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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 blocks AR activationA-D. LNCaP cells were cultured in serum-free media +/− 10 nM of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μmol Lapatinib for 24 h and AR, PSA and FKBP5 expression in total protein or mRNAwas assessed by western blot (A) or qRT-PCR (B). Means of representative experiments are shown ±s.d. For qRT-PCR target gene expression was normalized to GAPDH and untreated cells set to 1. For western blot analysis β-actin was used as a loading control. C. 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 or ENZ for 24 h. Fold induction of Probasin-Luc compared to untreated cells is shown. A representative experiment is shown ±s.d. D. LNCaP cells were cultured in serum-free media +/− 10 nmol of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib for 24 h and cell lysate was fractioned into nuclear and cytoplasmic extracts. Expression of AR and laminB1 or α-tubulin (as loading controls) were assessed by western blot. E. ChIP assay was performed on nuclear extracts from LNCaP cells cultured in serum-free media with or without 10 nmol R1881 for 12 h, followed by further incubation with or without 10 μmol Lapatinib for 1 h using 2.0 μg of rabbit IgG or anti-AR antibody. The quantitative RT-PCR was performed using immunoprecipitated DNAs, soluble chromatin and specific primer pairs for the PSA gene normalized to GAPDH. Untreated cells are set to 1. A representative experiment is shown ±s.d.
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Figure 3: Lapatinib blocks AR activationA-D. LNCaP cells were cultured in serum-free media +/− 10 nM of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μmol Lapatinib for 24 h and AR, PSA and FKBP5 expression in total protein or mRNAwas assessed by western blot (A) or qRT-PCR (B). Means of representative experiments are shown ±s.d. For qRT-PCR target gene expression was normalized to GAPDH and untreated cells set to 1. For western blot analysis β-actin was used as a loading control. C. 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 or ENZ for 24 h. Fold induction of Probasin-Luc compared to untreated cells is shown. A representative experiment is shown ±s.d. D. LNCaP cells were cultured in serum-free media +/− 10 nmol of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib for 24 h and cell lysate was fractioned into nuclear and cytoplasmic extracts. Expression of AR and laminB1 or α-tubulin (as loading controls) were assessed by western blot. E. ChIP assay was performed on nuclear extracts from LNCaP cells cultured in serum-free media with or without 10 nmol R1881 for 12 h, followed by further incubation with or without 10 μmol Lapatinib for 1 h using 2.0 μg of rabbit IgG or anti-AR antibody. The quantitative RT-PCR was performed using immunoprecipitated DNAs, soluble chromatin and specific primer pairs for the PSA gene normalized to GAPDH. Untreated cells are set to 1. A representative experiment is shown ±s.d.

Mentions: One important consequence of increased HER2 signaling in CRPC is re-activation of the AR [15, 26, 27], which also occurs during ENZ resistance [8]. Our results showing increased HER2 expression in ENZR cell lines suggest that as in CRPC, HER2 activation of the AR may be a mechanism of resistance to ENZ. Therefore, we examined the effect of Lapatinib, a dual EGFR/HER2 inhibitor, on AR signaling. As expected, we found that Lapatinib treatment induced a decrease of AR regulated genes at the protein and mRNA level in LNCaP cells (Fig. 3A-B) and PSA promoter activity was also significantly reduced in Lapatinib treated LNCaP cells after androgen stimulation (Fig. 3C). This was associated with decreased nuclear AR in Lapatinib treated cells (Fig. 3D). Accordingly, Lapatinib also inhibited androgen mediated AR binding to the androgen-responsive element (ARE) in the promoter region of PSA (PSA ARE+) (Fig. 3E). These results indicate that suppression of EGFR/HER2 signaling with Lapatinib inhibits AR activity in PCa cells and suggest the increased dependence of HER2 mediated AR activation in ENZR cells.


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 blocks AR activationA-D. LNCaP cells were cultured in serum-free media +/− 10 nM of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μmol Lapatinib for 24 h and AR, PSA and FKBP5 expression in total protein or mRNAwas assessed by western blot (A) or qRT-PCR (B). Means of representative experiments are shown ±s.d. For qRT-PCR target gene expression was normalized to GAPDH and untreated cells set to 1. For western blot analysis β-actin was used as a loading control. C. 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 or ENZ for 24 h. Fold induction of Probasin-Luc compared to untreated cells is shown. A representative experiment is shown ±s.d. D. LNCaP cells were cultured in serum-free media +/− 10 nmol of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib for 24 h and cell lysate was fractioned into nuclear and cytoplasmic extracts. Expression of AR and laminB1 or α-tubulin (as loading controls) were assessed by western blot. E. ChIP assay was performed on nuclear extracts from LNCaP cells cultured in serum-free media with or without 10 nmol R1881 for 12 h, followed by further incubation with or without 10 μmol Lapatinib for 1 h using 2.0 μg of rabbit IgG or anti-AR antibody. The quantitative RT-PCR was performed using immunoprecipitated DNAs, soluble chromatin and specific primer pairs for the PSA gene normalized to GAPDH. Untreated cells are set to 1. A representative experiment is shown ±s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496204&req=5

Figure 3: Lapatinib blocks AR activationA-D. LNCaP cells were cultured in serum-free media +/− 10 nM of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μmol Lapatinib for 24 h and AR, PSA and FKBP5 expression in total protein or mRNAwas assessed by western blot (A) or qRT-PCR (B). Means of representative experiments are shown ±s.d. For qRT-PCR target gene expression was normalized to GAPDH and untreated cells set to 1. For western blot analysis β-actin was used as a loading control. C. 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 or ENZ for 24 h. Fold induction of Probasin-Luc compared to untreated cells is shown. A representative experiment is shown ±s.d. D. LNCaP cells were cultured in serum-free media +/− 10 nmol of the synthetic androgen R1881 for 12 h, followed by further incubation +/− 10 μM Lapatinib for 24 h and cell lysate was fractioned into nuclear and cytoplasmic extracts. Expression of AR and laminB1 or α-tubulin (as loading controls) were assessed by western blot. E. ChIP assay was performed on nuclear extracts from LNCaP cells cultured in serum-free media with or without 10 nmol R1881 for 12 h, followed by further incubation with or without 10 μmol Lapatinib for 1 h using 2.0 μg of rabbit IgG or anti-AR antibody. The quantitative RT-PCR was performed using immunoprecipitated DNAs, soluble chromatin and specific primer pairs for the PSA gene normalized to GAPDH. Untreated cells are set to 1. A representative experiment is shown ±s.d.
Mentions: One important consequence of increased HER2 signaling in CRPC is re-activation of the AR [15, 26, 27], which also occurs during ENZ resistance [8]. Our results showing increased HER2 expression in ENZR cell lines suggest that as in CRPC, HER2 activation of the AR may be a mechanism of resistance to ENZ. Therefore, we examined the effect of Lapatinib, a dual EGFR/HER2 inhibitor, on AR signaling. As expected, we found that Lapatinib treatment induced a decrease of AR regulated genes at the protein and mRNA level in LNCaP cells (Fig. 3A-B) and PSA promoter activity was also significantly reduced in Lapatinib treated LNCaP cells after androgen stimulation (Fig. 3C). This was associated with decreased nuclear AR in Lapatinib treated cells (Fig. 3D). Accordingly, Lapatinib also inhibited androgen mediated AR binding to the androgen-responsive element (ARE) in the promoter region of PSA (PSA ARE+) (Fig. 3E). These results indicate that suppression of EGFR/HER2 signaling with Lapatinib inhibits AR activity in PCa cells and suggest the increased dependence of HER2 mediated AR activation in ENZR cells.

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