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

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.

Figure 1

pAkt^ser473 is elevated and phosphatase and tensin homolog lost in basal-like but not luminal-like xenografts. (A) Immunostaining of luminal-like (LL; top) and basal-like (BL; bottom) xenograft tumor sections from the vehicle control group, stained with anti-pAkt^ser473 (red) and anti-pan-Akt (green) antibodies, and secondary antibodies conjugated with near-infrared (NIR) fluorescent dyes (red channel, pAkt^ser473; green channel, pan-Akt). Negative control sections were stained with secondary antibodies alone (left two images in the panels). Tumors were isolated 3 days after the start of the treatment and 5 hours after the last supplementation of the respective drugs. All scale bars = 1 mm. (B) Quantification data from NIR immunofluorescence imaging (n = 4). The signal intensity of the total Akt immunostaining is similar in BL and LL xenografts, whereas the signal for pAkt ^ser473 is approximately fivefold higher in BL compared with LL xenografts. **P <0.0005. (C) Immunoblot analysis of the level of pAkt^ser473, total Akt and phosphatase and tensin homolog (PTEN) in lysates from two different BL and LL cancers. Level of β-actin included as a loading control. Numbers above the images and the lanes in the immunoblot refer to the individual tumor-bearing mouse. (D) Higher phosphatidylinositol 3-kinase (PI3K) signaling activity in BL xenografts was also confirmed by immunoblot analysis of pAkt^thr308 levels.

Figure 2

Three-day treatment of drugs targeting phosphatidylinositol 3-kinase pathway reduces active Akt level in basal-like xenografts. (A) Immunostaining of tissue sections from basal-like (BL; left) and luminal-like (LL; right) xenografts given vehicle control (upper panel), MK-2206 (middle panel) or BEZ253 (lower panel). pAkt^ser473 (red) and total Akt (green) are stained as in Figure 1. All scale bars = 1 mm. (B) Quantification data from near-infrared (NIR) immunofluorescence imaging (n = 4). The signal intensity was significantly reduced after treatment with MK-2206 and BEZ235 in BL but not in LL xenografts. *P <0.05, **P <0.0005. (C) Immunoblot analysis of tumor lysates for the level of pAkt^ser473 (upper panel) and β-actin loading control (lower panel). Lysates were prepared from tumors harvested 3 to 6 hours after the last administration of MK-2206 and BEZ235.

Figure 3

Plasma membrane-associated pAkt^ser473 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-pAkt^ser473 (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 pAkt^ser473 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.

Figure 4

pAkt^ser473 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 pAkt^ser473 (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 pAkt^ser473. The samples from the last three cases were classified as homogeneous basal-like breast cancer (BLBC). Scale bar = 5 mm. (B) Quantification of the pAkt^ser473 signal relative to the total Akt signal in the different clinical BLBC samples. For Case 129, the pAkt^ser473 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 pAkt^ser473 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.

Figure 5

Reduced pAkt^ser473 levels are correlated with tumor growth inhibition and reduced proliferation in basal-like xenografts. (A) Mitotic activity (phosphohistone H3 (PHH3) counts/10 fields of view) in basal-like and luminal-like xenografts after treatment with MK-2206 and BEZ235. (B) Correlation between pAkt^ser473 signal intensity and mitosis in basal-like xenografts treated with vehicle (open squares), MK-2206 (filled circles) and BEZ235 (open triangles). (C) Tumor volumes (relative to day 0) in basal-like xenografts treated with MK-2206 (filled circles) and BEZ235 (open triangles), compared with vehicle-treated controls (open squares). (D) Tumor volumes (relative to day 0) in luminal-like xenografts treated with MK-2206 (filled circles) and BEZ235 (open triangles), compared with vehicle-treated controls (open squares).

Figure 6

Phosphatidylinositol 3-kinase inhibition induces changes in glucose and choline metabolism. (A), (C) In basal-like xenografts, treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors increased the concentration of phosphocholine (PCho; both MK-2206 and BEZ235) and glycerophosphocholine (GPC; BEZ235 only). BEZ235 caused an increase in the glucose concentration, whereas both MK-2206 and BEZ235 reduced the lactate concentration. (B), (D) No treatment-related changes in these metabolites were observed in luminal-like xenografts. *Significantly different from control (P <0.05).

Figure 7

High-resolution magic angle spinning magnetic resonance spectroscopy demonstrates changes in glucose and choline metabolism. Example spectra from basal-like xenografts illustrate changes in (A) glucose and (B) choline metabolites. BEZ235 treatment (blue spectra) increased the glucose concentration compared with vehicle control (red spectra). A concomitant decrease in lactate concentration was observed. Both glycerophosphocholine (GPC) and phosphocholine (PCho) concentrations were also increased after BEZ235 treatment.