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Recent reports suggest that dysregulation of lipid metabolism is a key feature of the most invasive cancers. To identify potential biomarkers of tumor aggressiveness, we compared the proteomes of two experimental models of malignant mesothelioma in rats exhibiting different invasive properties. Quantitative changes between the most invasive, M5-T1, versus the least invasive, F4-T2, first led to a list of 424 proteins. A second step, cross-comparing this list with 433 proteins distinguishing invasive vs non-invasive tumors, led to identifying 88 proteins that specifically increased and 157 that decreased, respectively, characterizing the most aggressive M5-T1 tumor. Among the 15 mitochondrial proteins found in these lists, the very long-chain specific acyl-CoA dehydrogenase, encoded by the Acadvl gene and involved in fatty acid beta oxidation, appeared to play an important role in the metabolic reprogramming of the tumor microenvironment. Immunohistochemical staining of tumor sections confirmed increased expression of Acadvl in the M5-T1 tumor. Finally, the dramatic increase and decrease, observed in 25 and 17 proteins, respectively, suggested the existence of a strong link between mitochondrial events and modifications of the extracellular matrix, immune cell components or other subcellular compartments. These findings highlight some important aspects of the tumor microenvironment changes linked to aggressiveness.

Cross-species investigations on cancer invasiveness are a new approach that identified new biomarkers potentially useful for improving tumor diagnosis and prognosis in clinical medicine and veterinary science. In this study, we combined proteomic analysis of four experimental rat malignant mesothelioma (MM) tumors with analysis of ten patient-derived cell lines to identify common features associated to mitochondrial proteome rewiring. The comparison of significant abundance changes between invasive and non-invasive rat tumors rat tumors gave a list of 433 proteins, including twenty-six proteins reported to be exclusively located in mitochondria. Next, we analyzed the differential expression of genes encoding the mitochondrial proteins of interest in five primary epithelioid and five primary sarcomatoid human MM cell lines, and the most impressive increase was observed in the expression of the long-chain acyl coenzyme A dehydrogenase (ACADL). To evaluate the role of this enzyme in the migration/invasiveness, two epithelioid and two sarcomatoid human MM cell lines derived from patients with the highest and lowest overall survival were studied. Interestingly, sarcomatoid vs epithelioid cell lines were characterized by higher migration and fatty oxidation rates, in agreement with ACADL findings. These results suggest that evaluating mitochondrial proteins in MM specimens might identify tumors with higher invasiveness.

International audience; Over the past two decades, quantitative proteomics has emerged as an important tool for deciphering the complex molecular events involved in cancers. The number of references involving studies on the cancer metastatic process has doubled since 2010, while the last 5 years have seen the development of novel technologies combining deep proteome coverage capabilities with quantitative consistency and accuracy. To highlight key findings within this huge amount of information, the present review identified a list of tumor inva-sive biomarkers based on both the literature and data collected on a biocollection of experimental cell lines, tumor models of increasing invasiveness and tumor samples from patients with colorectal or breast cancer. Crossing these different data sources led to 76 proteins of interest out of 1,245 mentioned in the literature. Information on these proteins can potentially be translated into clinical prospects, since they represent potential targets for the development and evaluation of innovative therapies , alone or in combination. Herein, a systematical review of the biology of each of these proteins, including their specific subcellular/extracellular or multiple localizations is presented. Finally, as an important advantage of quantitative proteomics is the ability to provide data on all these molecules simultaneously in cell pellets, body fluids or paraffin-embedded sections of tumors/invaded tissues, the significance of some of their interconnections is discussed.

International audience; This study aimed to identify the proteomic changes produced by curcumin treatment following stimulation of the host immune system in a rat model of malignant mesothelioma. We analyzed the proteomes of secondary lymphoid organs from four normal rats, four untreated tumorbearing rats, and four tumor-bearing rats receiving repeated intraperitoneal administrations of curcumin. Cross-comparing proteome analyses of histological sections of the spleen from the three groups first identified a list of eighty-three biomarkers of interest, thirteen of which corresponded to proteins already reported in the literature and involved in the anticancer therapeutic effects of curcumin. In a second step, comparing these data with proteomic analyses of histological sections of mesenteric lymph nodes revealed eight common biomarkers showing a similar pattern of changes in both lymphoid organs. Additional findings included a partial reduction of the increase in spleencirculating biomarkers, a decrease in C-reactive protein and complement C3 in the spleen and lymphnodes, and an increase in lymph node purine nucleoside phosphorylase previously associated with liver immunodeficiency. Our results suggest some protein abundance changes could be related to the systemic, distant non-target antitumor effects produced by this phytochemical.

Introduction Le mesotheliome pleural malin (MPM), du notamment a l’exposition aux fibres minerales telles que l’amiante, est un cancer agressif, difficile a diagnostiquer et a traiter. Il apparait donc necessaire de rechercher de nouveaux marqueurs qui permettront un diagnostic plus precoce et le developpement de nouveaux traitements. Methodes Une analyse proteomique quantitative par LC-MS/MS (liquid chromatography coupled to tandem mass spectrometry) avec marquage iTRAQ (isobaric tagging reagents for quantitative proteomic analysis) a ete realisee sur un panel de 6 lignees cellulaires mesotheliales transformees de rat pour identifier des proteines d’interet. Differents tests fonctionnels in vitro de migration et invasion utilisant des siRNA specifiques ont ensuite ete effectues sur des lignees representatives de rat et humaine de MPM. D’autre part, la modulation de l’expression de cibles associees a la TEM (transition epithelio-mesenchymateuse) a egalement ete recherchee par western blotting. Resultats L’analyse proteomique quantitative a montre une diminution du taux de LPP (Lipoma Preferred Partner) dans les lignees mesotheliales les plus invasives. De plus, il a ete observe que l’invalidation de LPP induit une augmentation de l’invasion dans un modele de chambre de Boyden modifiee et de la migration dans un modele 2D en anneau, ceci en association avec l’apparition d’un phenotype plus mesenchymateux. Conclusion LPP jouerait un role limitant dans la progression du MPM en modulant les capacites migratoires et invasives des cellules mesotheliales via la regulation de certaines cibles de la TEM. LPP pourrait representer un nouveau marqueur diagnostic et pronostic du MPM.

International audience; Simple SummaryAggressive sarcomatoid tumors designed in inbred strains of immunocompetent rats represent useful tools for both the identification of biomarkers of invasiveness and evaluation of innovative therapies. Our aim was to investigate the molecular determinants of liver colonization and potential common biomarkers of sarcomas and sarcomatoid tumors, using the most invasive (M5-T1) of our four experimental models of peritoneal sarcomatoid malignant mesothelioma in the F344 rat. Using an advanced and robust technique of quantitative proteomics and a bank of paraffin-embedded tumor and tissue samples, we analyzed changes in the proteotype patterns of the liver from normal rats, adjacent non-tumorous liver from untreated tumor-bearing rats, and liver from tumor-bearing rats positively responding to repeated administrations of curcumin given intraperitoneally. The identification of proteome alterations accounting for the antitumor effects of curcumin and changes in the liver microenvironment, which favored the induction of an immune response, could be useful to the research community.AbstractInvestigations of liver metastatic colonization suggest that the microenvironment is preordained to be intrinsically hospitable to the invasive cancer cells. To identify molecular determinants of that organotropism and potential therapeutic targets, we conducted proteomic analyses of the liver in an aggressive model of sarcomatoid peritoneal mesothelioma (M5-T1). The quantitative changes between SWATH-MS (sequential window acquisition of all theoretical fragmentation spectra) proteotype patterns of the liver from normal rats (G1), adjacent non-tumorous liver from untreated tumor-bearing rats (G2), and liver from curcumin-treated rats without hepatic metastases (G3) were compared. The results identified 12 biomarkers of raised immune response against M5-T1 cells in G3 and 179 liver biomarker changes in (G2 vs. G1) and (G3 vs. G2) but not in (G3 vs. G1). Cross-comparing these 179 candidates with proteins showing abundance changes related to increasing invasiveness in four different rat mesothelioma tumor models identified seven biomarkers specific to the M5-T1 tumor. Finally, analysis of correlations between these seven biomarkers, purine nucleoside phosphorylase being the main biomarker of immune response, and the 179 previously identified proteins revealed a network orchestrating liver colonization and treatment efficacy. These results highlight the links between potential targets, raising interesting prospects for optimizing therapies against highly invasive cancer cells exhibiting a sarcomatoid phenotype and sarcoma cells.

Oncogenes or chemotherapy treatments trigger the induction of suppressive pathways such as apoptosis or senescence. Senescence was initially defined as a definitive arrest of cell proliferation but recent results have shown that this mechanism is also associated with cancer progression and chemotherapy resistance. Senescence is therefore much more heterogeneous than initially thought. How this response varies is not really understood, it has been proposed that its outcome relies on the secretome of senescent cells and on the maintenance of their epigenetic marks.Using experimental models of senescence escape, we now described that the stability of this suppression relies on specific tRNAs and aminoacyl-tRNA synthetases. Following chemotherapy treatment, the DNA binding of the type III RNA polymerase was reduced to prevent tRNA transcription and induce a complete cell cycle arrest. By contrast, during senescence escape, specific tRNAs such as tRNA-Leu-CAA and tRNA-Tyr-GTA were up-regulated. Reducing tRNA transcription appears necessary to control the strength of senescence since RNA pol III inhibition through BRF1 depletion maintained senescence and blocked cell persistence. mTOR inhibition also prevented CIS escape in association with a reduction of tRNA-Leu-CAA and tRNA-Tyr-GTA expression. Further confirming the role of the tRNA-Leu-CAA and tRNA-Tyr-GTA, their corresponding tRNA ligases, YARS and LARS, were necessary for senescence escape. This effect was specific since the CARS ligase had no effect on persistence. YARS and LARS inactivation reduced cell emergence and proteomic analysis indicated that this effect was associated with the down-regulation of E2F1 target genes.Overall, these findings highlight a new regulation of tRNA biology during senescence and suggest that specific tRNAs and ligases contribute to the strength and heterogeneity of this suppression.

International audience; Malignant mesothelioma (MM) still represents a devastating disease that is often detected too late, while the current effect of therapies on patient outcomes remains unsatisfactory. Invasiveness biomarkers may contribute to improving early diagnosis, prognosis, and treatment for patients, a task that could benefit from the development of high-throughput proteomics. To limit potential sources of bias when identifying such biomarkers, we conducted cross-species proteomic analyzes on three different MM sources. Data were collected firstly from two human MM cell lines, secondly from rat MM tumors of increasing invasiveness grown in immunocompetent rats and human MM tumors grown in immunodeficient mice, and thirdly from paraffin-embedded sections of patient MM tumors of the epithelioid and sarcomatoid subtypes. Our investigations identified three major invasiveness biomarkers common to the three tumor sources, CAPG, FABP4, and LAMB2, and an additional set of 25 candidate biomarkers shared by rat and patient tumors. Comparing the data to proteomic analyzes of preneoplastic and neoplastic rat mesothelial cell lines revealed the additional role of SBP1 in the carcinogenic process. These observations could provide new opportunities to identify highly vulnerable MM patients with poor survival outcomes, thereby improving the success of current and future therapeutic strategies.

Background: Estrogen positive/HER2 negative (ER+/HER2-) early (e) breast carcinoma (BC) is an heterogeneous entity, on the prognostic and predictive plan. Several prognostic multigene tests have been developed to identify patients in whom chemotherapy could be safely avoided. Proteomic is a complementary approach as variations in mRNA expression only account for ≈40% of the tumor-encoded protein range and it is taking into account, as other OMICs the tumoral microenvironment Sequential Windowed Acquisition of All Theoretical fragment (SWATH-MS) proteomic approach let an accurate and reproducible label-free quantification of large proteome. To our knowledge, no study has been conducted in a large cohort of luminal BC by SWATH-MS. The aim of this study was to establish a proteomic cartography of ER+/HER2- eBC and identify prognostic biomarkers. Methods: Frozen primary tumors were collected from 157 ER+/HER2- eBC treated in the ICO cancer center between 2006 and 2009. Patients were included if they fulfilled the following criteria: 1) ductal carcinoma; 2) unilateral; 3) first occurrence; 4) have received adjuvant chemotherapy. Clinicopathologic characteristics as outcomes were collected. Each sample was analyzed using SWATH-MS acquisition method as previously described (Aebersold et al, 2012). Peak extraction of the SWATH data was performed using either the Spectronaut software (ver 8.0, Biognosys, Switzerland). Peptide identification results were filtered with a q-value of < 1%. We performed clustering analysis (fuzzy clustering method) based on the 15% of most variant proteins. Functional annotation of clusters based on GO biological process terms enrichment (GOEA) was performed by means of ToppGene and GORILLA web tools. Results: The median of follow-up was 8.34 years. Respectively 32, 4 and 7 patients presented a metastatic, locoregional and controlateral recurrence. 684 among 4555 proteins represented the 15% of most variants proteins.Two ER+/HER2- eBC clusters were identified (C1 [23%] and C2 [77%]) by means of fuzzy clustering and GOEA. Two significant clinicopathological differences were observed between the two subgroups: more unifocal tumors in C1 (P = 0.0415) and mostly a clear better outcome in term of Disease Free Survival (DFS), Distant DFS (DDFS) and Overall survival (OS) in patients belonging to C2 (cf table 1). Functional annotation found that C1 was characterized by mRNA processing and protein synthesis (GO:0006396: RNA processing; GO:0008380: RNA splicing; GO:0016071: mRNA metabolic process; GO:0022613: ribonucleoprotein complex biogenesis), and C2 by a high immune response (GO:0002757: immune response-activating signal transduction; GO:0050778: positive regulation of immune response; GO:0002253: activation of immune response; GO:0050776: regulation of immune response). Differential protein expression according to the C1-C2 clusters will be presented at the meeting. Conclusion: Proteomic cartography by SWATH-MS can clearly distinguish two ER+/HER2- eBC subgroups with clear different prognosis with a better outcome for C2 patients compared to C1 patients. High immune response observed in C2 could underlie this difference with results that must be confirmed on external cohort. Nevertheless this approach could be considered as a complementary approach, helpful for clinical decision for administration of adjuvant treatment. All (n=157)Cluster 1 (n = 36)Cluster 2 (n = 121)PDisease-free survival72%55.5%77.6%0.0016Distant disease-free survival79.6%66.6%83.4%0.0063Overall survival81%61%87%0.0009Specific overall survival86%69%91%0.0012 Citation Format: Anne Patsouris, Hamza Lasla, Pascal Jézéquel, Philippe Juin, Alice Boissard, Cécile Henry, Mario Campone, Catherine Guette. High immune response identified as a good prognostic factor by proteomic SWATH-MS approach in 157 ER+/HER2- early breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-11.

International audience; Human olfactomedin-4 (OLFM4) is a secreted protein involved in a variety of cellular functions including proliferation, differentiation, apoptosis, and cell adhesion. OLFM4 expression has been studied in several tumor types including gastric, colorectal, lung, and endometrioid cancers where it has been suggested to be an independent favorable or unfavorable prognostic marker. For breast cancer, the clinical significance of OLFM4 is still unclear. In the present study, SWATH-MS is used as a tool for the robust identification and quantification of breast tissue proteins. SWATH-MS data show that OLFM4 expression is higher in DCIS than in invasive breast cancer. In-depth analysis of the breast tumor proteome show that OLFM4 is a favorable pronostic marker. Serum OLFM4 levels in peripheral blood are also analyzed by ELISA in 825 cases, including 94 cases of healthy individuals, 61 cases of non-invasive breast tumor (DCIS) and 670 cases of breast cancer (BC). It is found that serum OLFM4 levels are significantly higher in the DCIS cohort and in the breast cancer cohort compared with the healthy controls. This result suggests that circulating OLFM4 could be an interesting biomarker of early breast cancer. Data are available via ProteomeXchange with identifier PXD014194.

International audience; Heterogeneity and lack of targeted therapies represent the two main impediments to precision treatment of triple-negative breast cancer (TNBC). Therefore, molecular subtyping and identification of therapeutic pathways are required to optimize medical care. The aim of the present study is to define robust TNBC subtypes with clinical relevance by means of proteomics and transcriptomics. As a first step, unsupervised analyses are conducted in parallel on proteomics and transcriptomics data of 83 TNBC tumors. Proteomics data unsupervised analysis did not permit separation of TNBC into different subtypes, whereas transcriptomics data are able to clearly and robustly identify three subtypes: molecular apocrine (C1), basal-like immune-suppressed (C2), and basal-like immune response (C3). Supervised analysis of proteomics data are then conducted based on transcriptomics subtyping. Thirty out of 62 proteins differentially expressed between C1, C2, and C3 belonged to biological categories which characterized these TNBC clusters: luminal and androgen-regulated proteins (C1), basal, invasion, and extracellular matrix (C2), and basal and immune response (interferon pathway and immunoglobulins) (C3). Although proteomics unsupervised analysis of TNBC tumors is unsuccessful at identifying clusters, the integrated approach is promising. Identification and measurement of 30 proteins strengthen subtyping of TNBC based on robust transcriptomics unsupervised analysis.

International audience; In primary cells, senescence induces a permanent proliferative arrest to prevent the propagation of malignant cells. However, the outcome of senescence is more complex in advanced cancer cells where senescent states are heterogeneous. Here, this heterogeneity is discussed and it is proposed that proteomic analysis should be used to identify specific signatures of cancer cells that use this pathway as an adaptive mechanism. Since senescent cells produce an inflammatory secretome, MRM approaches and quantification with internal standards might be particularly suited to follow the expression of the corresponding markers in body fluids. Used in combination with imaging medical technics, a better characterization of senescence heterogeneity should help to monitor the response to chemotherapy treatment.

Senescence is a tumor suppressive mechanism that induces a permanent proliferative arrest in response to an oncogenic insult or to the genotoxic stress induced by chemotherapy. We have recently described that some cells can escape this arrest, either because senescence was incomplete or as a consequence of a phenotypic adaptation. Malignant cells which resisted senescence emerged as more transformed cells that resist anoikis and rely on survival pathways activated by Akt and Mcl-1. In this study, we further characterize senescence escape, investigating how emergent cells could reproliferate. During the initial step of chemotherapy-induced senescence (CIS), we found that cyclin D1 was upregulated and that cell emergence was prevented when its main partner cdk4 was inactivated. Results indicate that this kinase induced the upregulation of the EZH2 methylase, a component of the polycomb PRC2 complex. Downregulated during the early step of treatment, the methylase was reactivated in clones that escaped senescence. The inactivation of EZH2, either by siRNA or by specific inhibitors, led to a specific inhibition of cell emergence. We used quantitative proteomic analysis to identify new targets of the methylase involved in senescence escape. We identified proteins involved in receptor endocytosis and described new functions for the AP2M1 protein in the control of chemotherapy-mediated senescence. Our results indicate that AP2M1 is involved in the transmission of secreted signals produced by senescent cells, suggesting that this pathway might regulate specific receptors involved in the control of CIS escape. In light of these results, we therefore propose that the cdk4–EZH2–AP2M1 pathway plays an important role during chemotherapy resistance and senescence escape. Since targeted therapies are available against these proteins, we propose that they should be tested in the treatment of colorectal or breast cancers that become resistant to first-line genotoxic therapies.

International audience; To date, there is no available targeted therapy for patients who are diagnosed with triple-negative breast cancers (TNBC). The aim of this study was to identify a new specific target for specific treatments. Frozen primary tumors were collected from 83 adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling by iTRAQ-OFFGEL-LC-MS/MS approach in two series: a training cohort (n = 42) and a test set (n = 41). Patients who remains free of local or distant metastasis for a minimum of 5 years after surgery were classified in the no-relapse group; the others were in the relapse group. OPLS and Kaplan–Meier analyses were performed to select candidate markers, which were validated by immunohistochemistry. Three proteins were identified in the training set and validated in the test set by Kaplan–Meier method and immunohis-tochemistry (IHC): TrpRS as a good prognostic markers and DP and TSP1 as bad prognostic markers. We propose the establishment of an IHC test to calculate the score of TrpRS, DP, and TSP1 in TNBC tumors to evaluate the degree of aggressiveness of the tumors. Finally, we propose that DP and TSP1 could provide therapeutic targets for specific treatments.

International audience; Glioblastoma (GB) displays diffusely infiltrative growth patterns. Dispersive cells escape surgical resection and contribute to tumour recurrence within a few centimeters of the resection cavity in 90% of cases. We know that the non-neoplastic stromal compartment, in addition to infiltrative tumour cells, plays an active role in tumour recurrence. We isolated a new stromal cell population from the histologically normal surgical margins of GB by computer-guided stereotaxic biopsies and primary culture. These GB-associated stromal cells (GASCs) share phenotypic and functional properties with the cancer-associated fibroblasts (CAFs) described in the stroma of carcinomas. In particular, GASCs have tumour-promoting effects on glioma cells in vitro and in vivo. Here, we describe a quantitative proteomic analysis, using iTRAQ labelling and mass spectrometry, to compare GASCs with control stromal cells derived from non-GB peripheral brain tissues. A total of 1077 proteins were quantified and 67 proteins were found to differ between GASCs and control stromal cells. Several proteins changed in GASCs are related to a highly motile myofibroblast phenotype, and to wound healing and angiogenesis. The results for several selected proteins were validated by western blotting or flow cytometry. Furthermore, the effect of GASCs on angiogenesis was confirmed using the orthotopic U87MG glioma model. In conclusion, GASCs, isolated from GB histologically normal surgical margins and found mostly near blood vessels, could be a vascular niche constituent establishing a permissive environment, facilitating angiogenesis and possibly colonization of recurrence-initiating cells. We identify various proteins as being expressed in GASCs: some of these proteins may serve as prognostic factors for GB and/or targets for anti-glioma treatment.

International audience; Shotgun proteomic analyses are increasingly becoming methods of choice for complex samples. The development of effective methods for fractionating peptides to reduce the complexity of the sample before mass analysis is a key point in this strategy. The OFFGEL technology has recently become a tool of choice in proteomic analysis at peptide level. This OFFGEL electrophoresis (OGE) approach allows the in-solution separation of peptides from various biological sources by isoelectric focusing in highly resolved 24 fractions. It was also demonstrated that OGE technology is a filtering tool for pI-based validation of peptide identification. As peptide OGE is compatible with iTRAQ labeling, OGE is finding valuable applications in quantitative proteomics as well. The aim of this study is to explain a new 2D-OGE approach that improves the proteomic coverage of complex mixtures such as colorectal cell line lysates, and which is compatible with iTRAQ labeling.