tRNA biogenesis and specific aminoacyl-tRNA synthetases regulate senescence stability under the control of mTOR.

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 proliferative arrest 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 the generation of escaping cells. mTOR inhibition also prevented chemotherapy-induced senescence 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, results showed that their corresponding tRNA ligases, LARS and YARS, were necessary for senescence escape. This effect was specific since the CARS ligase had no effect on persistence. By contrast, the down-regulation of LARS and YARS reduced the emergence of persistent cells and this was associated with the modulation of E2F1 target genes expression. 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 tumor suppressive pathway.

Proteomes of Residual Tumors in Curcumin-Treated Rats Reveal Changes in Microenvironment/Malignant Cell Crosstalk in a Highly Invasive Model of Mesothelioma.

Curcumin exhibits both immunomodulatory properties and anticarcinogenic effects which have been investigated in different experimental tumor models and cancer types. Its interactions with multiple signaling pathways have been documented through proteomic studies on malignant cells in culture; however, in vivo approaches are scarce. In this study, we used a rat model of highly invasive peritoneal mesothelioma to analyze the residual tumor proteomes of curcumin-treated rats in comparison with untreated tumor-bearing rats (G1) and provide insights into the modifications in the tumor microenvironment/malignant cell crosstalk. The cross-comparing analyses of the histological sections of residual tumors from two groups of rats given curcumin twice on days 21 and 26 after the tumor challenge (G2) or four times on days 7, 9, 11 and 14 (G3), in comparison with G1, identified a common increase in caveolin-1 which linked with significant abundance changes affecting 115 other proteins. The comparison of G3 vs. G2 revealed additional features for 65 main proteins, including an increase in histidine-rich glycoprotein and highly significant abundance changes for 22 other proteins regulating the tumor microenvironment, linked with the presence of numerous activated T cells. These results highlight new features in the multiple actions of curcumin on tumor microenvironment components and cancer cell invasiveness.

Lymphoid Organ Proteomes Identify Therapeutic Efficacy Biomarkers Following the Intracavitary Administration of Curcumin in a Highly Invasive Rat Model of Peritoneal Mesothelioma.

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 tumor-bearing 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 spleen-circulating biomarkers, a decrease in C-reactive protein and complement C3 in the spleen and lymph nodes, 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.

iTRAQ-Based Quantitative Proteomic Analysis Strengthens Transcriptomic Subtyping of Triple-Negative Breast Cancer Tumors.

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.

Proteomics Approaches to Define Senescence Heterogeneity and Chemotherapy Response.

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.

OLFM4 Expression in Ductal Carcinoma In Situ and in Invasive Breast Cancer Cohorts by a SWATH-Based Proteomic Approach.

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.

Prediction of Recurrence and Survival for Triple-Negative Breast Cancer (TNBC) by a Protein Signature in Tissue Samples.

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 immunohistochemistry (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.

Gene-expression molecular subtyping of triple-negative breast cancer tumours: importance of immune response.

INTRODUCTION: Triple-negative breast cancers need to be refined in order to identify therapeutic subgroups of patients. METHODS: We conducted an unsupervised analysis of microarray gene-expression profiles of 107 triple-negative breast cancer patients and undertook robust functional annotation of the molecular entities found by means of numerous approaches including immunohistochemistry and gene-expression signatures. A triple-negative external cohort (n=87) was used for validation. RESULTS: Fuzzy clustering separated triple-negative tumours into three clusters: C1 (22.4%), C2 (44.9%) and C3 (32.7%). C1 patients were older (mean=64.6 years) than C2 (mean=56.8 years; P=0.03) and C3 patients (mean=51.9 years; P=0.0004). Histological grade and Nottingham prognostic index were higher in C2 and C3 than in C1 (P<0.0001 for both comparisons). Significant event-free survival (P=0.03) was found according to cluster membership: patients belonging to C3 had a better outcome than patients in C1 (P=0.01) and C2 (P=0.02). Event-free survival analysis results were confirmed when our cohort was pooled with the external cohort (n=194; P=0.01). Functional annotation showed that 22% of triple-negative patients were not basal-like (C1). C1 was enriched in luminal subtypes and positive androgen receptor (luminal androgen receptor). C2 could be considered as an almost pure basal-like cluster. C3, enriched in basal-like subtypes but to a lesser extent, included 26% of claudin-low subtypes. Dissection of immune response showed that high immune response and low M2-like macrophages were a hallmark of C3, and that these patients had a better event-free survival than C2 patients, characterized by low immune response and high M2-like macrophages: P=0.02 for our cohort, and P=0.03 for pooled cohorts. CONCLUSIONS: We identified three subtypes of triple-negative patients: luminal androgen receptor (22%), basal-like with low immune response and high M2-like macrophages (45%), and basal-enriched with high immune response and low M2-like macrophages (33%). We noted out that macrophages and other immune effectors offer a variety of therapeutic targets in breast cancer, and particularly in triple-negative basal-like tumours. Furthermore, we showed that CK5 antibody was better suited than CK5/6 antibody to subtype triple-negative patients.

Glioblastoma-associated stromal cells (GASCs) from histologically normal surgical margins have a myofibroblast phenotype and angiogenic properties.

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.

Proteomics of tumor and serum samples from isocitrate dehydrogenase-wildtype glioblastoma patients: is the detoxification of reactive oxygen species associated with shorter survival?

Proteomics has been little used for the identification of novel prognostic and/or therapeutic markers in isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GB). In this study, we analyzed 50 tumor and 30 serum samples from short- and long-term survivors of IDH-wildtype GB (STS and LTS, respectively) by data-independent acquisition mass spectrometry (DIA-MS)-based proteomics, with the aim of identifying such markers. DIA-MS identified 5422 and 826 normalized proteins in tumor and serum samples, respectively, with only three tumor proteins and 26 serum proteins displaying significant differential expression between the STS and LTS groups. These dysregulated proteins were principally associated with the detoxification of reactive oxygen species (ROS). In particular, GB patients in the STS group had high serum levels of malate dehydrogenase 1 (MDH1) and ribonuclease inhibitor 1 (RNH1) and low tumor levels of fatty acid-binding protein 7 (FABP7), which may have enabled them to maintain low ROS levels, counteracting the effects of the first-line treatment with radiotherapy plus concomitant and adjuvant temozolomide. A blood score built on the levels of MDH1 and RNH1 expression was found to be an independent prognostic factor for survival based on the serum proteome data for a cohort of 96 IDH-wildtype GB patients. This study highlights the utility of circulating MDH1 and RNH1 biomarkers for determining the prognosis of patients with IDH-wildtype GB. Furthermore, the pathways driven by these biomarkers, and the tumor FABP7 pathway, may constitute promising therapeutic targets for blocking ROS detoxification to overcome resistance to chemoradiotherapy in potential GB STS.

Two-step OFFGEL approach for effective peptide separation compatible with iTRAQ labeling.

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.

Comparison of spheroids formed by rat glioma stem cells and neural stem cells reveals differences in glucose metabolism and promising therapeutic applications.

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.

A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new nonmetastatic tumor marker.

Expression profiles represent new molecular tools that are useful to characterize the successive steps of tumor progression and the prediction of recurrence or chemotherapy response. In this study, we have used quantitative proteomic analysis to compare different stages of colorectal cancer. A combination of laser microdissection, OFFGEL separation, iTRAQ labeling, and MALDI-TOF/TOF MS was used to explore the proteome of 28 colorectal cancer tissues. Two software packages were used for identification and quantification of differentially expressed proteins: Protein Pilot and iQuantitator. Based on ∼1,190,702 MS/MS spectra, a total of 3138 proteins were identified, which represents the largest database of colorectal cancer realized to date and demonstrates the value of our quantitative proteomic approach. In this way, individual protein expression and variation have been identified for each patient and for each colorectal dysplasia and cancer stage (stages I-IV). A total of 555 proteins presenting a significant fold change were quantified in the different stages, and this differential expression correlated with immunohistochemistry results reported in the Human Protein Atlas database. To identify a candidate biomarker of the early stages of colorectal cancer, we focused our study on secreted proteins. In this way, we identified olfactomedin-4, which was overexpressed in adenomas and in early stages of colorectal tumors. This early stage overexpression was confirmed by immunohistochemistry in 126 paraffin-embedded tissues. Our results also indicate that OLFM4 is regulated by the Ras-NF-κB2 pathway, one of the main oncogenic pathways deregulated in colorectal tumors.

Long-Chain Acyl Coenzyme A Dehydrogenase, a Key Player in Metabolic Rewiring/Invasiveness in Experimental Tumors and Human Mesothelioma Cell Lines.

Cross-species investigations of cancer invasiveness are a new approach that has already identified new biomarkers which are 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 with mitochondrial proteome rewiring. A comparison of significant abundance changes between invasive and non-invasive rat tumors gave a list of 433 proteins, including 26 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; 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 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. Data are available via ProteomeXchange with the dataset identifier PXD042942.

Mitochondrial adaptation decreases drug sensitivity of persistent triple negative breast cancer cells surviving combinatory and sequential chemotherapy.

Triple negative breast cancer (TNBC) is an aggressive malignancy for which chemotherapy remains the standard treatment. However, between 3 and 5 years after chemotherapy, about half patients will relapse and it is essential to identify vulnerabilities of cancer cells surviving neoadujuvant therapy. In this study, we established persistent TNBC cell models after treating MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide, and then with paclitaxel, for a total of 18 weeks. The resulting chemo-persistent cell lines were more proliferative, both in vitro and in xenografted mice. Interestingly, MDA-MB-231 persistent cells became less sensitive to chemotherapeutic drugs, whereas SUM159-PT persistent cells kept similar sensitivity compared to control cells. The reduced sensitivity to chemotherapy in MDA-MB-231 persistent cells was found to be associated with an increased oxidative phosphorylation (OXPHOS) and modified levels of tricarboxylic acid cycle (TCA) intermediates. Integration of data from proteomics and metabolomics demonstrated TCA cycle among the most upregulated pathways in MDA-MB-231 persistent cells. The absence of glucose and pyruvate impeded OXPHOS in persistent cells, while the absence of glutamine did not. In contrast, OXPHOS was not modified in control cells independently of TCA substrates, indicating that MDA-MB-231 persistent cells evolved towards a more pyruvate dependent profile. Finally, the inhibition of pyruvate entry into mitochondria with UK-5099 reduced OXPHOS and re-sensitized persistent cells to therapeutic agents. Together, these findings suggest that targeting mitochondrial pyruvate metabolism may help to overcome mitochondrial adaptation of chemo-persistent TNBC.

Curcuminoids as Modulators of EMT in Invasive Cancers: A Review of Molecular Targets With the Contribution of Malignant Mesothelioma Studies.

Curcuminoids, which include natural acyclic diarylheptanoids and the synthetic analogs of curcumin, have considerable potential for fighting against all the characteristics of invasive cancers. The epithelial-to-mesenchymal transition (EMT) is a fundamental process for embryonic morphogenesis, however, the last decade has confirmed it orchestrates many features of cancer invasiveness, such as tumor cell stemness, metabolic rewiring, and drug resistance. A wealth of studies has revealed EMT in cancer is in fact driven by an increasing number of parameters, and thus understanding its complexity has now become a cornerstone for defining future therapeutic strategies dealing with cancer progression and metastasis. A specificity of curcuminoids is their ability to target multiple molecular targets, modulate several signaling pathways, modify tumor microenvironments and enhance the host's immune response. Although the effects of curcumin on these various parameters have been the subject of many reviews, the role of curcuminoids against EMT in the context of cancer have never been reviewed so far. This review first provides an updated overview of all EMT drivers, including signaling pathways, transcription factors, non-coding RNAs (ncRNAs) and tumor microenvironment components, with a special focus on the most recent findings. Secondly, for each of these drivers the effects of curcumin/curcuminoids on specific molecular targets are analyzed. Finally, we address some common findings observed between data reported in the literature and the results of investigations we conducted on experimental malignant mesothelioma, a model of invasive cancer representing a useful tool for studies on EMT and cancer.

Anterior gradient protein 2 is a marker of tumor aggressiveness in breast cancer and favors chemotherapy­induced senescence escape.

Among the different chemotherapies available, genotoxic drugs are widely used. In response to these drugs, particularly doxorubicin, tumor cells can enter into senescence. Chemotherapy­induced senescence (CIS) is a complex response. Long described as a definitive arrest of cell proliferation, the present authors and various groups have shown that this state may not be complete and could allow certain cells to reproliferate. The mechanism could be due to the activation of new signaling pathways. In the laboratory, the proteins involved in these pathways and triggering cell proliferation were studied. The present study determined a new role for anterior gradient protein 2 (AGR2) in vivo in patients and in vitro in a senescence escape model. AGR2's implication in breast cancer patients and proliferation of senescent cells was assessed based on a SWATH­MS proteomic study of patients' samples and RNA interference technology on cell lines. First, AGR2 was identified and it was found that its concentration is higher in the serum of patients with breast cancer and that this high concentration is associated with metastasis occurrence. An inverse correlation between intratumoral AGR2 expression and the senescence marker p16 was also observed. This observation led to the study of the role of AGR2 in the CIS escape model. In this model, it was found that AGR2 is overexpressed in cells during senescence escape and that its loss considerably reduces this phenomenon. Furthermore, it was shown that the extracellular form of AGR2 stimulated the reproliferation of senescent cells. The power of proteomic analysis based on the SWATH­MS approach allowed the present study to highlight the mammalian target of rapamycin (mTOR)/AKT signaling pathway in the senescence escape mechanism mediated by AGR2. Analysis of the two signaling pathways revealed that AGR2 modulated RICTOR and AKT phosphorylation. All these results showed that AGR2 expression in sera and tumors of breast cancer patients is a marker of tumor progression and metastasis occurrence. They also showed that its overexpression regulates CIS escape via activation of the mTOR/AKT signaling pathway.

A proteomic approach for plasma biomarker discovery with iTRAQ labelling and OFFGEL fractionation.

Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma "signatures," which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 microg of plasma proteins.

Biomarkers of tumor invasiveness in proteomics (Review).

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 invasive 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 paraffin­embedded sections of tumors/invaded tissues, the significance of some of their interconnections is discussed.

Curcumin Treatment Identifies Therapeutic Targets within Biomarkers of Liver Colonization by Highly Invasive Mesothelioma Cells-Potential Links with Sarcomas.

Investigations 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.