Sphingolipid distribution at mitochondria-associated membranes (MAMs) upon induction of apoptosis.

The levels and composition of sphingolipids and related metabolites are altered in aging and in common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC-MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified ER, mitochondria-associated membranes (MAMs), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, SM in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine-induced apoptosis in U251 cells. Ceramide (especially C16-ceramide) levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and SM, but sphingosine and lactosyl- and glycosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when SM levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and the ER during the early steps of apoptosis.

Adenosine Methylation Level of miR-125a-5p Promotes Anti-PD-1 Therapy Escape through the Regulation of IGSF11/VSIG3 Expression.

BACKGROUND: Despite encouraging anti-tumour activity in lung cancer, anti-PD-1 therapy has encountered increasing resistance to treatment. Several companion diagnostic assays have been performed to identify patients who may benefit from this immunotherapy and to adapt this therapy in case of acquired resistance. METHODS: A large panel of methods was used for the analysis of expression and methylation levels of miRNAs (qPCR, MemiRIP, …), protein/miRNA interactions (CLIP, oligo pull-down, …), and protein-protein interactions (CoIP) in cells and/or blood samples. RESULTS: Our work highlights that the saturation of PD-1 by anti-PD1 therapies induces an immune escape phenomenon due to the overexpression of IGSF11 following adenosine methylation of miR-125a-5p. Mechanistically, we identify METTL3/KHDRBS3 and HuR as two crucial players in the methylation and the loss of the repressive function of this miRNA. Finally, our work shows that the adenosine methylation of miR-125a-5p is analyzable from EVs/exosomes from longitudinal blood samples and that such EVs/exosomes modulate the IGSF11/VSIG3 expression in lung cancer cells to promote an immune escape phenomenon. CONCLUSIONS: Our data provide a biomarker (m6A-miR-125a-5p level) and two therapeutic solutions (anti-IGSF11 antibody and METTL3 inhibitor) that could potentially address the anti-PD1 therapy failure in the context of precision and personalized medicine.

Validation of tumor-associated macrophage ferritin light chain as a prognostic biomarker in node-negative breast cancer tumors: A multicentric 2004 national PHRC study.

Novel prognostic biomarkers are imperatively needed to help direct treatment decisions by typing subgroups of node-negative breast cancer patients. Large screening of different biological compartments, such as the proteome, by means of high throughput techniques may greatly help scientists to find such markers. The present retrospective multicentric study included 268 node-negative breast cancer patients. We used a proteomic approach of SELDI-TOF-MS screening to identify differentially expressed cytosolic proteins with prognostic impact. The screening cohort was composed of 198 patients. Seventy supplementary patients were included for validation. Immunohistochemistry (IHC) and immunoassay (IA) were run to confirm the prognostic role of the marker identified by SELDI-TOF-MS screening. IHC was also used to explore links between selected marker and epithelial-mesenchymal transition (EMT)-like, proliferation and macrophage markers. Ferritin light chain (FTL) was identified as an independent prognostic marker (HR = 1.30-95% CI: 1.10-1.50, p = 0.001). Validation step by means of IHC and IA confirmed the prognostic value of FTL level. CD68 IHC showed that FTL was stored in tumor-associated macrophages (TAM), which exhibit an M2-like phenotype. We report here, first, the validation of FTL as a breast tumor prognostic biomarker in node-negative patients, and second, the fact that FTL is stored in TAM.

TOM20-mediated transfer of Bcl2 from ER to MAM and mitochondria upon induction of apoptosis.

In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2-TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER-mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2-TOM20 interaction is thus an additional player in the control of apoptosis.

Liver X Receptor (LXR)-regulated Genes of Cholesterol Trafficking and Breast Cancer Severity.

BACKGROUND: Liver X receptor [LXR; nuclear receptor subfamily 1, group H, member 2 (NR1H2, alias LXRB)] can inhibit proliferation and induce apoptosis of cancer cells. Its relationship with disease severity is not known. MATERIALS AND METHODS: Expression of LXRB, ATP binding cassette subfamily A member 1 (ABCA1), ATP binding cassette subfamily G member 1 (ABCG1), apolipoprotein E (APOE) and paraoxonase 2 (PON2) were determined in 69 breast tumors and were related to clinical stages of the disease and tumor characteristics, as well as time to recurrence. RESULTS: ABCG1 expression differed with the tumor Scarff Bloom and Richardson (SBR) status (p=0.02), with a lower expression in SBRIII than in SBRII and SBRI. ABCG1 expression was significantly higher in estrogen receptor-positive tumors (N=63) (p=0.02). APOE expression was significantly lower in progesterone receptor-positive tumors (N=55) (p=0.03). No relationship with time to recurrence was observed. CONCLUSION: Expression of some LXR-dependent genes is related to breast tumor characteristics, but not time to recurrence. This may be due to a lack of study power or too short a follow-up time.

Leptin and adiponectin as new markers of undernutrition in cancer.

OBJECTIVES: To evaluate leptin and adiponectin as markers of undernutrition in cancer patients, and compare their performances with those of other biomarkers. DESIGN AND METHODS: This was a prospective and observational study of 132 patients with various types of cancer. Following the recommended professional criteria, we diagnosed undernutrition at the time of blood sampling for the biological analysis of leptin, adiponectin, paraoxonase (hydrolysis rate of three substrates: paraoxon (PON), phenylacetate (ARE) and thiolactone (LAC)), and the calculation of the Prognostic Inflammatory and Nutritional Index (PINI). Patients were monitored for one year to establish the mortality rate of the group. Relationships between biological variables and undernutrition were evaluated using univariate and multivariate logistic regression models. The Kaplan Meier method was used to analyse survival curves. Hazard ratios for death were calculated according to the quartiles of each biological variable. RESULTS: In the case of undernutrition, a decrease was observed in levels of leptin and in the lactonase activity (LAC) of paraoxonase, while adiponectin levels increased. Besides PINI, leptin was the only parameter that was independently related to undernutrition. While no relation was found between survival and leptin or adiponectin levels, evidence was found that PINI, LAC and ARE were associated with survival, even in multivariate analysis. CONCLUSIONS: Leptin and PINI are good markers of installed undernutrition, and PINI and ARE or LAC are reliable markers of the risk of death in patients suffering from cancer.

Sensitization of EGFR Wild-Type Non-Small Cell Lung Cancer Cells to EGFR-Tyrosine Kinase Inhibitor Erlotinib.

The benefit of EGFR-TKI in non-small cell lung cancer has been demonstrated in mutant EGFR tumors as first-line treatment but the benefit in wild-type EGFR tumors is marginal as well as restricted to maintenance therapy in pretreated patients. This work aimed at questioning the effects of cisplatin initial treatment on the EGFR pathway in non-small cell lung cancer and the functional consequences in vitro and in in vivo animal models of patient-derived xenografts (PDX). We establish here that cisplatin pretreatment specifically sensitizes wild-type EGFR-expressing cells to erlotinib, contrary to what happens in mutant EGFR cells and with a blocking EGFR antibody, both in vitro and in vivo The sensitization entails the activation of the kinase Src upstream of EGFR, thereafter transactivating EGFR through a ligand-independent activation. We propose a combination of markers that enable to discriminate between the tumors sensitized to erlotinib or not in PDX models, which should be worth testing in patients. These markers might be useful for the selection of patients who would benefit from erlotinib as a maintenance therapy. Mol Cancer Ther; 16(8); 1634-44. ©2017 AACR.

Paired measurement of serum amyloid A (SAA) and paraoxonase 1 (PON1) as useful markers in breast cancer recurrence.

OBJECTIVES: Paraoxonase 1 (PON1) and serum amyloid A (SAA) are carried by HDL. In case of inflammation, SAA and PON1 tend to change in opposite direction. In this study we determined if inflammation leads to altered PON1 activity using three different substrate hydrolysis rates, paraoxonase (PON), arylesterase (ARE) and lactonase (LAC) in breast cancer recurrence. DESIGN AND METHODS: 49 patients with a recurrence of breast cancer were analyzed for SAA, CRP, lipids, oxidized LDL, PON, ARE and LAC. Distribution of PON1 activities across the quartiles of CRP and SAA were compared by the Kruskal Wallis test. Non-parametric estimates of the survivor function were computed with Kaplan-Meier method. The association of SAA and ARE with short term death was assessed by logistic regression models. RESULTS: HDL and ARE decrease significantly across the quartiles of CRP. No significant differences were observed across SAA quartiles. The survival time was significantly related to the level of SAA (log rank: p<0.001) as well as the level of ARE (log rank: p=0.039). SAA and ARE were independently related to survival time below one year. CONCLUSIONS: PON1 does not seem to be directly affected by SAA, for any of the tested substrates, PON, ARE and LAC. The combined measurement of SAA and ARE could be a useful tool in this clinical situation, since they are independently related to short term death.

Paraoxonase 1 (PON1) as a marker of short term death in breast cancer recurrence.

OBJECTIVE: To relate paraoxonase (PON1) activity to survival time and short term death in breast cancer recurrence. DESIGN AND METHODS: PON1 activity was measured by its rate of hydrolysis of two different substrates, paraoxon (PON) and phenylacetate (ARE) in 50 patients with recurrence of breast cancer. Results were compared between patients surviving more than one year after the analysis (22) and those who died within one year (28). RESULTS: In a logistic regression analysis, ARE was negatively associated with early death (OR=0.10 [0.02-0.58], p=0.0109). PON did not reach significance (OR=0.43 [0.17-1.11], p=0.0826). In a multiple logistic regression analysis model, ARE was independently associated with early death (OR=0.12 [0.02-0.98], p=0.0476), besides interval time between diagnosis and recurrence (OR=0.54 [0.27-1.07], p=0.0781) and undernutrition (OR=3.95 [0.81-19.19], p=0.0883). CONCLUSION: Paraoxonase is a potential marker of survival in patients with breast cancer recurrence.

Surface-enhanced laser desorption/ionization time of flight mass spectrometry protein profiling identifies ubiquitin and ferritin light chain as prognostic biomarkers in node-negative breast cancer tumors.

Novel prognostic biomarkers are imperatively needed to help direct treatment decisions by typing subgroups of node-negative breast cancer patients. The current study has used a proteomic approach of SELDI-TOF-MS screening to identify differentially cytosolic expressed proteins with a prognostic impact in 30 node-negative breast cancer patients with no relapse versus 30 patients with metastatic relapse. The data analysis took into account 73 peaks, among which 2 proved, by means of univariate Cox regression, to have a good cumulative prognostic-informative power. Repeated random sampling (n = 500) was performed to ensure the reliability of the peaks. Optimized thresholds were then computed to use both peaks as risk factors and, adding them to the St. Gallen ones, improve the prognostic classification of node-negative breast cancer patients. Identification of ubiquitin and ferritin light chain (FLC), corresponding to the two peaks of interest, was obtained using ProteinChip LDI-Qq-TOF-MS. Differential expression of the two proteins was further confirmed by Western blotting analyses and immunohistochemistry. SELDI-TOF-MS protein profiling clearly showed that a high level of cytosolic ubiquitin and/or a low level of FLC were associated with a good prognosis in breast cancer.