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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 inhibits breast cancer cell proliferation and functions synergistically with lapatinib. (A-B) MDA-MB-468 (A) and SK-BR-3 (B) cells were treated with different concentrations of perhexiline for 72 hours, and cell viability was measured using the MTS assay. Each experimental point was performed in triplicate, and results represent mean ± SEM of three independent experiments. (C) The synergistic effect of perhexiline and lapatinib on inhibiting HER3-mediated Akt signaling. MDA-MB 468 cells treated with 5 μM perhexiline alone or in combination with 100 nM lapatinib for 2 or 36 hours. Cell lysates were analyzed for the phosphorylation status of endogenous HER3 and Akt as well as total HER3 and Akt expression. (D-E) Perhexiline and lapatinib synergistically inhibit MDA-MB-468 (D) and SK-BR-3 (E) cell growth. Cells were treated with increasing concentrations of perhexiline and lapatinib alone or in combination for 72 hours. MTS assays were performed to measure cell viability. Values are expressed as a percentage of DMSO-treated cells. Results are presented as the mean ± SEM. Combinational index (CI) quantifies the degree of synergism in a drug combination. CI is obtained using the method of Chou and Martin in the software CompuSyn. CI <1 indicates drug synergy. ED50, ED75, ED90 and ED95 are the effective doses at which 50%, 75%, 90% and 95% cells are killed, respectively. DMSO, dimethyl sulfoxide; HER3, human epidermal growth factor receptor 3; SEM, standard error of the mean.
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Fig4: Perhexiline inhibits breast cancer cell proliferation and functions synergistically with lapatinib. (A-B) MDA-MB-468 (A) and SK-BR-3 (B) cells were treated with different concentrations of perhexiline for 72 hours, and cell viability was measured using the MTS assay. Each experimental point was performed in triplicate, and results represent mean ± SEM of three independent experiments. (C) The synergistic effect of perhexiline and lapatinib on inhibiting HER3-mediated Akt signaling. MDA-MB 468 cells treated with 5 μM perhexiline alone or in combination with 100 nM lapatinib for 2 or 36 hours. Cell lysates were analyzed for the phosphorylation status of endogenous HER3 and Akt as well as total HER3 and Akt expression. (D-E) Perhexiline and lapatinib synergistically inhibit MDA-MB-468 (D) and SK-BR-3 (E) cell growth. Cells were treated with increasing concentrations of perhexiline and lapatinib alone or in combination for 72 hours. MTS assays were performed to measure cell viability. Values are expressed as a percentage of DMSO-treated cells. Results are presented as the mean ± SEM. Combinational index (CI) quantifies the degree of synergism in a drug combination. CI is obtained using the method of Chou and Martin in the software CompuSyn. CI <1 indicates drug synergy. ED50, ED75, ED90 and ED95 are the effective doses at which 50%, 75%, 90% and 95% cells are killed, respectively. DMSO, dimethyl sulfoxide; HER3, human epidermal growth factor receptor 3; SEM, standard error of the mean.

Mentions: To further determine the effect of perhexiline on breast cancer cell proliferation, MDA-MB-468 and SK-BR-3 cells were treated with different concentrations of perhexiline for 72 hours in regular growth medium. Perhexiline treatment effectively inhibited cell proliferation in both cell lines with IC50 of 2.7 ± 0.07 and 4.8 ± 0.2 μM for MDA-MB-468 and SK-BR-3, respectively (Figure 4A-B). These IC50 values were similar to those needed to induce internalization and degradation of HER3 receptors. Given upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors (for example lapatinib, gefitinib, erlotinib) [12-14], we next assessed the effect of perhexiline combined with lapatinib, an agent commonly used to treat HER2-positive breast cancer. Treatment of MDA-MB-468 cells with 100 nM lapatinib for 2 hours led to decreased phosphorylation and activation of HER3 and downstream Akt signaling (Figure 4C). However, lapatinib treatment after 36 hours led to the recovery of HER3 and AKT phosphorylation, a result similar to the original report describing lapatinib resistance mediated by HER3 (Figure 4C) [12]. In contrast, the inhibition of HER3 and downstream Akt signaling remained effective in cells treated with a combination of 5 μM perhexiline and 100 nM lapatinib, suggesting that perhexiline can potentially be used to prevent the resistance to HER family tyrosine kinase inhibitors (Figure 4C).Figure 4


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 inhibits breast cancer cell proliferation and functions synergistically with lapatinib. (A-B) MDA-MB-468 (A) and SK-BR-3 (B) cells were treated with different concentrations of perhexiline for 72 hours, and cell viability was measured using the MTS assay. Each experimental point was performed in triplicate, and results represent mean ± SEM of three independent experiments. (C) The synergistic effect of perhexiline and lapatinib on inhibiting HER3-mediated Akt signaling. MDA-MB 468 cells treated with 5 μM perhexiline alone or in combination with 100 nM lapatinib for 2 or 36 hours. Cell lysates were analyzed for the phosphorylation status of endogenous HER3 and Akt as well as total HER3 and Akt expression. (D-E) Perhexiline and lapatinib synergistically inhibit MDA-MB-468 (D) and SK-BR-3 (E) cell growth. Cells were treated with increasing concentrations of perhexiline and lapatinib alone or in combination for 72 hours. MTS assays were performed to measure cell viability. Values are expressed as a percentage of DMSO-treated cells. Results are presented as the mean ± SEM. Combinational index (CI) quantifies the degree of synergism in a drug combination. CI is obtained using the method of Chou and Martin in the software CompuSyn. CI <1 indicates drug synergy. ED50, ED75, ED90 and ED95 are the effective doses at which 50%, 75%, 90% and 95% cells are killed, respectively. DMSO, dimethyl sulfoxide; HER3, human epidermal growth factor receptor 3; SEM, standard error of the mean.
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Fig4: Perhexiline inhibits breast cancer cell proliferation and functions synergistically with lapatinib. (A-B) MDA-MB-468 (A) and SK-BR-3 (B) cells were treated with different concentrations of perhexiline for 72 hours, and cell viability was measured using the MTS assay. Each experimental point was performed in triplicate, and results represent mean ± SEM of three independent experiments. (C) The synergistic effect of perhexiline and lapatinib on inhibiting HER3-mediated Akt signaling. MDA-MB 468 cells treated with 5 μM perhexiline alone or in combination with 100 nM lapatinib for 2 or 36 hours. Cell lysates were analyzed for the phosphorylation status of endogenous HER3 and Akt as well as total HER3 and Akt expression. (D-E) Perhexiline and lapatinib synergistically inhibit MDA-MB-468 (D) and SK-BR-3 (E) cell growth. Cells were treated with increasing concentrations of perhexiline and lapatinib alone or in combination for 72 hours. MTS assays were performed to measure cell viability. Values are expressed as a percentage of DMSO-treated cells. Results are presented as the mean ± SEM. Combinational index (CI) quantifies the degree of synergism in a drug combination. CI is obtained using the method of Chou and Martin in the software CompuSyn. CI <1 indicates drug synergy. ED50, ED75, ED90 and ED95 are the effective doses at which 50%, 75%, 90% and 95% cells are killed, respectively. DMSO, dimethyl sulfoxide; HER3, human epidermal growth factor receptor 3; SEM, standard error of the mean.
Mentions: To further determine the effect of perhexiline on breast cancer cell proliferation, MDA-MB-468 and SK-BR-3 cells were treated with different concentrations of perhexiline for 72 hours in regular growth medium. Perhexiline treatment effectively inhibited cell proliferation in both cell lines with IC50 of 2.7 ± 0.07 and 4.8 ± 0.2 μM for MDA-MB-468 and SK-BR-3, respectively (Figure 4A-B). These IC50 values were similar to those needed to induce internalization and degradation of HER3 receptors. Given upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors (for example lapatinib, gefitinib, erlotinib) [12-14], we next assessed the effect of perhexiline combined with lapatinib, an agent commonly used to treat HER2-positive breast cancer. Treatment of MDA-MB-468 cells with 100 nM lapatinib for 2 hours led to decreased phosphorylation and activation of HER3 and downstream Akt signaling (Figure 4C). However, lapatinib treatment after 36 hours led to the recovery of HER3 and AKT phosphorylation, a result similar to the original report describing lapatinib resistance mediated by HER3 (Figure 4C) [12]. In contrast, the inhibition of HER3 and downstream Akt signaling remained effective in cells treated with a combination of 5 μM perhexiline and 100 nM lapatinib, suggesting that perhexiline can potentially be used to prevent the resistance to HER family tyrosine kinase inhibitors (Figure 4C).Figure 4

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