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Effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on NEU/HER2 overexpressing mammary tumours in MMTV-NEU-NT mice monitored by Magnetic Resonance Spectroscopy.

Rodrigues LM, Chung YL, Al Saffar NM, Sharp SY, Jackson LE, Banerji U, Stubbs M, Leach MO, Griffiths JR, Workman P - BMC Res Notes (2012)

Bottom Line: At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose.Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment.The data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, UK.

ABSTRACT

Background: The importance of ERBB2/NEU/HER2 in the response of breast tumours to the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin) has been demonstrated in the clinic. ERBB2 is an oncoprotein client that is highly dependent on HSP90. This and other oncogenic client proteins (e.g. B-RAF, C-RAF, ALK and CDK4) are depleted by 17-AAG in both animal tumours and patients. Here we investigate by Magnetic Resonance Spectroscopy (MRS) the metabolic response of 17-AAG in spontaneous, NEU/HER2 driven mammary tumours in transgenic MMTV-NEU-NT mice and in cells isolated and cultured from these tumours.

Methods: Mammary tumours were monitored by 31P MRS in vivo and in tumour extracts, comparing control and 17-AAG treated mice. A cell line derived from NEU/HER2 mammary tumours was also cultured and the effect of 17-AAG was measured by 31P MRS in cell extracts. Molecular biomarkers were assessed by immunoblotting in extracts from cells and tumours. For comparison of tumour volume, metabolite concentrations and Western blot band intensities, two-tailed unpaired t-tests were used.

Results: The NEU/HER2 mammary tumours were very sensitive to 17-AAG and responded in a dose-dependent manner to 3 daily doses of 20, 40 and 80mg/kg of 17-AAG, all of which caused significant regression. At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose. Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment. Western blots confirmed the expected action of 17-AAG in inducing HSP72 and significantly depleting HSP90 client proteins, including NEU/HER2 both in tumours and in isolated cells.

Conclusions: The data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment.

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Effect of 17-AAG on cultured cells isolated from a MMTV-NEU-NT solid tumour. Cells treated for 24hr at 37°C with vehicle (control) or 1.12μM 17-AAG. A) Western blots of NEU/HER2, B-RAF, C-RAF, HSP72, CDK4 and cyclin D1 in vehicle treated (lanes 1,3,5) and 17-AAG treated (lanes 2,4,6) cells. Intensity of bands were quantitated relative to GAPDH, (n = 5). B)31P MR spectra of cell extracts from control cells (bottom), or cells following 17-AAG treatment (top). Peak assignments are: phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate (Pi), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (α-, β-, γ-NTP). Results were confirmed in six separate experiments, and representative spectra are shown. C) Percentage changes (relative to control) in cell number, phosphoethanolamine (PE), phosphocholine (PC), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (β-NTP), phosphomonoesters (PME = PE + PC), PC/GPC, phosphodiesters (PDE = GPE + GPC) and PDE/βNTP levels in MMTV-NEU-NT tumour cells following treatment with 17-AAG (1.12 μM) for 24h. Results are presented as the mean ± SEM (error bars) of six separate experiments. *p ≤ 0.05, **p < 0.01, two-tailed unpaired t test was used for all comparisons.
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Figure 5: Effect of 17-AAG on cultured cells isolated from a MMTV-NEU-NT solid tumour. Cells treated for 24hr at 37°C with vehicle (control) or 1.12μM 17-AAG. A) Western blots of NEU/HER2, B-RAF, C-RAF, HSP72, CDK4 and cyclin D1 in vehicle treated (lanes 1,3,5) and 17-AAG treated (lanes 2,4,6) cells. Intensity of bands were quantitated relative to GAPDH, (n = 5). B)31P MR spectra of cell extracts from control cells (bottom), or cells following 17-AAG treatment (top). Peak assignments are: phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate (Pi), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (α-, β-, γ-NTP). Results were confirmed in six separate experiments, and representative spectra are shown. C) Percentage changes (relative to control) in cell number, phosphoethanolamine (PE), phosphocholine (PC), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (β-NTP), phosphomonoesters (PME = PE + PC), PC/GPC, phosphodiesters (PDE = GPE + GPC) and PDE/βNTP levels in MMTV-NEU-NT tumour cells following treatment with 17-AAG (1.12 μM) for 24h. Results are presented as the mean ± SEM (error bars) of six separate experiments. *p ≤ 0.05, **p < 0.01, two-tailed unpaired t test was used for all comparisons.

Mentions: The MMTV-NEU-NT tumour cell line continued to overexpress NEU/HER2 in cell culture and this expression was largely abolished by 17-AAG treatment, as shown by western blotting (Figure 5A). The mean normalized band intensity of NEU/HER2 decreased from 0.34 ± 0.04 to 0.05 ± 0.005 (p < 0.001). There were also significant decreases in the normalized band intensity of C-RAF, from 0.53 ± 0.11 to 0.21 ± 0.07 (p < 0.05), and of cyclin D1, from 1.21 ± 0.1 to 0.85 ± 0.04 (p < 0.03). Normalized intensity changes of B-RAF (1.96 ± 0.16 to 1.84 ± 0.12) and CDK4 (0.88 ± 0.1 to 0.79 ± 0.05) were not significant. Treatment with 17-AAG also induced HSP72 expression, the normalized band intensity of which increased from 0.35 ± 0.04 to 0.97 ± 0.05 (p < 0.0001). Quantitation of band intensity is expressed relative to GAPDH, mean ± sem, n = 5 (Figure 5A). These results provide molecular evidence that HSP90 was inhibited in the 17-AAG-treated cultured mammary tumour cells, including marked depletion of NEU/HER2.


Effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on NEU/HER2 overexpressing mammary tumours in MMTV-NEU-NT mice monitored by Magnetic Resonance Spectroscopy.

Rodrigues LM, Chung YL, Al Saffar NM, Sharp SY, Jackson LE, Banerji U, Stubbs M, Leach MO, Griffiths JR, Workman P - BMC Res Notes (2012)

Effect of 17-AAG on cultured cells isolated from a MMTV-NEU-NT solid tumour. Cells treated for 24hr at 37°C with vehicle (control) or 1.12μM 17-AAG. A) Western blots of NEU/HER2, B-RAF, C-RAF, HSP72, CDK4 and cyclin D1 in vehicle treated (lanes 1,3,5) and 17-AAG treated (lanes 2,4,6) cells. Intensity of bands were quantitated relative to GAPDH, (n = 5). B)31P MR spectra of cell extracts from control cells (bottom), or cells following 17-AAG treatment (top). Peak assignments are: phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate (Pi), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (α-, β-, γ-NTP). Results were confirmed in six separate experiments, and representative spectra are shown. C) Percentage changes (relative to control) in cell number, phosphoethanolamine (PE), phosphocholine (PC), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (β-NTP), phosphomonoesters (PME = PE + PC), PC/GPC, phosphodiesters (PDE = GPE + GPC) and PDE/βNTP levels in MMTV-NEU-NT tumour cells following treatment with 17-AAG (1.12 μM) for 24h. Results are presented as the mean ± SEM (error bars) of six separate experiments. *p ≤ 0.05, **p < 0.01, two-tailed unpaired t test was used for all comparisons.
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Figure 5: Effect of 17-AAG on cultured cells isolated from a MMTV-NEU-NT solid tumour. Cells treated for 24hr at 37°C with vehicle (control) or 1.12μM 17-AAG. A) Western blots of NEU/HER2, B-RAF, C-RAF, HSP72, CDK4 and cyclin D1 in vehicle treated (lanes 1,3,5) and 17-AAG treated (lanes 2,4,6) cells. Intensity of bands were quantitated relative to GAPDH, (n = 5). B)31P MR spectra of cell extracts from control cells (bottom), or cells following 17-AAG treatment (top). Peak assignments are: phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate (Pi), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (α-, β-, γ-NTP). Results were confirmed in six separate experiments, and representative spectra are shown. C) Percentage changes (relative to control) in cell number, phosphoethanolamine (PE), phosphocholine (PC), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), phosphocreatine (PCr) and nucleotide triphosphate (β-NTP), phosphomonoesters (PME = PE + PC), PC/GPC, phosphodiesters (PDE = GPE + GPC) and PDE/βNTP levels in MMTV-NEU-NT tumour cells following treatment with 17-AAG (1.12 μM) for 24h. Results are presented as the mean ± SEM (error bars) of six separate experiments. *p ≤ 0.05, **p < 0.01, two-tailed unpaired t test was used for all comparisons.
Mentions: The MMTV-NEU-NT tumour cell line continued to overexpress NEU/HER2 in cell culture and this expression was largely abolished by 17-AAG treatment, as shown by western blotting (Figure 5A). The mean normalized band intensity of NEU/HER2 decreased from 0.34 ± 0.04 to 0.05 ± 0.005 (p < 0.001). There were also significant decreases in the normalized band intensity of C-RAF, from 0.53 ± 0.11 to 0.21 ± 0.07 (p < 0.05), and of cyclin D1, from 1.21 ± 0.1 to 0.85 ± 0.04 (p < 0.03). Normalized intensity changes of B-RAF (1.96 ± 0.16 to 1.84 ± 0.12) and CDK4 (0.88 ± 0.1 to 0.79 ± 0.05) were not significant. Treatment with 17-AAG also induced HSP72 expression, the normalized band intensity of which increased from 0.35 ± 0.04 to 0.97 ± 0.05 (p < 0.0001). Quantitation of band intensity is expressed relative to GAPDH, mean ± sem, n = 5 (Figure 5A). These results provide molecular evidence that HSP90 was inhibited in the 17-AAG-treated cultured mammary tumour cells, including marked depletion of NEU/HER2.

Bottom Line: At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose.Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment.The data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, UK.

ABSTRACT

Background: The importance of ERBB2/NEU/HER2 in the response of breast tumours to the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin) has been demonstrated in the clinic. ERBB2 is an oncoprotein client that is highly dependent on HSP90. This and other oncogenic client proteins (e.g. B-RAF, C-RAF, ALK and CDK4) are depleted by 17-AAG in both animal tumours and patients. Here we investigate by Magnetic Resonance Spectroscopy (MRS) the metabolic response of 17-AAG in spontaneous, NEU/HER2 driven mammary tumours in transgenic MMTV-NEU-NT mice and in cells isolated and cultured from these tumours.

Methods: Mammary tumours were monitored by 31P MRS in vivo and in tumour extracts, comparing control and 17-AAG treated mice. A cell line derived from NEU/HER2 mammary tumours was also cultured and the effect of 17-AAG was measured by 31P MRS in cell extracts. Molecular biomarkers were assessed by immunoblotting in extracts from cells and tumours. For comparison of tumour volume, metabolite concentrations and Western blot band intensities, two-tailed unpaired t-tests were used.

Results: The NEU/HER2 mammary tumours were very sensitive to 17-AAG and responded in a dose-dependent manner to 3 daily doses of 20, 40 and 80mg/kg of 17-AAG, all of which caused significant regression. At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose. Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment. Western blots confirmed the expected action of 17-AAG in inducing HSP72 and significantly depleting HSP90 client proteins, including NEU/HER2 both in tumours and in isolated cells.

Conclusions: The data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment.

Show MeSH
Related in: MedlinePlus