Cytosolic Phospholipase A2 Alpha Regulates TLR Signaling and Migration in Metastatic 4T1 Cells.

Metastatic disease is the leading cause of death in breast cancer patients. Disrupting the cancer cell's ability to migrate may be a strategy for hindering metastasis. Cytosolic phospholipase A2 α (cPLA2α), along with downstream proinflammatory and promigratory metabolites, has been implicated in several aspects of tumorigenesis, as well as metastasis, in various types of cancer. In this study, we aim to characterize the response to reduced cPLA2α activity in metastatic versus non-metastatic cells. We employ an isogenic murine cell line pair displaying metastatic (4T1) and non-metastatic (67NR) phenotype to investigate the role of cPLA2α on migration. Furthermore, we elucidate the effect of reduced cPLA2α activity on global gene expression in the metastatic cell line. Enzyme inhibition is achieved by using a competitive pharmacological inhibitor, cPLA2α inhibitor X (CIX). Our data show that 4T1 expresses significantly higher cPLA2α levels as compared to 67NR, and the two cell lines show different sensitivity to the CIX treatment with regards to metabolism and proliferation. Inhibition of cPLA2α at nontoxic concentrations attenuates migration of highly metastatic 4T1 cells, but not non-metastatic 67NR cells. Gene expression analysis indicates that processes such as interferon type I (IFN-I) signaling and cell cycle regulation are key processes regulated by cPLA2a in metastatic 4T1 cells, supporting the findings from the biological assays. This study demonstrates that two isogenic cancer cell lines with different metastatic potential respond differently to reduced cPLA2α activity. In conclusion, we argue that cPLA2α is a potential therapeutic target in cancer and that enzyme inhibition may inhibit metastasis through an anti-migratory mechanism, possibly involving Toll-like receptor signaling and type I interferons.

Glutamine to proline conversion is associated with response to glutaminase inhibition in breast cancer.

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. 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.

Detection and Differentiation of Breast Cancer Sub-Types using a cPLA2α Activatable Fluorophore.

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.

Patterns of Exchange of Multiplying Onion (Allium cepa L. Aggregatum-Group) in Fennoscandian Home Gardens.

Multiplying onion (Allium cepa L. Aggregatum-Group), commonly known as shallot or potato onion, has a long tradition of cultivation in Fennoscandian home gardens. During the last decades, more than 80 accessions, maintained as vegetatively propagated clones, have been gathered from home gardens in all Fennoscandian countries. A genetic analysis showed regional patterns of accessions belonging to the same genetic group. However, accessions belonging to the same genetic group could originate in any of the countries. These results suggested both short- and long-distance exchange of set onions, which was confirmed by several survey responses. Some of the most common genetic groups also resembled different modern varieties. The morphological characterization illustrated that most characters were strongly influenced by environment and set onion properties. The only reliably scorable trait was bulb skin color. Neither our morphological nor genetic results support a division between potato onions and shallots. Instead, naming seems to follow linguistic traditions. An ethnobotanical survey tells of the Fennoscandian multiplying onions as being a crop with reliable harvest, excellent storage ability, and good taste. An increased cultivation of this material on both household and commercial scale should be possible.

Anti-vascular effects of the cytosolic phospholipase A2 inhibitor AVX235 in a patient-derived basal-like breast cancer model.

BACKGROUND: Group IVA cytosolic phospholipase A2 (cPLA2α) plays an important role in tumorigenesis and angiogenesis. It is overexpressed in basal-like breast cancer (BLBC), which is aggressive and usually triple-negative, making it unresponsive to current targeted therapies. Here, we evaluated the anti-angiogenic effects of a specific cPLA2α inhibitor, AVX235, in a patient-derived triple-negative BLBC model. METHODS: Mice bearing orthotopic xenografts received i.p. injections of AVX235 or DMSO vehicle daily for 1 week and then every other day for up to 19 days. Six treated and six control mice were terminated after 2 days of treatment, and the tumors excised for high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) and prostaglandin E2 (PGE2) enzyme immunoassay (EIA) analysis. A 1-week imaging study was performed on a separate cohort of mice. Longitudinal dynamic contrast enhanced (DCE)-MRI was performed before, after 4 days, and after 1 week of treatment. The mice were then perfused with a radiopaque vascular casting agent, and the tumors excised for micro-CT angiography. Subsequently, tumors were sectioned and stained with lectin and for Ki67 or α-smooth muscle actin to quantify endothelial cell proliferation and vessel maturity, respectively. Partial least squares discriminant analysis was performed on the multivariate HR MAS MRS data, and non-parametric univariate analyses using Mann-Whitney U tests (α = 0.05) were performed on all other data. RESULTS: Glycerophosphocholine and PGE2 levels, measured by HR MAS MRS and EIA, respectively, were lower in treated tumors after 2 days of treatment. These molecular changes are expected downstream effects of cPLA2α inhibition and were followed by significant tumor growth inhibition after 8 days of treatment. DCE-MRI revealed that AVX235 treatment caused a decrease in tumor perfusion. Concordantly, micro-CT angiography showed that vessel volume fraction, density, and caliber were reduced in treated tumors. Moreover, histology showed decreased endothelial cell proliferation and fewer immature vessels in treated tumors. CONCLUSIONS: These results demonstrate that cPLA2α inhibition with AVX235 resulted in decreased vascularization and perfusion and subsequent inhibition of tumor growth. Thus, cPLA2α inhibition may be a potential new therapeutic option for triple-negative basal-like breast cancer.