Limits...
Metabolic biomarkers for response to PI3K inhibition in basal-like breast cancer.

Moestue SA, Dam CG, Gorad SS, Kristian A, Bofin A, Mælandsmo GM, Engebråten O, Gribbestad IS, Bjørkøy G - Breast Cancer Res. (2013)

Bottom Line: Following treatment, basal-like xenografts demonstrated reduced levels of pAktser473 and decreased proliferation.This correlated with metabolic changes, as both MK-2206 and BEZ235 reduced lactate concentration and increased phosphocholine concentration in the basal-like tumors.Long-term treatment with MK-2206 or BEZ235 resulted in significant growth inhibition in basal-like, but not luminal-like, xenografts.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in cancer cells through numerous mutations and epigenetic changes. The recent development of inhibitors targeting different components of the PI3K pathway may represent a valuable treatment alternative. However, predicting efficacy of these drugs is challenging, and methods for therapy monitoring are needed. Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype, frequently associated with PI3K pathway activation. The objectives of this study were to quantify the PI3K pathway activity in tissue sections from xenografts representing basal-like and luminal-like breast cancer before and immediately after treatment with PI3K inhibitors, and to identify metabolic biomarkers for treatment response.

Methods: Tumor-bearing animals (n = 8 per treatment group) received MK-2206 (120 mg/kg/day) or BEZ235 (50 mg/kg/day) for 3 days. Activity in the PI3K/Akt/mammalian target of rapamycin pathway in xenografts and human biopsies was evaluated using a novel method for semiquantitative assessment of Aktser473 phosphorylation. Metabolic changes were assessed by ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy.

Results: Using a novel dual near-infrared immunofluorescent imaging method, basal-like xenografts had a 4.5-fold higher baseline level of pAktser473 than luminal-like xenografts. Following treatment, basal-like xenografts demonstrated reduced levels of pAktser473 and decreased proliferation. This correlated with metabolic changes, as both MK-2206 and BEZ235 reduced lactate concentration and increased phosphocholine concentration in the basal-like tumors. BEZ235 also caused increased glucose and glycerophosphocholine concentrations. No response to treatment or change in metabolic profile was seen in luminal-like xenografts. Analyzing tumor sections from five patients with BLBC demonstrated that two of these patients had an elevated pAktser473 level.

Conclusion: The activity of the PI3K pathway can be determined in tissue sections by quantitative imaging using an antibody towards pAktser473. Long-term treatment with MK-2206 or BEZ235 resulted in significant growth inhibition in basal-like, but not luminal-like, xenografts. This indicates that PI3K inhibitors may have selective efficacy in basal-like breast cancer with increased PI3K signaling, and identifies lactate, phosphocholine and glycerophosphocholine as potential metabolic biomarkers for early therapy monitoring. In human biopsies, variable pAktser473 levels were observed, suggesting heterogeneous PI3K signaling activity in BLBC.

Show MeSH

Related in: MedlinePlus

Plasma membrane-associated pAktser473 is lost in response to targeted inhibition of the phosphatidylinositol 3-kinase pathway. Tumor sections from vehicle control (two upper panels) or MK-2206-treated (lower left panel) or BEZ253-treated (lower right panel) mice were stained with secondary antibodies alone (negative control; upper left panel) or with anti-pAktser473 (red) and total Akt (green). DNA was visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Images were obtained using identical settings in the confocal microscope using a 20× objective. The sections were first imaged with a near-infrared (NIR) scanner and subsequently imaged by confocal microscopy to detect the fluorescent-labeled antibodies in the visible light area. Treatment with MK-2206 or BEZ235 causes a marked reduction of pAktser473 levels, and loss of membrane localization compared with vehicle-treated controls. Numbers above the images refer to the individual tumor bearing mouse. All scale bars = 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3672699&req=5

Figure 3: Plasma membrane-associated pAktser473 is lost in response to targeted inhibition of the phosphatidylinositol 3-kinase pathway. Tumor sections from vehicle control (two upper panels) or MK-2206-treated (lower left panel) or BEZ253-treated (lower right panel) mice were stained with secondary antibodies alone (negative control; upper left panel) or with anti-pAktser473 (red) and total Akt (green). DNA was visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Images were obtained using identical settings in the confocal microscope using a 20× objective. The sections were first imaged with a near-infrared (NIR) scanner and subsequently imaged by confocal microscopy to detect the fluorescent-labeled antibodies in the visible light area. Treatment with MK-2206 or BEZ235 causes a marked reduction of pAktser473 levels, and loss of membrane localization compared with vehicle-treated controls. Numbers above the images refer to the individual tumor bearing mouse. All scale bars = 20 µm.

Mentions: The use of NIR dyes conjugated to the respective secondary antibodies allowed co-staining with secondary antibodies that can be imaged by conventional confocal microscopy. Based on the finding that basal-like xenografts had a significantly elevated pAktser473 level, the subcellular localization of pAktser473 was examined by confocal microscopy. In basal-like control tumors, a clearly elevated plasma membrane-enriched pAktser473 signal was observed. In response to treatment with MK-2206 and BEZ235, this signal was clearly reduced (Figure 3). As for the NIR scanning, we observed an unspecific signal in the 800 nm channel for total Akt that probably represents binding of anti-mouse IgG secondary antibodies to xenograft host immunoglobulins. This unspecific staining seemed to be limited to extracellular space consistent with binding of the secondary antibody to host immunoglobulins. However, there was still a detectable specific intracellular signal for total Akt that was enriched in the plasma membrane in tumors from untreated animals but more diffuse in the cytosol after treatment. No nuclear staining of pAktser473 was observed.


Metabolic biomarkers for response to PI3K inhibition in basal-like breast cancer.

Moestue SA, Dam CG, Gorad SS, Kristian A, Bofin A, Mælandsmo GM, Engebråten O, Gribbestad IS, Bjørkøy G - Breast Cancer Res. (2013)

Plasma membrane-associated pAktser473 is lost in response to targeted inhibition of the phosphatidylinositol 3-kinase pathway. Tumor sections from vehicle control (two upper panels) or MK-2206-treated (lower left panel) or BEZ253-treated (lower right panel) mice were stained with secondary antibodies alone (negative control; upper left panel) or with anti-pAktser473 (red) and total Akt (green). DNA was visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Images were obtained using identical settings in the confocal microscope using a 20× objective. The sections were first imaged with a near-infrared (NIR) scanner and subsequently imaged by confocal microscopy to detect the fluorescent-labeled antibodies in the visible light area. Treatment with MK-2206 or BEZ235 causes a marked reduction of pAktser473 levels, and loss of membrane localization compared with vehicle-treated controls. Numbers above the images refer to the individual tumor bearing mouse. All scale bars = 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672699&req=5

Figure 3: Plasma membrane-associated pAktser473 is lost in response to targeted inhibition of the phosphatidylinositol 3-kinase pathway. Tumor sections from vehicle control (two upper panels) or MK-2206-treated (lower left panel) or BEZ253-treated (lower right panel) mice were stained with secondary antibodies alone (negative control; upper left panel) or with anti-pAktser473 (red) and total Akt (green). DNA was visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Images were obtained using identical settings in the confocal microscope using a 20× objective. The sections were first imaged with a near-infrared (NIR) scanner and subsequently imaged by confocal microscopy to detect the fluorescent-labeled antibodies in the visible light area. Treatment with MK-2206 or BEZ235 causes a marked reduction of pAktser473 levels, and loss of membrane localization compared with vehicle-treated controls. Numbers above the images refer to the individual tumor bearing mouse. All scale bars = 20 µm.
Mentions: The use of NIR dyes conjugated to the respective secondary antibodies allowed co-staining with secondary antibodies that can be imaged by conventional confocal microscopy. Based on the finding that basal-like xenografts had a significantly elevated pAktser473 level, the subcellular localization of pAktser473 was examined by confocal microscopy. In basal-like control tumors, a clearly elevated plasma membrane-enriched pAktser473 signal was observed. In response to treatment with MK-2206 and BEZ235, this signal was clearly reduced (Figure 3). As for the NIR scanning, we observed an unspecific signal in the 800 nm channel for total Akt that probably represents binding of anti-mouse IgG secondary antibodies to xenograft host immunoglobulins. This unspecific staining seemed to be limited to extracellular space consistent with binding of the secondary antibody to host immunoglobulins. However, there was still a detectable specific intracellular signal for total Akt that was enriched in the plasma membrane in tumors from untreated animals but more diffuse in the cytosol after treatment. No nuclear staining of pAktser473 was observed.

Bottom Line: Following treatment, basal-like xenografts demonstrated reduced levels of pAktser473 and decreased proliferation.This correlated with metabolic changes, as both MK-2206 and BEZ235 reduced lactate concentration and increased phosphocholine concentration in the basal-like tumors.Long-term treatment with MK-2206 or BEZ235 resulted in significant growth inhibition in basal-like, but not luminal-like, xenografts.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in cancer cells through numerous mutations and epigenetic changes. The recent development of inhibitors targeting different components of the PI3K pathway may represent a valuable treatment alternative. However, predicting efficacy of these drugs is challenging, and methods for therapy monitoring are needed. Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype, frequently associated with PI3K pathway activation. The objectives of this study were to quantify the PI3K pathway activity in tissue sections from xenografts representing basal-like and luminal-like breast cancer before and immediately after treatment with PI3K inhibitors, and to identify metabolic biomarkers for treatment response.

Methods: Tumor-bearing animals (n = 8 per treatment group) received MK-2206 (120 mg/kg/day) or BEZ235 (50 mg/kg/day) for 3 days. Activity in the PI3K/Akt/mammalian target of rapamycin pathway in xenografts and human biopsies was evaluated using a novel method for semiquantitative assessment of Aktser473 phosphorylation. Metabolic changes were assessed by ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy.

Results: Using a novel dual near-infrared immunofluorescent imaging method, basal-like xenografts had a 4.5-fold higher baseline level of pAktser473 than luminal-like xenografts. Following treatment, basal-like xenografts demonstrated reduced levels of pAktser473 and decreased proliferation. This correlated with metabolic changes, as both MK-2206 and BEZ235 reduced lactate concentration and increased phosphocholine concentration in the basal-like tumors. BEZ235 also caused increased glucose and glycerophosphocholine concentrations. No response to treatment or change in metabolic profile was seen in luminal-like xenografts. Analyzing tumor sections from five patients with BLBC demonstrated that two of these patients had an elevated pAktser473 level.

Conclusion: The activity of the PI3K pathway can be determined in tissue sections by quantitative imaging using an antibody towards pAktser473. Long-term treatment with MK-2206 or BEZ235 resulted in significant growth inhibition in basal-like, but not luminal-like, xenografts. This indicates that PI3K inhibitors may have selective efficacy in basal-like breast cancer with increased PI3K signaling, and identifies lactate, phosphocholine and glycerophosphocholine as potential metabolic biomarkers for early therapy monitoring. In human biopsies, variable pAktser473 levels were observed, suggesting heterogeneous PI3K signaling activity in BLBC.

Show MeSH
Related in: MedlinePlus