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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.

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pAktser473 is elevated in a subset of clinical samples of basal-like breast cancer. (A) Sections from five clinical samples classified as basal-like breast cancer were immunostained for total Akt (green, 800 nm) or pAktser473 (red, 700 nm) and scanned using the near-infrared (NIR) scanner. The sample from Case 129 contains both normal tissue (NT) and cancerous tissue (CT). In Case 72, there was a longitudinal structure in the section that contained skin and resulted in a structure that is faintly visible in the negative control and also demonstrated elevated signal of both total Akt and pAktser473. The samples from the last three cases were classified as homogeneous basal-like breast cancer (BLBC). Scale bar = 5 mm. (B) Quantification of the pAktser473 signal relative to the total Akt signal in the different clinical BLBC samples. For Case 129, the pAktser473 was quantified both in the NT and in the CT. Quantifications were done in three to five randomly selected circular regions of interest of the tumors and presented as the mean relative intensity in the different areas with standard deviations. (C) The pAktser473 signal is mainly located to the plasma membrane of the cancer cells in BLBC. Imaged area is from the area labeled with an arrowhead in (A). Scale bar = 20 μm.
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Figure 4: pAktser473 is elevated in a subset of clinical samples of basal-like breast cancer. (A) Sections from five clinical samples classified as basal-like breast cancer were immunostained for total Akt (green, 800 nm) or pAktser473 (red, 700 nm) and scanned using the near-infrared (NIR) scanner. The sample from Case 129 contains both normal tissue (NT) and cancerous tissue (CT). In Case 72, there was a longitudinal structure in the section that contained skin and resulted in a structure that is faintly visible in the negative control and also demonstrated elevated signal of both total Akt and pAktser473. The samples from the last three cases were classified as homogeneous basal-like breast cancer (BLBC). Scale bar = 5 mm. (B) Quantification of the pAktser473 signal relative to the total Akt signal in the different clinical BLBC samples. For Case 129, the pAktser473 was quantified both in the NT and in the CT. Quantifications were done in three to five randomly selected circular regions of interest of the tumors and presented as the mean relative intensity in the different areas with standard deviations. (C) The pAktser473 signal is mainly located to the plasma membrane of the cancer cells in BLBC. Imaged area is from the area labeled with an arrowhead in (A). Scale bar = 20 μm.

Mentions: NIR scanning and confocal microscopy demonstrated an elevated level of pAktser473 in the MAS98.12 basal-like xenograft model. To see whether this animal model is representative for BLBC, we determined the level of pAktser473 in tumor sections from five human BLBC biopsies. As expected, we observed that there was very little unspecific signal in the absence of the primary antibodies (Figure 4A). This highly reduced background in the clinical BLBC samples is very probably due to the absence of mouse immunoglobulins that can bind the secondary antibodies and give an unspecific signal. The sample that demonstrated strongest pAktser473 signal contained both normal and cancerous tissue. Importantly, the pAktser473 signal was found elevated only in the part of the sample that contained tumor cells.


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)

pAktser473 is elevated in a subset of clinical samples of basal-like breast cancer. (A) Sections from five clinical samples classified as basal-like breast cancer were immunostained for total Akt (green, 800 nm) or pAktser473 (red, 700 nm) and scanned using the near-infrared (NIR) scanner. The sample from Case 129 contains both normal tissue (NT) and cancerous tissue (CT). In Case 72, there was a longitudinal structure in the section that contained skin and resulted in a structure that is faintly visible in the negative control and also demonstrated elevated signal of both total Akt and pAktser473. The samples from the last three cases were classified as homogeneous basal-like breast cancer (BLBC). Scale bar = 5 mm. (B) Quantification of the pAktser473 signal relative to the total Akt signal in the different clinical BLBC samples. For Case 129, the pAktser473 was quantified both in the NT and in the CT. Quantifications were done in three to five randomly selected circular regions of interest of the tumors and presented as the mean relative intensity in the different areas with standard deviations. (C) The pAktser473 signal is mainly located to the plasma membrane of the cancer cells in BLBC. Imaged area is from the area labeled with an arrowhead in (A). Scale bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: pAktser473 is elevated in a subset of clinical samples of basal-like breast cancer. (A) Sections from five clinical samples classified as basal-like breast cancer were immunostained for total Akt (green, 800 nm) or pAktser473 (red, 700 nm) and scanned using the near-infrared (NIR) scanner. The sample from Case 129 contains both normal tissue (NT) and cancerous tissue (CT). In Case 72, there was a longitudinal structure in the section that contained skin and resulted in a structure that is faintly visible in the negative control and also demonstrated elevated signal of both total Akt and pAktser473. The samples from the last three cases were classified as homogeneous basal-like breast cancer (BLBC). Scale bar = 5 mm. (B) Quantification of the pAktser473 signal relative to the total Akt signal in the different clinical BLBC samples. For Case 129, the pAktser473 was quantified both in the NT and in the CT. Quantifications were done in three to five randomly selected circular regions of interest of the tumors and presented as the mean relative intensity in the different areas with standard deviations. (C) The pAktser473 signal is mainly located to the plasma membrane of the cancer cells in BLBC. Imaged area is from the area labeled with an arrowhead in (A). Scale bar = 20 μm.
Mentions: NIR scanning and confocal microscopy demonstrated an elevated level of pAktser473 in the MAS98.12 basal-like xenograft model. To see whether this animal model is representative for BLBC, we determined the level of pAktser473 in tumor sections from five human BLBC biopsies. As expected, we observed that there was very little unspecific signal in the absence of the primary antibodies (Figure 4A). This highly reduced background in the clinical BLBC samples is very probably due to the absence of mouse immunoglobulins that can bind the secondary antibodies and give an unspecific signal. The sample that demonstrated strongest pAktser473 signal contained both normal and cancerous tissue. Importantly, the pAktser473 signal was found elevated only in the part of the sample that contained tumor cells.

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