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In this study, we probed the importance of O-GlcNAc transferase (OGT) activity for the survival of tamoxifen-sensitive (TamS) and tamoxifen-resistant (TamR) breast cancer cells. Tamoxifen is an antagonist of estrogen receptor (ERα), a transcription factor expressed in over 50% of breast cancers. ERα-positive breast cancers are successfully treated with tamoxifen; however, a significant number of patients develop tamoxifen-resistant disease. We show that in vitro development of tamoxifen-resistance is associated with increased sensitivity to the OGT small molecule inhibitor OSMI-1. Global transcriptome profiling revealed that TamS cells adapt to OSMI-1 treatment by increasing the expression of histone genes. This is known to mediate chromatin compaction. In contrast, TamR cells respond to OGT inhibition by activating the unfolded protein response and by significantly increasing ERRFI1 expression. ERRFI1 is an endogenous inhibitor of ERBB-signaling, which is a known driver of tamoxifen-resistance. We show that ERRFI1 is selectively downregulated in ERα-positive breast cancers and breast cancers driven by ERBB2. This likely occurs via promoter methylation. Finally, we show that increased ERRFI1 expression is associated with extended survival in patients with ERα-positive tumors (p = 9.2e−8). In summary, we show that tamoxifen-resistance is associated with sensitivity to OSMI-1, and propose that this is explained in part through an epigenetic activation of the tumor-suppressor ERRFI1 in response to OSMI-1 treatment. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Epithelial-to-mesenchymal transition (EMT) is fundamental to both normal tissue development and cancer progression. We hypothesized that EMT plasticity defines a range of metabolic phenotypes and that individual breast epithelial metabolic phenotypes are likely to fall within this phenotypic landscape. To determine EMT metabolic phenotypes, the metabolism of EMT was described within genome-scale metabolic models (GSMMs) using either transcriptomic or proteomic data from the breast epithelial EMT cell culture model D492. The ability of the different data types to describe breast epithelial metabolism was assessed using constraint-based modeling which was subsequently verified using 13C isotope tracer analysis. The application of proteomic data to GSMMs provided relatively higher accuracy in flux predictions compared to the transcriptomic data. Furthermore, the proteomic GSMMs predicted altered cholesterol metabolism and increased dependency on argininosuccinate lyase (ASL) following EMT which were confirmed in vitro using drug assays and siRNA knockdown experiments. The successful verification of the proteomic GSMMs afforded iBreast2886, a breast GSMM that encompasses the metabolic plasticity of EMT as defined by the D492 EMT cell culture model. Analysis of breast tumor proteomic data using iBreast2886 identified vulnerabilities within arginine metabolism that allowed prognostic discrimination of breast cancer patients on a subtype-specific level. Taken together, we demonstrate that the metabolic reconstruction iBreast2886 formalizes the metabolism of breast epithelial cell development and can be utilized as a tool for the functional interpretation of high throughput clinical data.

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects.

The association between lipid metabolism and long-term outcomes is relevant for tumor diagnosis and therapy. Archival material such as formalin-fixed and paraffin embedded (FFPE) tissues is a highly valuable resource for this aim as it is linked to long-term clinical follow-up. Therefore, there is a need to develop robust methodologies able to detect lipids in FFPE material and correlate them with clinical outcomes. In this work, lipidic alterations were investigated in patient-derived xenograft of breast cancer by using a matrix-assisted laser desorption ionization mass spectrometry (MALDI MSI) based workflow that included antigen retrieval as a sample preparation step. We evaluated technical reproducibility, spatial metabolic differentiation within tissue compartments, and treatment response induced by a glutaminase inhibitor (CB-839). This protocol shows a good inter-day robustness (CV = 26 ± 12%). Several lipids could reliably distinguish necrotic and tumor regions across the technical replicates. Moreover, this protocol identified distinct alterations in the tissue lipidome of xenograft treated with glutaminase inhibitors. In conclusion, lipidic alterations in FFPE tissue of breast cancer xenograft observed in this study are a step-forward to a robust and reproducible MALDI-MSI based workflow for pre-clinical and clinical applications.

The editors are publishing this note to alert readers to concerns about [this article][1] ([1][2]). The editors were made aware of duplicated Western blot bands in Fig. 4A. Specifically, two sets of Western blot bands are duplicates: (i) T47D CCTα in the top right panel and T47D cadherin in the

Post-translational modification of intracellular proteins with a single N-acetylglucosamine sugar (O-GlcNAcylation) regulates signaling, proliferation, metabolism and protein stability. In breast cancer, expression of the enzyme that catalyzes O-GlcNAcylation – O-GlcNAc-transferase (OGT), and the extent of protein O-GlcNAcylation, are upregulated in tumor tissue, and correlate with cancer progression. Here we compare the significance of O-GlcNAcylation in a panel of breast cancer cells of different phenotypes. We find a greater dependency on OGT among triple-negative breast cancer (TNBC) cell lines, which respond to OGT inhibition by undergoing cell cycle arrest and apoptosis. Searching for the cause of this response, we evaluate the changes in the proteome that occur after OGT inhibition or knock-down, employing a reverse-phase protein array (RPPA). We identify transcriptional repressor - hairy and enhancer of split-1 (HES1) - as a mediator of the OGT inhibition response in the TNBC cells. Inhibition of OGT as well as the loss of HES1 results in potent cytotoxicity and apoptosis. The study raises a possibility of using OGT inhibition to potentiate DNA damage in the TNBC cells. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Introduction Glutaminase inhibitors target cancer cells by blocking the conversion of glutamine to glutamate, thereby potentially interfering with anaplerosis and synthesis of amino acids and glutathione. The drug CB-839 has shown promising effects in preclinical experiments and is currently undergoing clinical trials in several human malignancies, including triple-negative breast cancer (TNBC). However, response to glutaminase inhibitors is variable and there is a need for identification of predictive response biomarkers. The aim of this study was to determine how glutamine is utilized in two patient-derived xenograft (PDX) models of breast cancer representing luminal-like/ER+ (MAS98.06) and basal-like/triple-negative (MAS98.12) breast cancer and to explore the metabolic effects of CB-839 treatment. Experimental MAS98.06 and MAS98.12 PDX mice received CB-839 (200 mg/kg) or drug vehicle two times daily p.o. for up to 28 days (n = 5 per group), and the effect on tumor growth was evaluated. Expression of 60 genes and seven glutaminolysis key enzymes were determined using gene expression microarray analysis and immunohistochemistry (IHC), respectively, in untreated tumors. Uptake and conversion of glutamine were determined in the PDX models using HR MAS MRS after i.v. infusion of [5-13C] glutamine when the models had received CB-839 (200 mg/kg) or vehicle for 2 days (n = 5 per group). Results Tumor growth measurements showed that CB-839 significantly inhibited tumor growth in MAS98.06 tumors, but not in MAS98.12 tumors. Gene expression and IHC analysis indicated a higher proline synthesis from glutamine in untreated MAS98.06 tumors. This was confirmed by HR MAS MRS of untreated tumors demonstrating that MAS98.06 used glutamine to produce proline, glutamate, and alanine, and MAS98.12 to produce glutamate and lactate. In both models, treatment with CB-839 resulted in accumulation of glutamine. In addition, CB-839 caused depletion of alanine, proline, and glutamate ([1-13C] glutamate) in the MAS98.06 model. Conclusion Our findings indicate that TNBCs may not be universally sensitive to glutaminase inhibitors. The major difference in the metabolic fate of glutamine between responding MAS98.06 xenografts and non-responding MAS98.12 xenografts is the utilization of glutamine for production of proline. We therefore suggest that addiction to proline synthesis from glutamine is associated with response to CB-839 in breast cancer. Graphical abstract The effect of glutaminase inhibition in two breast cancer patient-derived xenograft (PDX) models. 13C HR MAS MRS analysis of tumor tissue from CB-839-treated and untreated models receiving 13C-labeled glutamine ([5-13C] Gln) shows that the glutaminase inhibitor CB-839 is causing an accumulation of glutamine (arrow up) in two PDX models representing luminal-like breast cancer (MAS98.06) and basal-like breast cancer (MAS98.12). In MAS98.06 tumors, CB-839 is in addition causing depletion of proline ([5-13C] Pro), alanine ([1-13C] Ala), and glutamate ([1-13C] Glu), which could explain why CB-839 causes tumor growth inhibition in MAS98.06 tumors, but not in MAS98.12 tumors.

Purpose To compare the effects of two anti-angiogenic drugs, bevacizumab and a cytosolic phospholipase A2-α inhibitor (AVX235), on the relationship between vascular structure and dynamic contrast enhanced (DCE)-MRI measurements in a patient-derived breast cancer xenograft model. Methods Mice bearing MAS98.12 tumors were randomized into three groups: bevacizumab-treated (n = 9), AVX235-treated (n = 9), and control (n = 8). DCE-MRI was performed pre- and post-treatment. Median initial area under the concentration-time curve (IAUC60 ) and volume transfer constant (Ktrans ) were computed for each tumor. Tumors were excised for ex vivo micro-CT (computed tomography) angiography, from which the vascular surface area (VSA) and fractional blood volume (FBV) were computed. Spearman correlation coefficients (ρ) were computed to evaluate the associations between the DCE-MRI and micro-CT parameters. Results With the groups pooled, IAUC60 and Ktrans correlated significantly with VSA (ρ = 0.475 and 0.527; P = 0.019 and 0.008). There were no significant correlations within the control group. There were various significant correlations within the treatment groups, but the correlations in the bevacizumab group were of opposite sign, for example, Ktrans versus FBV: AVX235, ρ = 0.800 (P = 0.014); bevacizumab, ρ = -0.786 (P = 0.023). Conclusion DCE-MRI measurements can highly depend on vascular structure. The relationship between vascular structure and function changed markedly after anti-angiogenic treatment. Magn Reson Med 78:1513-1522, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Estrogen receptor α (ERα) is a key regulator of breast growth and breast cancer development. Here, we report how ERα impacts these processes by reprogramming metabolism in malignant breast cells. We employed an integrated approach, combining genome-wide mapping of chromatin-bound ERα with estrogen-induced transcript and metabolic profiling, to demonstrate that ERα reprograms metabolism upon estrogen stimulation, including changes in aerobic glycolysis, nucleotide and amino acid synthesis, and choline (Cho) metabolism. Cho phosphotransferase CHPT1, identified as a direct ERα-regulated gene, was required for estrogen-induced effects on Cho metabolism, including increased phosphatidylcholine synthesis. CHPT1 silencing inhibited anchorage-independent growth and cell proliferation, also suppressing early-stage metastasis of tamoxifen-resistant breast cancer cells in a zebrafish xenograft model. Our results showed that ERα promotes metabolic alterations in breast cancer cells mediated by its target CHPT1, which this study implicates as a candidate therapeutic target. Cancer Res; 76(19); 5634–46. ©2016 AACR.

Cytosolic phospholipase A2α (cPLA2α) has been shown to be elevated in breast cancer and is a potential biomarker in the differentiation of molecular sub-types. Using a cPLA2α activatable fluorophore, DDAO arachidonate, we explore its ability to function as a contrast agent in fluorescence-guided surgery. In cell lines ranging in cPLA2α expression and representing varying breast cancer sub-types, we show DDAO arachidonate activates with a high correlation to cPLA2α expression level. Using a control probe, DDAO palmitate, in addition to cPLA2α inhibition and genetic knockdown, we show that this activation is a result of cPLA2α activity. In mouse models, using an ex vivo tumor painting technique, we show that DDAO arachidonate activates to a high degree in basal-like versus luminal-like breast tumors and healthy mammary tissue. Finally, we show that using an in vivo model, orthotopic basal-like tumors give significantly high probe activation compared to healthy mammary fat pads and surrounding tissue. Together we conclude that cPLA2α activatable fluorophores such as DDAO arachidonate may serve as a useful contrast agent for the visualization of tumor margins in the fluorescence-guided surgery of basal-like breast cancer. © 2019 The Author(s). Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License

Patients with triple-negative breast cancer (TNBC) are unresponsive to endocrine and anti-HER2 pharmacotherapy, limiting their therapeutic options to chemotherapy. TNBC is frequently associated with abnormalities in the PI3K/AKT/mTOR signaling pathway; drugs targeting this pathway are currently being evaluated in these patients. However, the response is variable, partly due to heterogeneity within TNBC, conferring a need to identify biomarkers predicting response and resistance to targeted therapy. In this study, we used a metabolomics approach to assess response to the mTOR inhibitor everolimus in a panel of TNBC patient-derived xenografts (PDX) (n = 103 animals). Tumor metabolic profiles were acquired using high-resolution magic angle spinning magnetic resonance spectroscopy. Partial least-squares-discriminant analysis on relative metabolite concentrations discriminated treated xenografts from untreated controls with an accuracy of 67% (p = 0.003). Multilevel linear mixed-effects models (LMM) indicated reduced glycolytic lactate production and glutaminolysis after treatment, consistent with PI3K/AKT/mTOR pathway inhibition. Although inherent metabolic heterogeneity between different PDX models seemed to hinder prediction of treatment response, the metabolic effects following treatment were more pronounced in responding xenografts compared to nonresponders. Additionally, the metabolic information predicted p53 mutation status, which may provide complementary insight into the interplay between PI3K signaling and other drivers of disease progression. Copyright © 2017 American Chemical Society. This is the authors' accepted and refereed manuscript to the article. Locked until 14 March 2018 due to copyright restrictions

To study cancer associated with abnormal metabolism of phospholipids, of which several have been proposed as biomarkers for malignancy or to monitor response to anticancer therapy. We explored 3D (31) P magnetic resonance spectroscopic imaging (MRSI) at high magnetic field for in vivo assessment of individual phospholipids in two patient-derived breast cancer xenografts representing good and poor prognosis (luminal- and basal-like tumors).Metabolic profiles from luminal-like and basal-like xenograft tumors were obtained in vivo using 3D (31) P MRSI at 11.7T and from tissue extracts in vitro at 14.1T. Gene expression analysis was performed in order to support metabolic differences between the two xenografts.In vivo (31) P MR spectra were obtained in which the prominent resonances from phospholipid metabolites were detected at a high signal-to-noise ratio (SNR >7.5). Metabolic profiles obtained in vivo were in agreement with those obtained in vitro and could be used to discriminate between the two xenograft models, based on the levels of phosphocholine, phosphoethanolamine, glycerophosphocholine, and glycerophosphoethanolamine. The differences in phospholipid metabolite concentration could partly be explained by gene expression profiles.Noninvasive metabolic profiling by 3D (31) P MRSI can discriminate between subtypes of breast cancer based on different concentrations of choline- and ethanolamine-containing phospholipids.J. Magn. Reson. Imaging 2014. © 2014 Wiley Periodicals, Inc.

Purpose To investigate the effect of bevacizumab treatment on vascular architecture and function in two xenograft models with different angiogenic properties using diffusion-weighted magnetic resonance imaging (DW-MRI) and dynamic contrast-enhanced MRI (DCE-MRI). Materials and Methods Mice carrying basal-like (MAS98.12) or luminal-like (MAS98.06) orthotopic breast cancer xenografts were treated with bevacizumab (5 mg/kg), doxorubicin (8 mg/kg), or both drugs in combination. DW-MRI and DCE-MRI were performed before and 3 days after treatment using a Bruker 7T preclinical scanner. Mean microvessel density (MVD) and proliferating microvessel density (pMVD) in the tumors were determined for evaluation of vascular response to bevacizumab treatment. Results No changes in DCE-MRI or DW-MRI parameters were observed in untreated controls during the experiment period. DW-MRI showed increased apparent diffusion coefficient (ADC) values in all treatment groups in both basal-like and luminal-like xenografts. DCE-MRI showed increased contrast agent uptake, particularly in central regions of the tumors, after bevacizumab/combination treatment in both xenograft models. This was accompanied by decreased MVD and pMVD in basal-like xenografts. Doxorubicin treatment had no effect on DCE-MRI parameters in any of the xenograft models. Conclusion Both DW-MRI and DCE-MRI demonstrated an early response to bevacizumab treatment in the xenograft tumors. Increased contrast agent uptake and reduced MVD/pMVD is consistent with a normalization of vascular function. J. Magn. Reson. Imaging 2013;38:1043–1053. © 2013 Wiley Periodicals, Inc.

Background: The phosphoinositide-3 kinase (PI3K) pathway is an attractive therapeutic target. However, difficulty in predicting therapeutic response limits the clinical implementation of PI3K inhibitors. This study evaluates the utility of clinically relevant magnetic resonance imaging (MRI) biomarkers for noninvasively assessing the in vivo response to the dual PI3K/mTOR inhibitor BEZ235 in two ovarian cancer models with differential PI3K pathway activity. Methods: The PI3K signalling activity of TOV-21G and TOV-112D human ovarian cancer cells was investigated in vitro. Cellular and vascular response of the xenografts to BEZ235 treatment (65 mg kg−1, 3 days) was assessed in vivo using diffusion-weighted (DW) and dynamic contrast-enhanced (DCE)-MRI. Micro-computed tomography was performed to investigate changes in vascular morphology. Results: The TOV-21G cells showed higher PI3K signalling activity than TOV-112D cells in vitro and in vivo. Treated TOV-21G xenografts decreased in volume and DW-MRI revealed an increased water diffusivity that was not found in TOV-112D xenografts. Treatment-induced improvement in vascular functionality was detected with DCE-MRI in both models. Changes in vascular morphology were not found. Conclusions: Our results suggest that DW- and DCE-MRI can detect cellular and vascular response to PI3K/mTOR inhibition in vivo. However, only DW-MRI could discriminate between a strong and weak response to BEZ235. From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/

O-linked N-acetyl-glucosamine transferase (OGT) is an enzyme that catalyzes addition of the O-GlcNAc modification to a wide range of intracellular proteins. The O-GlcNAc modification is a product of the hexosamine biosynthetic pathway, which requires glucose and glutamine as substrates. Uptake of both of these nutrients is often up-regulated in cancer, which in turn leads to an increase in the total protein O-GlcNAcylation. Increased OGT expression has also been reported in most cancer types, including the most frequently diagnosed cancer in women, breast cancer. Many of the breast cancers rely on estrogen receptor alpha (ERα) for proliferation and have shown a strong response to the ERα inhibition, most commonly achieved by treatment with tamoxifen. However, while efficient, prolonged exposure to tamoxifen commonly causes resistance and relapse of the disease. It is therefore vital to uncover mechanisms which contribute to the resistance in order to develop adequate treatment strategy for these patients. Here, we have investigated the effect of targeting OGT in an isogenic pair of ERα-positive tamoxifen-sensitive MCF7 and tamoxifen-resistant TAMR breast cancer cell lines. OGT inhibition decreased viability and triggered cell death in both cell lines. These responses were associated with over 50% reduction in ERα expression in both MCF7 and TAMR cells. Reduced O-GlcNAcylation has previously been reported to induce endoplasmic reticulum stress and activation of transcription factor C/EBP homologous protein (CHOP), which promotes cell death. Targeting OGT resulted in a strong increase of CHOP expression, which appeared more prominent in the TAMR cells. Finally, targeting OGT induced a very pronounced cell cycle arrest in the G2/M phase in the TAMR cells, while the MCF7 cell lined showed a very modest response. Taken together, these results indicate that targeting OGT leads to a differential response in the tamoxifen-sensitive and resistant breast cancer cells. Currently, we are using an expanded panel of tamoxifen-resistant cell lines to perform expression microarrays, metabolic flux assays and DNA damage response analysis in order to uncover the cause of the differential response to OGT targeting. This may help us identify potential therapeutic combinations that might be suitable for treatment of tamoxifen-resistant cancers. Citation Format: Anna Barkovskaya, Lina Prasmickaite, Ian G. Mills, Gunhild M. Mælandsmo, Siver A. Moestue, Harri M. Itkonen. Inhibition of O-GlcNAc transferase in tamoxifen resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3737.

Stewart et al. (1) recently reported that EDI3 is the glycerophosphodiesterase responsible for breaking down glycerophosphocholine (GPC) to choline and G3P in mammalian cells. The authors further claim that this is of relevance in breast cancer, as this disease, according to the authors, is associated with reduced levels of GPC and a low GPC/phosphocholine (PCho) ratio. By implication, inhibition of EDI3 could increase the concentration of GPC, restore a normal GPC/PCho ratio, and reduce metastatic potential. In our opinion, this hypothesis, which is based on studies of cultured breast cancer cells, does not take recent clinical findings into account. In a cohort of 156 patients, our group has found that estrogen receptor-negative breast cancer is associated with low PCho and high GPC concentrations (2). Furthermore, we have demonstrated that a basal-like patient-derived animal model had a remarkably high GPC/PCho ratio (3), which was corroborated by analysis of tumor tissue from patients with triple-negative breast cancer. These findings clearly indicate that the most aggressive breast cancer subtypes have higher GPC concentration than subtypes with a better prognosis. Furthermore, we have studied the role of GPC as a biomarker for response to therapy. In breast cancer biopsy specimens collected from patients receiving neoadjuvant chemotherapy, we have found that a favorable response to treatment, in fact, is associated with a reduction in GPC concentration during the course of treatment (4). Finally, we have shown that reduction in GPC concentration after neoadjuvant chemotherapy is associated with long-term survival in this patient group (5). In summary, high GPC concentration and a GPC/PCho ratio >1 is associated with aggressive subtypes of breast cancer. Furthermore, a reduction in GPC levels can be predictive of response to treatment. These findings are not consistent with the hypothesis that restoration of a high GPC/PCho ratio would be beneficial in breast cancer, which forms the basis for the work of Stewart et al. (1). The abnormal metabolism of breast cancer, not surprisingly, reflects the heterogeneity of the disease. The complex role of GPC remains poorly understood, but it has become increasingly clear that high, rather than low, GPC concentrations are associated with poor prognosis in breast cancer. Thus, in our opinion, the available body of data is not taken into account. Pursuing the findings in the article (1) can, of course, lead to discovery of novel drug targets. However, research based on a hypothesis that is not consistent with clinical findings may be considered a suboptimal approach for targeting the abnormal choline metabolism in breast cancer. Inhibition of EDI3 could surely have inhibitory effects on breast cancer cells, as interference with choline phospholipid metabolism repeatedly has been associated with such effects. We therefore await further progress from Stewart et al. in this field in great anticipation, and hope that further elucidation of GPC metabolism can lead to findings of new prognostic biomarkers or drug targets.

Source at https://doi.org/10.1016/j.molonc.2012.10.005. Accepted manuscript version, licensed CC BY-NC-ND 4.0. Antiangiogenic therapy with bevacizumab has shown varying results in breast cancer clinical trials. Identifying robust biomarkers for selecting patients who may benefit from such treatment and for monitoring response is important for the future use of bevacizumab. Two established xenograft models representing basal‐like and luminal‐like breast cancer were used to study bevacizumab treatment response on the metabolic and gene expression levels. Tumor samples were obtained from mice treated with bevacizumab, doxorubicin or a combination of the two drugs, and high resolution magic angle spinning magnetic resonance spectroscopy and gene expression microarray analysis was performed. Combination treatment with bevacizumab showed the strongest growth inhibiting effect in basal‐like tumors, and this was reflected by a significant change in the metabolomic and transcriptomic profiles. In the luminal‐like xenografts, addition of bevacizumab did not improve the effect of doxorubicin. On the global transcriptomic level, the largest gene expression changes were observed for the most efficient treatment in both models. Glycerophosphocholine showed opposite response in the treated xenografts compared with untreated controls; lower in basal‐like and higher in luminal‐like tumors. Comparing combination therapy with doxorubicin monotherapy in basal‐like xenografts, 14 genes showed significant differential expression, including very low density lipoprotein receptor (VLDLR) and hemoglobin, theta 1 (HBQ1). Bevacizumab‐treated tumors were associated with a more hypoxic phenotype, while no evidence was found for associations between bevacizumab treatment and vascular invasion or tumor grade. This study underlines the importance of characterizing biological differences between subtypes of breast cancer to identify personalized biomarkers for improved patient stratification and evaluation of response to therapy.

Breast cancer is the most frequent cancer among women in Norway. The outcome of this disease is heterogeneous. Some patients have slowly growing tumors which stay confined within the mammary gland, whereas others have aggressive tumors that grow rapidly and metastasize to distant tissues. Based on gene expression profiles, breast cancer has been divided into at least five different subtypes with differences in clinical characteristics and prognosis. Improved understanding of the biology of these subtypes and how they should be treated is needed to benefit from this molecular subtyping in breast cancer diagnosis and treatment. The motivation for this work has been to map differences in breast cancer metabolism and vascularization using different MR imaging and spectroscopy methods. The work shows how different MR methods can be used to study the biology of animal breast cancer models representing the molecular subgroups luminal-like and basal-like breast cancer. The findings represent MR characteristics in breast cancer with good and poor prognosis, respectively. MR spectroscopy can be used to study metabolism in cells and tissues. Since cancer cells have a large need for energy and molecular building blocks in order to grow fast, they have metabolic properties that differ markedly from healthy cells. The thesis describes how MR-spectroscopy can detect differences in choline metabolism between luminal-like and basal-like breast cancer, and demonstrates how metabolic patterns found in the animal models are representative for findings in clinical samples. Since CCCs are proposed as biomarkers in diagnosis and evaluation of response to therapy in breast cancer, differences in choline metabolism between different molecular subtypes may be of clinical relevance. The thesis also describes how glucose metabolism can be studied using 13C-labeled glucose. Using a stable, MR-detectable isotope allows assessment of the metabolic fate of glucose. Abnormal glucose metabolism is a typical feature in cancer, and is the basis for PET imaging using FDG. There is also great interest for use of hyperpolarized MR spectroscopy for measurement of lactate production in cancer. The method that is established in this work allows ex vivo studies of metabolism and the relationship between gene expression and metabolic rates. In the luminal-like and basal-like animal models, it was found that the least aggressive model had the highest glycolytic rate, suggesting that tumor growth rate and aggressiveness not necessarily is directly linked to glycolytic rate. MR imaging can be used for anatomical imaging of the body, but also for studies of functional properties such as perfusion and cellular density. In tumors, the blood vessels are typically leaky, poorly organized and have suboptimal function. Without sufficient blood supply, tumor growth is limited. The tumor cells’ ability to stimulate growth of new vessels has impact on disease outcome. Vascularization and neoangiogenesis was studied in the luminal-like and basal-like models. Using dynamic contrast-enhanced MR imaging and immunohistochemistry, it was found that the basal-like xenograft model had higher contrast uptake, that it is better vascularised and has more active angiogenesis than the luminal-like xenograft. In a follow-up study, the effect of the antiangiogenic agent bevacizumab was studied. This drug inhibits the formation of new blood vessels and can therefore slow down tumor growth. Shortly after treatment, MR imaging demonstrated increased contrast agent uptake in the tumors. Immunohistochemistry showed a reduction in the number of microvessels and angiogenic activity. These findings were interpreted as normalization of blood vessels and improved vascular function. MR imaging of vascular normalization may have impact on the use of bevacizumab together with other cytotoxic drugs or radiotherapy. The thesis consists of four papers which all describe use of MR in animal xenograft models of luminal-like and basal-like breast cancer. In the two first papers, high resolution ex vivo MR spectroscopy has been combined with gene expression analysis for description of tumor-specific metabolic properties. In the last two papers, dynamic contrast-enhanced MR imaging and pharmacokinetic analysis of contrast agent distribution has been used to describe tumor vascularization and how this respond to antiangiogenic treatment. All four papers describe how different MR techniques can distinguish between animal models of varying aggressiveness. They also show how the MR parameters are associated with molecular or structural properties of the tumors. The thesis may therefore contribute to improved understanding of MR findings in breast cancer diagnosis and treatment. In addition, it forms a basis for further studies of these animal xenograft models of luminal-like and basal-like breast cancer, especially with regard to identification of new treatment regimens and MR imaging for therapy response monitoring.

Background Increased concentrations of choline-containing compounds are frequently observed in breast carcinomas, and may serve as biomarkers for both diagnostic and treatment monitoring purposes. However, underlying mechanisms for the abnormal choline metabolism are poorly understood. Methods The concentrations of choline-derived metabolites were determined in xenografted primary human breast carcinomas, representing basal-like and luminal-like subtypes. Quantification of metabolites in fresh frozen tissue was performed using high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS). The expression of genes involved in phosphatidylcholine (PtdCho) metabolism was retrieved from whole genome expression microarray analyses. The metabolite profiles from xenografts were compared with profiles from human breast cancer, sampled from patients with estrogen/progesterone receptor positive (ER+/PgR+) or triple negative (ER-/PgR-/HER2-) breast cancer. Results In basal-like xenografts, glycerophosphocholine (GPC) concentrations were higher than phosphocholine (PCho) concentrations, whereas this pattern was reversed in luminal-like xenografts. These differences may be explained by lower choline kinase (CHKA, CHKB) expression as well as higher PtdCho degradation mediated by higher expression of phospholipase A2 group 4A (PLA2G4A) and phospholipase B1 (PLB1) in the basal-like model. The glycine concentration was higher in the basal-like model. Although glycine could be derived from energy metabolism pathways, the gene expression data suggested a metabolic shift from PtdCho synthesis to glycine formation in basal-like xenografts. In agreement with results from the xenograft models, tissue samples from triple negative breast carcinomas had higher GPC/PCho ratio than samples from ER+/PgR+ carcinomas, suggesting that the choline metabolism in the experimental models is representative for luminal-like and basal-like human breast cancer. Conclusions The differences in choline metabolite concentrations corresponded well with differences in gene expression, demonstrating distinct metabolic profiles in the xenograft models representing basal-like and luminal-like breast cancer. The same characteristics of choline metabolite profiles were also observed in patient material from ER+/PgR+ and triple-negative breast cancer, suggesting that the xenografts are relevant model systems for studies of choline metabolism in luminal-like and basal-like breast cancer.

Introduction: Basal-like breast cancer is frequently associated with triple negative phenotype, and there is a need for novel therapeutic strategies for this patient population. Inhibitors of cytosolic phospholipase A2 (cPLA2) have been suggested to block both MAPK and PI3K signalling and have a high potential for activity in basal-like breast cancer (Lin 1993, Wen 2013). In this study, we compared the expression of PLA2G4A between luminal B and basal-like breast cancer, both in patient-derived xenograft models (PDX) and human cancer tissue. In addition, we studied the effect of the novel cPLA2 inhibitor AVX235 on tumor growth in a basal-like PDX model. Materials and methods: Tumor tissue specimens were obtained from PDX models (n = 26) and a clinical breast cancer biobank (n = 32). Gene expression analysis was carried out on Agilent 8×60K microarrays. The expression of 54 genes directly involved in choline metabolism was examined. Differential expression of choline genes between basal-like and luminal B tumors was calculated by subtraction of log2 expression values. Mice carrying bilateral MAS98.12 basal-like xenografts (Bergamaschi 2009) were treated with the cPLA2 inhibitor AVX235 (30 mg/kg i.p. daily for 7 days then every second day for 14 days, n = 6) or drug-free vehicle (control group, n = 6). Results: The PDX models were subtyped into basal-like (n = 19) and luminal B (n = 7) subtypes based on the expression of 500 intrinsic genes [Sørlie 2003]. For the human cancer tissue, there were 18 basal-like and 14 luminal B tumors. There was a significant correlation between differential choline gene expression in basal-like vs luminal B tumors in PDX models and human cancer tissue (p Conclusion: PLA2G4A is higher expressed in basal-like than in luminal B breast cancer. Treatment with the cPLA2 inhibitor AVX235 significantly inhibits tumor growth. These data suggest that cPLA2 inhibitors may be of particular value in treatment of basal-like breast cancer. References: Lin LL et al: cPLA2 is phosphorylated and activated by MAP kinase. Cell 1993; 72; 269-278. Wen ZH et al: Critical role of arachidonic acid-activated mTOR signaling in breast carcinogenesis and angiogenesis. Oncogene 2013; 32; 160-170. Bergamaschi A et al: Molecular profiling and characterization of luminal-like and basal-like in vivo breast cancer xenograft models. Mol Oncol 2009; 3; 469-482. Sørlie T et al: Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 2003; 100; 8418-8423. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-08.

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.