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Inhibition of the HER2-YB1-AR axis with Lapatinib synergistically enhances Enzalutamide anti-tumor efficacy in castration resistant prostate cancer.

Shiota M, Bishop JL, Takeuchi A, Nip KM, Cordonnier T, Beraldi E, Kuruma H, Gleave ME, Zoubeidi A - Oncotarget (2015)

Bottom Line: Potent therapies that prevent AR signaling, such as Enzalutamide (ENZ), are mainstay treatments for CRPC; however patients eventually progress with ENZ resistant (ENZR) disease.Despite efficacy in vitro, in vivo monotherapy with Lapatinib did not prevent ENZR tumor growth.However, combination treatment of Lapatinib with ENZ most effectively induced cell death in LNCaP cells in vitro and was more effective than ENZ alone in preventing tumor growth in an in vivo model of CRPC.

View Article: PubMed Central - PubMed

Affiliation: The Vancouver Prostate Centre and Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
Incurable castration-resistant prostate cancer (CRPC) is driven by androgen receptor (AR) activation. Potent therapies that prevent AR signaling, such as Enzalutamide (ENZ), are mainstay treatments for CRPC; however patients eventually progress with ENZ resistant (ENZR) disease. In this study, we investigated one mechanism of ENZ resistance, and tried to improve therapeutic efficiency of ENZ. We found HER2 expression is increased in ENZR tumors and cell lines, and is induced by ENZ treatment of LNCaP cells. ENZ-induced HER2 overexpression was dependent on AKT-YB1 activation and modulated AR activity. HER2 dependent AR activation in LNCaP and ENZR cells was effectively blocked by treatment with the EGFR/HER2 inhibitor Lapatinib, which reduced cell viability and increased apoptosis. Despite efficacy in vitro, in vivo monotherapy with Lapatinib did not prevent ENZR tumor growth. However, combination treatment of Lapatinib with ENZ most effectively induced cell death in LNCaP cells in vitro and was more effective than ENZ alone in preventing tumor growth in an in vivo model of CRPC. These results suggest that while HER2 overexpression and subsequent AR activation is a targetable mechanism of resistance to ENZ, therapy using Lapatinib is only a rational therapeutic approach when used in combination with ENZ in CRPC.

No MeSH data available.


Related in: MedlinePlus

Lapatinib augments therapeutic effect of ENZ in vitro and in vivoA. LNCaP cells were cultured in serum-free media +/− 10 nM of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib and/or 10uM ENZ for 24 h and AR and PSA was assessed by western blot. B. LNCaP cells were transfected with 0.5 μg/mL of PSA–Luc plasmid and 0.05 μg/mL of pRL-TK. After 12 h, media was changed into serum-free media with or without 10 nmol R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib and/or ENZ for 24 h. Fold induction of Probasin–Luc compared to untreated cells is shown. A representative experiment is shown ±s.d. C. LNCaP cells were treated with 10 μM of ENZ and/or Lapatinib. After incubation for 48 h, the cells were stained with propidium iodide and analyzed by flow cytometry for cell cycle fractions. Means from a representative experiment are shown, error bars ± s.d. D. Whole-cell extracts from LNCaP cells treated with 20 μmol/L of ENZ and/or Lapatinib for 48 h were analyzed by western blot analysis for cleaved PARP and β-actin (loading control). E. (Right) LNCaP cells were treated with various concentrations Lapatinib for 72 h and cell survival was analyzed by crystal violet assay. Means from a representative experiment are shown, error bars ± s.d. (Left) Dose-dependent effects of Lapatinib, ENZ or Lapatinib+ENZ (right panel) and CI values for ED50, ED75 and ED90 of Lapatinib+ENZ (left panel) calculated by CalcuSyn software were assessed. Cell growth in the absence of inhibitor corresponds to 1. F. After establishment of LNCaP tumors (200mm3) in castrated mice, mice were treated with 100mg/kg Lapatinib or vehicle alone, n=10. Representative data of tumor volume over 12 weeks is shown.
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Figure 5: Lapatinib augments therapeutic effect of ENZ in vitro and in vivoA. LNCaP cells were cultured in serum-free media +/− 10 nM of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib and/or 10uM ENZ for 24 h and AR and PSA was assessed by western blot. B. LNCaP cells were transfected with 0.5 μg/mL of PSA–Luc plasmid and 0.05 μg/mL of pRL-TK. After 12 h, media was changed into serum-free media with or without 10 nmol R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib and/or ENZ for 24 h. Fold induction of Probasin–Luc compared to untreated cells is shown. A representative experiment is shown ±s.d. C. LNCaP cells were treated with 10 μM of ENZ and/or Lapatinib. After incubation for 48 h, the cells were stained with propidium iodide and analyzed by flow cytometry for cell cycle fractions. Means from a representative experiment are shown, error bars ± s.d. D. Whole-cell extracts from LNCaP cells treated with 20 μmol/L of ENZ and/or Lapatinib for 48 h were analyzed by western blot analysis for cleaved PARP and β-actin (loading control). E. (Right) LNCaP cells were treated with various concentrations Lapatinib for 72 h and cell survival was analyzed by crystal violet assay. Means from a representative experiment are shown, error bars ± s.d. (Left) Dose-dependent effects of Lapatinib, ENZ or Lapatinib+ENZ (right panel) and CI values for ED50, ED75 and ED90 of Lapatinib+ENZ (left panel) calculated by CalcuSyn software were assessed. Cell growth in the absence of inhibitor corresponds to 1. F. After establishment of LNCaP tumors (200mm3) in castrated mice, mice were treated with 100mg/kg Lapatinib or vehicle alone, n=10. Representative data of tumor volume over 12 weeks is shown.

Mentions: As an alternative to monotherapy, combination therapies targeting novel signaling pathways along-side the AR may be more viable alternatives for patients with anti-androgen resistant PCa [28]. To investigate this possibility, we assessed the therapeutic combination effect of ENZ with Lapatinib on AR signaling in LNCaP cells. As shown in Fig. 5A, combination treatment of Lapatinib with ENZ enhanced the reduction of AR and PSA expression (Fig. 4A) as well as AR transactivation in LNCaP cells (Fig. 4B) more so than ENZ or Lapatinib treatment alone. Accordingly, combination of ENZ with Lapatinib also showed the greatest efficacy in increasing the fraction of cells in the SubG0 phase of cell cycle (Fig. 4C) and induction of PARP cleavage (Fig. 4D) compared to ENZ or Lapatinib alone. Moreover, combination treatment of ENZ and Lapatinib showed the greatest reduction in cell viability compared to monotherapy with Lapatinib. Calculated combination indices determined at ED50, ED75 and ED90 revealed values below 1 (Fig. 5E), indicating ENZ and Lapatinib are strongly synergistic in LNCaP cells. These results suggest that HER2 signaling is important for growth and survival in the context of anti-androgen treatment and co-targeting this pathway and the AR may have synergistic effects in CRPC.


Inhibition of the HER2-YB1-AR axis with Lapatinib synergistically enhances Enzalutamide anti-tumor efficacy in castration resistant prostate cancer.

Shiota M, Bishop JL, Takeuchi A, Nip KM, Cordonnier T, Beraldi E, Kuruma H, Gleave ME, Zoubeidi A - Oncotarget (2015)

Lapatinib augments therapeutic effect of ENZ in vitro and in vivoA. LNCaP cells were cultured in serum-free media +/− 10 nM of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib and/or 10uM ENZ for 24 h and AR and PSA was assessed by western blot. B. LNCaP cells were transfected with 0.5 μg/mL of PSA–Luc plasmid and 0.05 μg/mL of pRL-TK. After 12 h, media was changed into serum-free media with or without 10 nmol R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib and/or ENZ for 24 h. Fold induction of Probasin–Luc compared to untreated cells is shown. A representative experiment is shown ±s.d. C. LNCaP cells were treated with 10 μM of ENZ and/or Lapatinib. After incubation for 48 h, the cells were stained with propidium iodide and analyzed by flow cytometry for cell cycle fractions. Means from a representative experiment are shown, error bars ± s.d. D. Whole-cell extracts from LNCaP cells treated with 20 μmol/L of ENZ and/or Lapatinib for 48 h were analyzed by western blot analysis for cleaved PARP and β-actin (loading control). E. (Right) LNCaP cells were treated with various concentrations Lapatinib for 72 h and cell survival was analyzed by crystal violet assay. Means from a representative experiment are shown, error bars ± s.d. (Left) Dose-dependent effects of Lapatinib, ENZ or Lapatinib+ENZ (right panel) and CI values for ED50, ED75 and ED90 of Lapatinib+ENZ (left panel) calculated by CalcuSyn software were assessed. Cell growth in the absence of inhibitor corresponds to 1. F. After establishment of LNCaP tumors (200mm3) in castrated mice, mice were treated with 100mg/kg Lapatinib or vehicle alone, n=10. Representative data of tumor volume over 12 weeks is shown.
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Figure 5: Lapatinib augments therapeutic effect of ENZ in vitro and in vivoA. LNCaP cells were cultured in serum-free media +/− 10 nM of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib and/or 10uM ENZ for 24 h and AR and PSA was assessed by western blot. B. LNCaP cells were transfected with 0.5 μg/mL of PSA–Luc plasmid and 0.05 μg/mL of pRL-TK. After 12 h, media was changed into serum-free media with or without 10 nmol R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib and/or ENZ for 24 h. Fold induction of Probasin–Luc compared to untreated cells is shown. A representative experiment is shown ±s.d. C. LNCaP cells were treated with 10 μM of ENZ and/or Lapatinib. After incubation for 48 h, the cells were stained with propidium iodide and analyzed by flow cytometry for cell cycle fractions. Means from a representative experiment are shown, error bars ± s.d. D. Whole-cell extracts from LNCaP cells treated with 20 μmol/L of ENZ and/or Lapatinib for 48 h were analyzed by western blot analysis for cleaved PARP and β-actin (loading control). E. (Right) LNCaP cells were treated with various concentrations Lapatinib for 72 h and cell survival was analyzed by crystal violet assay. Means from a representative experiment are shown, error bars ± s.d. (Left) Dose-dependent effects of Lapatinib, ENZ or Lapatinib+ENZ (right panel) and CI values for ED50, ED75 and ED90 of Lapatinib+ENZ (left panel) calculated by CalcuSyn software were assessed. Cell growth in the absence of inhibitor corresponds to 1. F. After establishment of LNCaP tumors (200mm3) in castrated mice, mice were treated with 100mg/kg Lapatinib or vehicle alone, n=10. Representative data of tumor volume over 12 weeks is shown.
Mentions: As an alternative to monotherapy, combination therapies targeting novel signaling pathways along-side the AR may be more viable alternatives for patients with anti-androgen resistant PCa [28]. To investigate this possibility, we assessed the therapeutic combination effect of ENZ with Lapatinib on AR signaling in LNCaP cells. As shown in Fig. 5A, combination treatment of Lapatinib with ENZ enhanced the reduction of AR and PSA expression (Fig. 4A) as well as AR transactivation in LNCaP cells (Fig. 4B) more so than ENZ or Lapatinib treatment alone. Accordingly, combination of ENZ with Lapatinib also showed the greatest efficacy in increasing the fraction of cells in the SubG0 phase of cell cycle (Fig. 4C) and induction of PARP cleavage (Fig. 4D) compared to ENZ or Lapatinib alone. Moreover, combination treatment of ENZ and Lapatinib showed the greatest reduction in cell viability compared to monotherapy with Lapatinib. Calculated combination indices determined at ED50, ED75 and ED90 revealed values below 1 (Fig. 5E), indicating ENZ and Lapatinib are strongly synergistic in LNCaP cells. These results suggest that HER2 signaling is important for growth and survival in the context of anti-androgen treatment and co-targeting this pathway and the AR may have synergistic effects in CRPC.

Bottom Line: Potent therapies that prevent AR signaling, such as Enzalutamide (ENZ), are mainstay treatments for CRPC; however patients eventually progress with ENZ resistant (ENZR) disease.Despite efficacy in vitro, in vivo monotherapy with Lapatinib did not prevent ENZR tumor growth.However, combination treatment of Lapatinib with ENZ most effectively induced cell death in LNCaP cells in vitro and was more effective than ENZ alone in preventing tumor growth in an in vivo model of CRPC.

View Article: PubMed Central - PubMed

Affiliation: The Vancouver Prostate Centre and Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
Incurable castration-resistant prostate cancer (CRPC) is driven by androgen receptor (AR) activation. Potent therapies that prevent AR signaling, such as Enzalutamide (ENZ), are mainstay treatments for CRPC; however patients eventually progress with ENZ resistant (ENZR) disease. In this study, we investigated one mechanism of ENZ resistance, and tried to improve therapeutic efficiency of ENZ. We found HER2 expression is increased in ENZR tumors and cell lines, and is induced by ENZ treatment of LNCaP cells. ENZ-induced HER2 overexpression was dependent on AKT-YB1 activation and modulated AR activity. HER2 dependent AR activation in LNCaP and ENZR cells was effectively blocked by treatment with the EGFR/HER2 inhibitor Lapatinib, which reduced cell viability and increased apoptosis. Despite efficacy in vitro, in vivo monotherapy with Lapatinib did not prevent ENZR tumor growth. However, combination treatment of Lapatinib with ENZ most effectively induced cell death in LNCaP cells in vitro and was more effective than ENZ alone in preventing tumor growth in an in vivo model of CRPC. These results suggest that while HER2 overexpression and subsequent AR activation is a targetable mechanism of resistance to ENZ, therapy using Lapatinib is only a rational therapeutic approach when used in combination with ENZ in CRPC.

No MeSH data available.


Related in: MedlinePlus