 3; NRGβ1, neuregulin β1; PBS, phosphate-buffered saline; SEM, standard error of the mean.
Mentions: To determine the effect of perhexiline on HER3-mediated oncogenic signaling in breast cancer cells, serum-starved MDA-MB-468 cells were pretreated with perhexiline and subsequently stimulated with NRGβ1. As shown in Figure 3A, perhexiline treatment significantly decreased NRGβ1-induced phosphorylation of Akt and ERK. This inhibition of signaling coincided with perhexiline-mediated downregulation of HER3 (Figure 3A). In addition, MTS assays were performed to assess the effect of perhexiline on cell proliferation. Results showed that NRGβ1 treatment resulted in a 33.5% increase in cell proliferation consistent with HER3’s role in promoting tumor growth. Treating cells with perhexiline decreased cell proliferation by 11.5% and 36.3% in serum-free and NRGβ1-containing medium, respectively (Figure 3B).Figure 3

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