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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 phospholipid related metabolites and Pi by31P MRS in extracts of MMTV-NEU-NT tumors: 3 daily doses of 17-AAG (20, 40, 80 mg/kg) or vehicle only (control) and tumours sampled at 24 hr post 3rddose. Data are expressed as mean ± SEM., n = 3 to 6, **p < 0.05, *p = 0.06 when compared to controls. Phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate (Pi), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC).
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Figure 3: Effect of 17-AAG on phospholipid related metabolites and Pi by31P MRS in extracts of MMTV-NEU-NT tumors: 3 daily doses of 17-AAG (20, 40, 80 mg/kg) or vehicle only (control) and tumours sampled at 24 hr post 3rddose. Data are expressed as mean ± SEM., n = 3 to 6, **p < 0.05, *p = 0.06 when compared to controls. Phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate (Pi), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC).

Mentions: 31P MRS of extracts allows better resolution of the signals than spectra acquired from in vivo tissue. Spectra of extracts of tumours from mice that had received 3 daily doses of 17-AAG were acquired, and individual phospholipid metabolites, i.e. PC, PE, GPC and GPE, were observed. Tumours from mice treated with 3 doses of 80mg/kg 17-AAG showed decreases in PE (p = 0.01), PC (p = 0.06) and [PE + PC] (p = 0.001). At the 40mg/kg dose the sum of [PE + PC] was significantly lower (Figure 3) when compared with vehicle-treated controls (p = 0.05). GPC and GPE levels were not significantly different at any of the 17-AAG dose levels when compared with vehicle-treated controls (Figure 3). 1H MRS of the extracts confirmed a significant decrease in PC in a cohort of tumours 24 hrs after treatment with 40mg/kg 17-AAG, but no other significant changes were observed (data not shown).


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 phospholipid related metabolites and Pi by31P MRS in extracts of MMTV-NEU-NT tumors: 3 daily doses of 17-AAG (20, 40, 80 mg/kg) or vehicle only (control) and tumours sampled at 24 hr post 3rddose. Data are expressed as mean ± SEM., n = 3 to 6, **p < 0.05, *p = 0.06 when compared to controls. Phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate (Pi), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3412754&req=5

Figure 3: Effect of 17-AAG on phospholipid related metabolites and Pi by31P MRS in extracts of MMTV-NEU-NT tumors: 3 daily doses of 17-AAG (20, 40, 80 mg/kg) or vehicle only (control) and tumours sampled at 24 hr post 3rddose. Data are expressed as mean ± SEM., n = 3 to 6, **p < 0.05, *p = 0.06 when compared to controls. Phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate (Pi), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC).
Mentions: 31P MRS of extracts allows better resolution of the signals than spectra acquired from in vivo tissue. Spectra of extracts of tumours from mice that had received 3 daily doses of 17-AAG were acquired, and individual phospholipid metabolites, i.e. PC, PE, GPC and GPE, were observed. Tumours from mice treated with 3 doses of 80mg/kg 17-AAG showed decreases in PE (p = 0.01), PC (p = 0.06) and [PE + PC] (p = 0.001). At the 40mg/kg dose the sum of [PE + PC] was significantly lower (Figure 3) when compared with vehicle-treated controls (p = 0.05). GPC and GPE levels were not significantly different at any of the 17-AAG dose levels when compared with vehicle-treated controls (Figure 3). 1H MRS of the extracts confirmed a significant decrease in PC in a cohort of tumours 24 hrs after treatment with 40mg/kg 17-AAG, but no other significant changes were observed (data not shown).

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