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La chirurgie est la modalité de traitement curatif la plus fréquemment utilisée dans les cancers de la tête et du cou. Elle est intégrée dans des schémas de stratification thérapeutique précis, mais la conduite de l’acte chirurgical et son évaluation ne tiennent, la plupart du temps, pas compte de la biologie tumorale. Nous présentons dans cette revue plusieurs études qui montrent comment les analyses de la biologie tumorale pourraient préciser les indications et le contour d’une résection chirurgicale, personnaliser la prise en charge péri-opératoire du patient, et faciliter la détection des récurrences tumorales. Ces études apportent ainsi une preuve de principe qu’une chirurgie de précision, c’est-à-dire adossée à la biologie tumorale, à la façon de la médecine de précision pour d’autres cancers, est applicable aux cancers de la tête et du cou.

RESUME Objectifs: les inhibiteurs de point de controle immunitaires (IPCI) entrainent frequemment des effets indesirables immunologiques (irAEs, Immune Related Adverse Events), dont certains ont ete associes a de meilleures reponses tumorales. Dans cette etude, nous avons evalue l’impact des effets indesirables rhumatologiques sur la survie globale (SG) et sur la reponse tumorale chez des patients traites par IPCI. Methodes: Il s’agissait d’une etude retrospective, observationnelle et monocentrique ayant pour objectif l’analyse de la survie globale et de la reponse tumorale de patients traites par IPCI et ayant presente un effet indesirable (EI) rhumatologique, en comparaison avec les patients n’ayant presente aucun EI. Resultats: Entre decembre 2010 et septembre 2018, 264 patients atteints de cancer de tous types ont ete inclus. Quarante-trois patients (16,3 %) ont presente au moins un EI rhumatologique. La SG mediane des patients ayant presente un EI rhumatologique etait significativement plus elevee que celle des patients sans EI : 131,6 semaines (intervalle de confiance a 95 % [IC 95 %] [69,3 — non atteint]) vs. 42,7 semaines (IC95 % [25,6 — non atteint]), respectivement (P Conclusions: La survenue d’EI rhumatologique chez les patients traites par IPCI est associee a une meilleure SG et a une meilleure reponse tumorale. Il est donc essentiel de detecter ces EI rhumatologiques le plus precocement possible, afin de permettre une prise en charge rapide et optimale des patients, compte tenu de leur potentiel de reponse a long terme.

Background: The coagulome, defined as the repertoire of genes that locally regulate coagulation and fibrinolysis, is a key determinant of vascular thromboembolic complications of cancer. In addition to vascular complications, the coagulome may also regulate the tumor microenvironment (TME). Glucocorticoids are key hormones that mediate cellular responses to various stresses and exert anti-inflammatory effects. We addressed the effects of glucocorticoids on the coagulome of human tumors by investigating interactions with Oral Squamous Cell Carcinoma, Lung Adenocarcinoma, and Pancreatic Adenocarcinoma tumor types. Methods: We analyzed the regulation of three essential coagulome components, i.e., the tissue factor (TF), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) in cancer cell lines exposed to specific agonists of the glucocorticoid receptor (GR) (dexamethasone and hydrocortisone). We used QPCR, immunoblots, small-interfering RNA, Chromatin immunoprecipitation sequencing (ChIPseq) and genomic data from whole tumor and single-cell analyses. Results: Glucocorticoids modulate the coagulome of cancer cells through a combination of indirect and direct transcriptional effects. Dexamethasone directly increased PAI-1 expression in a GR-dependent manner. We confirmed the relevance of these findings in human tumors, where high GR activity/high SERPINE1 expression corresponded to a TME enriched in active fibroblasts and with a high TGF-β response. Conclusion: The transcriptional regulation of the coagulome by glucocorticoids that we report may have vascular consequences and account for some of the effects of glucocorticoids on the TME.

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Surgical resection is the most frequent curative treatment proposed to patients with head and neck cancers. It is currently integrated into specific therapeutic schemes and therapeutic stratification, but the surgical procedure itself as well as its evaluation do not rely on tumor biology. Here, we present a number of recent studies, mostly based on system analyses and genomics, that show how tumor analyses could help to: i) define the indications and the extent of surgical resections; ii) personalize the perioperative management; iii) facilitate the detection of post-surgical tumor recurrence. Overall, these studies provide a proof of principle that precision surgery, i.e. based on tumor biology, similarly to precision medicine, is applicable to head and neck cancers.Principe et applicabilité de la chirurgie de précision aux cancers de la tête et du cou.La chirurgie est la modalité de traitement curatif la plus fréquemment utilisée dans les cancers de la tête et du cou. Elle est intégrée dans des schémas de stratification thérapeutique précis, mais la conduite de l’acte chirurgical et son évaluation ne tiennent, la plupart du temps, pas compte de la biologie tumorale. Nous présentons dans cette revue plusieurs études qui montrent comment les analyses de la biologie tumorale pourraient préciser les indications et le contour d’une résection chirurgicale, personnaliser la prise en charge péri-opératoire du patient, et faciliter la détection des récurrences tumorales. Ces études apportent ainsi une preuve de principe qu’une chirurgie de précision, c’est-à-dire adossée à la biologie tumorale, à la façon de la médecine de précision pour d’autres cancers, est applicable aux cancers de la tête et du cou.

Background Oral squamous cell carcinoma (OSCC) is the most frequent type of tumor arising from the oral cavity. Surgery is the cornerstone of the treatment of these cancers. Tumor biology has long been overlooked as an important contributor to the outcome of surgical procedures, but recent studies are challenging this concept. Molecular analyses of tumor DNA or RNA provide a rich source of information about the biology of OSCC. Methods We searched for relevant articles using PubMed. We examined in particular the prospect of applying molecular methods for minimally invasive exploration of OSCC biology. Results We examined five potential applications of genomics to the surgical management and study of OSCC: i) assessing oral potentially malignant lesions; ii) tumor staging prior to surgery; iii) predicting postoperative risk in locally advanced tumors; iv) measuring minimal residual disease and optimizing the longitudinal monitoring of OSCC; and v) predicting the efficacy of medical treatment. Conclusions Genomic information can be harnessed in order to identify new biomarkers that could improve the staging, choice of therapy and management of OSCC. The identification of new biomarkers is awaited for better personalization of the surgical treatment of OSCC.

Simple Summary Cancer is associated with a wide spectrum of hemostatic complications that range from thrombotic events to hemorrhage. The tumor coagulomes, i.e., the essential actors that locally regulate coagulation and fibrinolysis, play a key role in these complications. They might also play a regulatory role in various cell types of the tumor microenvironment. Here, we explored the coagulome of Oral Squamous Cell Carcinoma (OSCC) across tumor types, between OSCC tumors and within individual tumors. The coagulome of OSCC is characterized by a high expression of antipodal activators of coagulation and fibrinolysis, and subpopulations of pro-coagulant and pro-fibrinolytic cancer cells coexist within individual tumors. Importantly, we noted that dendritic cells within OSCC with a procoagulant profile express high levels of key immune checkpoint molecules. Further studies examining a possible negative modulation of the tumor’s adaptive immune response by the coagulation process are warranted. Abstract Background: Hemostatic complications, ranging from thromboembolism to bleeding, are a significant source of morbidity and mortality in cancer patients. The tumor coagulome represents the multiple genes and proteins that locally contribute to the equilibrium between coagulation and fibrinolysis. We aimed to study the coagulome of Oral Squamous Cell Carcinoma (OSCC) and examine its link to the tumor microenvironment (TME). Methods: We used data from bulk tumor DNA/RNA-seq (The Cancer Genome Atlas), single-cell RNA-seq data and OSCC cells in culture. Results: Among all tumor types, OSCC was identified as the tumor with the highest mRNA expression levels of F3 (Tissue Factor, TF) and PLAU (urokinase type-plasminogen activator, uPA). Great inter- and intra-tumor heterogeneity were observed. Single-cell analyses showed the coexistence of subpopulations of pro-coagulant and pro-fibrinolytic cancer cells within individual tumors. Interestingly, OSCC with high F3 expressed higher levels of the key immune checkpoint molecules CD274/PD-L1, PDCD1LG2/PD-L2 and CD80, especially in tumor dendritic cells. In vitro studies confirmed the particularity of the OSCC coagulome and suggested that thrombin exerts indirect effects on OSCC cells. Conclusions: OSCC presents a specific coagulome. Further studies examining a possible negative modulation of the tumor’s adaptive immune response by the coagulation process are warranted.

After allogeneic hematopoietic stem-cell transplantation (alloHSCT), the chimerism assay is used to monitor cell engraftment and quantify the respective proportions of donor/recipient cells in blood or bone-marrow samples. Here, we aimed to better assess the utility of determining CD3

Highlights • PD-L1 is an important immune checkpoint molecule expressed by HNSCC. • 5-FU induces PD-L1 expression in HNSCC cells. • PD-L1 upregulation is DNA damage Response- and JAK-dependent. • 5-FU potentiates the effect of the inflammatory cytokine Ifn-γ. • Targeting EGFR with cetuximab blunts PD-L1 expression induced by 5-FU., Objectives The immune checkpoint molecule PD-L1 (CD274) is a crucial regulator of the tumor immune response. Its expression has been reported in the therapeutic context in Head and Neck Squamous Cell Carcinoma (HNSCC), but it remains unclear how therapeutically approved molecules regulate PD-L1 expression in HNSCC cells. Materials and methods Three HNSCC cell lines (BICR6, PE/CA-PJ34 and PE/CA-PJ41) were used to analyze PD-L1 expression by immunoblotting, immunofluorescence and QPCR. Freely-available single cell RNAseq data from HNSCC were also used. Results 5-Fluorouracil (5-FU) increased the expression of PD-L1 with high efficacy in HNSCC cells. Single cell RNAseq data suggested the specificity of the regulation of PD-L1 in this context. The effect of 5-FU on PD-L1 expression was related to its genotoxic effect and was prevented by extracellular application of thymidine or using a chemical inhibitor of the DNA damage Response kinases ATM/ATR. We found that the effect of 5-FU was additive or synergistic with IFN-γ, the canonical inducer of PD-L1 in epithelial cells. QPCR analysis confirmed this finding and identified JAK-dependent transcriptional activation of PD-L1/CD274 as the underlying mechanism. The induction of PD-L1 by 5-FU was partially prevented by Epidermal Growth Factor Receptor (EGFR) inhibition with cetuximab. Conclusion Our study highlights the specific DNA Damage Response- and JAK- dependent induction of PD-L1 by 5-FU in HNSCC cells. This induction is regulated by the cytokine context and is potentially therapeutically actionable.

International audience; Background/Aim: Multiple myeloma (MM) is characterized by high production of immunoglobulins resulting in a constant source of endoplasmic reticulum (ER)-stress. Mesencephalic astrocyte-derived neurotrophic factor (MANF) was identified as a possible circulating biomarker that could help in monitoring ER-stress mediated diseases. Materials and Methods: To assess the relevance of MANF in MM, we performed in silico and in vitro analysis in malignant cell lines including the myeloma cell line RPMI 8226. Serum MANF concentration was compared between healthy subjects (n=60), patients with MM (n=68), or those with monoclonal gammopathy of undetermined significance (MGUS) (n=73). Results: MANF mRNA expression was upregulated in the RPMI 8226 cell line, and higher secretion of MANF was measured in RPMI 8226 supernatant. Serum MANF levels were not significantly different between MM or MGUS patients and those in age-and sex-matched healthy controls. Conclusion: MANF was not validated as a biomarker of interest in MM patients. Its potential implication in myeloma pathogenesis should be investigated.

International audience; Highlights- A hypercoagulable state established within the tumor accounts for the frequent hemostatic complications that occur in cancer patients, including venous thromboembolism.- The coagulation cascades exert multiple direct and indirect effects on cancer cells and the components of the tumor microenvironment, both within and outside of the vascular space.- The recent application of systems biology and single-cell approaches provides a comprehensive and deeper view of the coagulome and highlights its link to the tumor microenvironment.- The study of the coagulome provides unprecedented opportunities to explore the tumor microenvironment, to better predict the hemostatic complications, revisit the significance of clinical biomarkers, and it might ultimately enable us to steer the tumor immune response or vascular integrity.Human tumors often trigger a hypercoagulable state that promotes hemostatic complications, including venous thromboembolism. The recent application of systems biology to the study of the coagulome highlighted its link to shaping the tumor microenvironment (TME), both within and outside of the vascular space. Addressing this link provides the opportunity to revisit the significance of biomarkers of hemostasis and assess the communication between vasculature and tumor parenchyma, an important topic considering the advent of immune checkpoint inhibitors and vascular normalization strategies. Understanding how the coagulome and TME influence each other offers exciting new prospects for predicting hemostatic complications and boosting the effectiveness of cancer treatment.

Immune checkpoint inhibitors (ICIs) frequently induce immune related adverse events (irAEs) that may be associated with more favorable clinical outcomes. We aimed to evaluate the impact of all types of rheumatic adverse events (AEs) on overall survival (OS) and tumor response in patients treated with ICIs.We performed a single-center retrospective observational study to analyze the OS and tumor response in patients receiving ICIs who experienced a rheumatic AE compared to those who did not experience any AE.From December 2010 to September 2018, 264 patients with any cancer type were included. Forty-three patients (16.3%) presented with at least one rheumatic AE. The median OS of patients with rheumatic AEs was significantly higher than that of patients without AEs, with 132 weeks (95% CI [69.3-not reached]) and 42.7 weeks (95% CI [25.6-not reached]), respectively (P0.01). This result remained significant after multivariate analysis (HR 0.54, 95% CI [0.30-0.97], P0.05). Also, tumor response was better in patients with rheumatic AEs.The occurrence of rheumatic AEs in patients treated with ICIs is associated with better survival and tumor response. Therefore, it seems essential to detect rheumatic AEs as early as possible to allow rapid and optimal management, given the long-term response potential of these patients.

Solid tumors often establish a procoagulable state that can lead to venous thromboembolism (VTE). Although some of the key genes involved in this process are known, no previous study has compared the “coagulome”, i.e., the expression of coagulation/fibrinolysis genes, across different primary tumor types. It is also unclear whether the coagulome is associated with specific characteristics of the tumor microenvironment (TME). We aimed to address this question. We analyzed the expression of the genes F3, PLAU, PLAT, PLAUR, SERPINB2, and SERPINE1 in 32 cancer types using data from The Cancer Genome Atlas (TCGA) and other freely available resources. We identified specific expression patterns of procoagulant and fibrinolytic genes. The expression of the Tissue Factor (F3) was found to be tumor type dependent, with the highest expression in glioblastoma (GBM), a highly procoagulable tumor type. Conversely, high expression of the fibrinolysis gene cluster PLAU, PLAUR, SERPINE1 was consistently linked to the characteristics of the TME (monocytic infiltration) and high expression of important checkpoints of the immune response, such as PD-L2 and CD276/B7-H3. These tumor-specific patterns of expression might partially explain the differences in VTE risk among tumor types. We propose that biomarkers of coagulation fibrinolysis might provide valuable information about the TME in cancer patients.

The identification of the biological determinants that shape the response of tumors to medical therapies offers perspectives for better patient stratification and therapeutic targeting. Here, we discuss how genomics could help to improve the surgical treatment of head and neck squamous cell carcinoma (HNSCC). We examine the potential use of genomic analyses for: i) refining and standardizing the indications for surgery, ii) the choice of surgical procedure, and iii) the follow-up of patients with resected tumors. We highlight the studies that used genomics to explore the contribution of tumor biology to the outcome of surgery. We discuss the important developments that are challenging current surgical practice in HNSCC, such as neoadjuvant immunotherapy and the analysis of circulating DNA. Genomic analyses provide practical tools that could help improve the pathological diagnosis and staging of HNSCC, and increase the appreciation of the importance of tumor biology in the outcome of surgery. Identification of biomarkers will likely contribute to a move toward precision surgery of HNSCC, i.e. the personalization of surgical practice based on tumor biology.

Purpose of review Perineural invasion (PNI) is a pathological feature frequently observed in head and neck squamous cell carcinoma (HNSCC). The difficulties of pathological standardization and the lack of a simple validated experimental model to study PNI render its analysis complex. Here, we aim to summarize the recent advances in the understanding of the biology of PNI in HNSCC and their potential clinical implications. Recent findings PNI is a multistep process leading to a dialogue between cancer cells and nerve fibers in HNSCC. Recent studies have identified some of the active molecular mechanisms involved in PNI. Comprehensive studies addressing the transcriptional regulation of PNI bring interesting perspectives for a standardized molecular diagnosis of PNI and a better assessment of its contribution to the aggressiveness of HNSCC. Summary Perineural invasion is a complex process that reflects specific tumor biology. In addition to unveiling new fundamental concepts about the tumor microenvironment, research on PNI promises to identify new biomarkers, enabling progress in therapeutic development against HNSCC.

Perineural invasion (PNI) is a common histopathological finding in head and neck squamous cell carcinoma (HNSCC). We aimed to explore the molecular mechanisms involved in PNI and the role of PNI as an aggressive pathological feature.We used data from The Cancer Genome Atlas (TCGA) to relate the histological presentation of 528 HNSCC tumours to clinical, whole genome expression and proteomic data.We identified a specific gene expression profile highly enriched in genes related to muscle differentiation/function and associated with PNI in HNSCC. We explored the clinical significance of this profile in three groups of HNSCC tumours stratified according to their low, intermediate or high risk of post-surgical recurrence. In the "low-risk" group, defined as tumours indicated for surgery without adjuvant radiotherapy (n = 51), the PNI gene expression profile identified a subset of HNSCC with a higher rate of tumour recurrence, decreased Disease Free Survival (DFS) and Overall Survival (OS) (p 0.0001 and p = 0.0064, respectively). Comparable results were observed in "intermediate risk" tumours (n = 112), but not in "high risk" tumours (n = 147), whose prognosis was driven by the presence of lymph node extracapsular spread. Finally, we found that tumours with histological PNI had increased activation levels of the Akt/PKB and mTOR (mammalian Target Of Rapamycin) kinases.PNI is characterised by a specific gene expression profile and distinct biological characteristics. Analysing the PNI gene expression profile holds potential for therapeutic stratification of HNSCC and identification of a subset of tumours with a higher risk of recurrence.

Les progres de la decennie passee ont rendu l’etude du traitement medical des cancers plus efficace, mais ils ont aussi revele la complexite des mecanismes de la cancerogenese. Pour la plupart des tumeurs humaines, on connait plusieurs cibles therapeutiques et des medicaments potentiels. Malheureusement, les acteurs identifies comme des cibles therapeutiques fonctionnent en effet souvent en reseaux, ce qui engendre des suppleances fonctionnelles et des proprietes de resilience/resistance au ciblage therapeutique. Le recours a la modelisation mathematique permet de realiser des etudes de systeme dans l’optique d’ameliorer le ciblage therapeutique des tumeurs solides. Nous presentons les principaux types de modeles mathematiques et des exemples recents illustrant leur interet en oncobiologie moleculaire.

// Cedric Boudot 1, * , Lucie Henaut 1, * , Ursula Thiem 1 , Sandra Geraci 2 , Mariangela Galante 1 , Paulo Saldanha 1 , Zuzana Saidak 1 , Isabelle Six 1 , Philippe Clezardin 2 , Said Kamel 1 and Romuald Mentaverri 1 1 Inserm U1088, Centre Universitaire de Recherche en Sante, Universite de Picardie Jules Verne, Amiens, France 2 Inserm UMR 1033, LYOS, Lyon, France * These authors have contributed equally to this work Correspondence to: Said Kamel, email: said.kamel@u-picardie.fr Keywords: bone metastasis, breast cancer, calcium-sensing receptor, epiregulin, osteolysis Received: July 15, 2016 Accepted: March 14, 2017 Published: April 10, 2017 ABSTRACT Introduction and Aims: Osteolytic bone metastases are observed in advanced cases of breast cancer. In vitro data suggest that the activity of the calcium-sensing receptor (CaSR) expressed by metastatic cells could potentiate their osteolytic potential. This study aimed to demonstrate in vivo the involvement of the CaSR in breast cancer cells osteolytic potential and to identify potential targets linked to CaSR activity. Methods and Results: MDA-MB-231 stably transfected with plasmids containing either a full-length wild-type CaSR (CaSR-WT), or a functionally inactive dominant negative mutant (CaSR-DN) or an empty vector (EV) were intratibially injected into Balb/c-Nude mice. X-ray analysis performed 19 days after injection showed a dramatic increase of osteolytic lesions in mice injected with CaSR-WT-transfected cells as compared to mice injected with EV- or CaSR-DN-transfected cells. This was associated with decreased BV/TV ratio and increased tumor burden. Epiregulin, an EGF-like ligand, was identified by a DNA microarray as a possible candidate involved in CaSR-mediated osteolysis. Indeed, in vitro , CaSR overexpression increased both epiregulin expression and secretion as compared to EV- or CaSR-DN-transfected cells. Increased epiregulin expression was also detected in osteolytic bone lesions from mice injected with CaSR-WT-transfected MDA-MB-231. In vitro , exposure of osteoblastic cells (HOB and SaOS2) to exogenous epiregulin significantly decreased OPG mRNA expression. Exposure of osteoblastic cells to conditioned media prepared from CaSR-WT-transfected cells also decreased OPG expression. This effect was partially blocked after addition of an anti-epiregulin antibody. Conclusions: Overexpression of a functional CaSR in metastatic breast cancer cells dramatically amplifies their osteolytic potential through epiregulin-mediated OPG downregulation.

The tumor microenvironment is an important determinant of glioblastoma (GBM) progression and response to treatment. How oncogenic signaling in GBM cells modulates the composition of the tumor microenvironment and its activation is unclear. We aimed to explore the potential local immunoregulatory function of ERK1/2 signaling in GBM. Using proteomic and transcriptomic data (RNA seq) available for GBM tumors from The Cancer Genome Atlas (TCGA), we show that GBM with high levels of phosphorylated ERK1/2 have increased infiltration of tumor-associated macrophages (TAM) with a non-inflammatory M2 polarization. Using three human GBM cell lines in culture, we confirmed the existence of ERK1/2-dependent regulation of the production of the macrophage chemoattractant CCL2/MCP1. In contrast with this positive regulation of TAM recruitment, we found no evidence of a direct effect of ERK1/2 signaling on two other important aspects of TAM regulation by GBM cells: (1) the expression of the immune checkpoint ligands PD-L1 and PD-L2, expressed at high mRNA levels in GBM compared with other solid tumors; (2) the production of the tumor metabolite lactate recently reported to dampen tumor immunity by interacting with the receptor GPR65 present on the surface of TAM. Taken together, our observations suggest that ERK1/2 signaling regulates the recruitment of TAM in the GBM microenvironment. These findings highlight some potentially important particularities of the immune microenvironment in GBM and could provide an explanation for the recent observation that GBM with activated ERK1/2 signaling may respond better to anti-PD1 therapeutics.

International audience; Objectives: Achieving complete tumour resection is one of the main goals of surgery for head and neck squamous cell carcinoma (HNSCC) tumours. Whether biological characteristics of tumours contribute to the surgical resectability and the presence of positive surgical margins (SM) after resection of HNSCC is unclear. We aimed to address this issue.Materials and methods: We used data from The Cancer Genome Atlas (TCGA) to relate the SM status of 356 HNSCC tumours covering five major primary locations (tongue, larynx, tonsils, floor of mouth and buccal mucosa) with data from multiple omics approaches (transcriptomic, genomic and proteomic analyses).Results: We identified three differentially expressed genes whose expression was significantly associated with the presence of positive SM in tongue tumours (n = 144). The three genes (CCDC66, ZRANB2 and VCPKMT) displayed significantly higher mRNA levels in tongue tumours with positive SM compared to tumours with negative SM. The corresponding gene expression signature identified tongue tumours with a positive SM with high sensitivity and specificity (85% and 76%, respectively, Area Under the Curve (AUC) = 0.84). Tongue tumours with this signature were characterised by a high grade, elevated proliferation levels and a tumour stroma with fewer fibroblasts and endothelial cells.Conclusion: Positive SM were found to be strikingly associated with tumour biology in tongue tumours. These findings offer interesting perspectives for biomarker identification and precision surgery in these tumours.

International audience

Uric acid (UA) is the end product of the catabolism of purines, and its serum levels are commonly increased in cancer patients. We aimed to explore the transcriptional regulation of tumour uricogenesis in human tumours, and relate uricogenesis with tumour pathological and pharmacological findings. Using data from The Cancer Genome Atlas (TCGA), we analysed the expression levels of xanthine dehydrogenase (XDH) and adenine phosphoribosyltransferase (APRT), two key enzymes in UA production and the purine salvage pathway, respectively. We found large differences between tumour types and individual tumours in their expression of XDH and APRT. Variations in locus-specific DNA methylation and gene copy number correlated with the expression levels of XDH and APRT in human tumours respectively. We explored the consequences of this differential regulation of uricogenesis. Tumours with high levels of XDH mRNA were characterised by higher expression of several genes encoding pro-inflammatory and immune cytokines, and increased levels of tumour infiltration with immune cells. Finally, we studied cancer drug sensitivity using data from the National Cancer Institute-60 (NCI-60) database. A specific correlation was found between the expression levels of APRT and cell sensitivity to the chemotherapeutic agent 5-fluorouracil (5-FU). Our findings underline the existence of great differences in uricogenesis between different types of human tumours. The study of uricogenesis offers promising perspectives for the identification of clinically relevant molecular biomarkers and for tumour stratification in the therapeutic context.

International audience; Sorafenib is the standard of care for the treatment of advanced hepatocellular carcinoma (HCC). However, identifying secreted biomarkers that predict sorafenib efficacy in all HCC patients remains challenging. It was recently reported that sorafenib interferes with protein homeostasis and inhibits global translation in tumour cells. A likely consequence of this inhibition would be the interruption of autocrine loops. The aim of the present study was to investigate the effect of sorafenib on two growth factors implicated in autocrine loops and HCC tumour invasion: amphiregulin (AREG) and vascular endothelial growth factor (VEGF). ELISA, quantitative polymerase chain reaction analysis, western blotting and a cytokine array were performed on HCC cell lines and the prognostic role of these two biomarkers in HCC patients was evaluated. Serum AREG and VEGF levels were assayed by ELISA in 55 patients with advanced HCC treated with sorafenib. It was observed that sorafenib decreased AREG, VEGF and cytokine expression at the transcriptional and post-transcriptional levels. All HCC patients in our cohort had detectable concentrations of AREG and VEGF both at baseline and after sorafenib treatment. The decreased serum levels of AREG and VEGF after 15 days of sorafenib treatment were significantly associated with better overall and progression-free survival. The results of the multivariate analysis demonstrated that a decrease in AREG was an independent prognostic indicator of overall survival (hazard ratio, 0.208; 95% confidence interval, 0.173-0.673; P=0.0003). These results suggest that sorafenib inhibits autocrine loops and that early decrease in serum AREG or VEGF levels predicts sorafenib efficacy in HCC patients.

Background/aim The squamous cell carcinoma antigen (SCCA), encoded by the genes SERPINB3/B4, is a tumour marker produced by head and neck squamous cell carcinoma (HNSCC). We aimed to examine SERPINB3/B4 mRNA levels and its clinical significance in the therapeutic context. Materials and methods We retrieved mRNA expression levels, clinical, pathological and genomic data for 520 HNSCC from The Cancer Genome Atlas (TCGA). Results HNSCC tumours express high levels of SERPINB3/B4 mRNA. SERPINB3 expression differs depending on Human papillomavirus (HPV) infection status, primary tumour location, grade and differentiation, extension to lymph nodes and extracapsular spread. Interestingly, we observed an association between SERPINB3/B4 and the presence of tumour immune infiltrate as well as the expression of the immune checkpoint regulators PD-L1/PD-L2 that depended on HPV status. Conclusion Our findings point to potential interest of SERPINB3/B4 for the stratification of HNSCC patients in the therapeutic context.

Recent progress in biology has made the study of the medical treatment of cancer more effective, but it has also revealed the large complexity of carcinogenesis and cell signaling. For many types of cancer, several therapeutic targets are known and in some cases drugs against these targets exist. Unfortunately, the target proteins often work in networks, resulting in functional adaptation and the development of resilience/resistance to medical treatment. The use of mathematical modeling makes it possible to carry out system-level analyses for improved study of therapeutic targeting in solid tumours. We present the main types of mathematical models used in cancer research and we provide examples illustrating the relevance of these approaches in molecular oncobiology.

The squamous cell carcinoma antigen (SCCA), encoded by the genes SERPINB3/B4, is a tumour marker produced by head and neck squamous cell carcinoma (HNSCC). We aimed to examine SERPINB3/B4 mRNA levels and its clinical significance in the therapeutic context.We retrieved mRNA expression levels, clinical, pathological and genomic data for 520 HNSCC from The Cancer Genome Atlas (TCGA).HNSCC tumours express high levels of SERPINB3/B4 mRNA. SERPINB3 expression differs depending on Human papillomavirus (HPV) infection status, primary tumour location, grade and differentiation, extension to lymph nodes and extracapsular spread. Interestingly, we observed an association between SERPINB3/B4 and the presence of tumour immune infiltrate as well as the expression of the immune checkpoint regulators PD-L1/PD-L2 that depended on HPV status.Our findings point to potential interest of SERPINB3/B4 for the stratification of HNSCC patients in the therapeutic context.

Cell-cell and cell-matrix interactions mediated by cell adhesion molecules are important mechanisms controlling cell fate and function. Here, we review recent advances in the implication of the cell adhesion molecules integrins and cadherins in the control of osteoblastogenesis and bone formation. We discuss emerging evidence indicating that signaling pathways mediated by integrins and cadherins and their crosstalk with the Wnt/β-catenin signaling pathway regulate osteogenic differentiation and mechanotransduction. We also offer a comprehensive view of the mechanisms by which some integrins and cadherins control the differentiation of cells of the osteoblast lineage in bone marrow niches. Understanding how specific integrins or cadherins may promote osteogenic cell differentiation, bone formation, and repair may lead to novel therapeutic strategies.

Age-related bone loss is characterized by reduced osteoblastogenesis and excessive bone marrow adipogenesis. The mechanisms governing bone marrow mesenchymal stromal cell (BMSC) differentiation into adipocytes or osteoblasts during aging are unknown. We show here that overexpressing N-cadherin (Cadh2) in osteoblasts increased BMSC adipocyte differentiation and reduced osteoblast differentiation in young transgenic (Tg) mice whereas this phenotype was fully reversed with aging. The reversed phenotype with age was associated with enhanced Wnt5a and Wnt10b expression in osteoblasts and a concomitant increase in BMSC osteogenic differentiation. Consistent with this mechanism, conditioned media from young wild type osteoblasts inhibited adipogenesis and promoted osteoblast differentiation in BMSC from old Cadh2 Tg mice, and this response was abolished by Wnt5a and Wnt10b silencing. Transplantation of BMSC from old Cadh2 Tg mice into young Tg recipients increased Wnt5a and Wnt10b expression and rescued BMSC osteogenic differentiation. In senescent osteopenic mice, blocking the CADH2-Wnt interaction using an antagonist peptide increased Wnt5a and Wnt10b expression, bone formation, and bone mass. The data indicate that Cadh2/Wnt interaction in osteoblasts regulates BMSC lineage determination, bone formation, and bone mass and suggest a therapeutic target for promoting bone formation in the aging skeleton.

Intermittent administration of parathyroid hormone (PTH) 1–34 at a standard dose has been shown to induce anabolic effects in bone. However, whether low-dose PTH promotes bone formation during senescence is unknown. To address this issue, we determined the effects of low-dose PTH and analysed the underlying mechanisms in prematurely senescent mice that display osteopenia. Treatment of 9-week-oldSamp6mice for 6 weeks with PTH at a standard dose (100 μg/kg per day) increased vertebral and femoral bone mass and improved bone microarchitecture as a result of increased bone-forming surfaces and mineral apposition rate (MAR). At a tenfold lower dose (10 μg/kg per day), PTH increased axial bone volume and trabecular thickness, as detected by bone histomorphometry but not by micro-computed tomography analysis. This anabolic effect resulted from increased osteoblast activity, as reflected by increased serum N-terminal propeptide of type 1 procollagen (P1NP) levels and MAR, with unchanged bone-forming surface or osteoblast surface. Mechanistically, low-dose PTH increased the expression of osteoblast markers in bone marrow stromal cells and mature osteoblasts, which was associated with increased expression of the Wnt effectorWisp1. Moreover, low-dose PTH decreased the expression of theMef2ctranscription factor, resulting in decreasedSostexpression in osteoblasts/osteocytes. These results indicate that PTH at a low dose is effective at promoting bone formation and increased bone volume in senescent osteopenic mice through increased osteoblast activity and modulation of specific Wnt effectors, which raises the potential therapeutic use of intermittent PTH at low dose to increase bone forming activity and bone mass in skeletal senescence.

The RAS-RAF-MEK-ERK cascade is a key oncogenic signal transduction pathway activated in many types of tumours in humans. Sorafenib, the medical treatment of reference against advanced stages of hepatocellular carcinoma (HCC), inhibits the RAF-MEK-ERK cascade in HCC cells. Based on previous studies suggesting that this cascade is an important target of sorafenib in HCC cells, we explored its regulation using mathematical modelling and ordinary differential equations. We analysed the dynamic regulation of the core components of the RAF-MEK-ERK cascade in three human HCC cell lines (Huh7, Hep3B and PLC/PRF5) with heterogeneous responses to sorafenib. In silico predictions derived from our mathematical model suggested that the disappearance of phosphorylated MEK and ERK proteins catalysed by cellular phosphatases is an essential mechanism underlying the anti-ERK efficacy of sorafenib in HCC cells. This prediction was experimentally validated using specific inhibitors of the phosphatases PP2A (Protein Phosphatase 2A) and DUSP1/6 (Dual-specificity phosphatases 1/6). These findings highlight an unexpected mode of action of sorafenib on the kinome of HCC cells, and open new perspectives regarding the therapeutic targeting of the RAF-MEK-ERK cascade in this context.

Background Sorafenib, a kinase inhibitor active against various solid tumours, induces oxidative stress and ferroptosis, a new form of oxidative necrosis, in some cancer cells. Clinically-applicable biomarkers that reflect the impact of sorafenib on the redox metabolism of cancer cells are lacking. Methods We used gene expression microarrays, real-time PCR, immunoblot, protein-specific ELISA, and gene reporter constructs encoding the enzyme luciferase to study the response of a panel of cancer cells to sorafenib. Tumour explants prepared from surgical hepatocellular carcinoma (HCC) samples and serum samples obtained from HCC patients receiving sorafenib were also used. Results We observed that genes of the metallothionein-1 (MT1) family are induced in the HCC cell line Huh7 exposed to sorafenib. Sorafenib increased the expression of MT1G mRNA in a panel of human cancer cells, an effect that was not observed with eight other clinically-approved kinase inhibitors. We identified the minimal region of the MT1G promoter that confers inducibility by sorafenib to a 133 base pair region containing an Anti-oxidant Response Element (ARE) and showed the essential role of the transcription factor NRF2 (Nuclear factor erythroid 2-Related Factor 2). We examined the clinical relevance of our findings by analysing the regulation of MT1G in five tumour explants prepared from surgical HCC samples. Finally, we showed that the protein levels of MT1 increase in the serum of some HCC patients receiving sorafenib, and found an association with reduced overall survival. Conclusion These findings indicate that MT1 constitute a biomarker adapted for exploring the impact of sorafenib on the redox metabolism of cancer cells. Electronic supplementary material The online version of this article (doi:10.1186/s12943-016-0526-2) contains supplementary material, which is available to authorized users.

With aging, bone marrow mesenchymal stromal cell (MSC) osteoblast differentiation decreases whereas MSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. Here, we investigated whether activation of cell signaling by strontium ranelate (SrRan) can reverse the excessive adipogenic differentiation associated with aging. In murine MSC cultures, SrRan increased Runx2 expression and matrix mineralization and decreased PPARγ2 expression and adipogenesis. This effect was associated with increased expression of the Wnt noncanonical representative Wnt5a and adipogenic modulator Maf and was abrogated by Wnt- and nuclear factor of activated T-cells (NFAT)c antagonists, implying a role for Wnt and NFATc/Maf signaling in the switch in osteoblastogenesis to adipogenesis induced by SrRan. To confirm this finding, we investigated the effect of SrRan in SAMP6 senescent mice, which exhibit decreased osteoblastogenesis, increased adipogenesis, and osteopenia. SrRan administration at a clinically relevant dose level increased bone mineral density, bone volume, trabecular thickness and number, as shown by densitometric, microscanning, and histomorphometric analyses in long bones and vertebrae. This attenuation of bone loss was related to increased osteoblast surface and bone formation rate and decreased bone marrow adipocyte volume and size. The restoration of osteoblast and adipocyte balance induced by SrRan was linked to increased Wnt5a and Maf expression in the bone marrow. The results indicate that SrRan acts on lineage allocation of MSCs by antagonizing the age-related switch in osteoblast to adipocyte differentiation via mechanisms involving NFATc/Maf and Wnt signaling, resulting in increased bone formation and attenuation of bone loss in senescent osteopenic mice.

The proteins of the B-cell lymphoma 2 (Bcl-2) family are important regulators of apoptosis under normal and pathological conditions. Chemical compounds that block the antiapoptotic proteins of this family have been introduced, such as 4-[4-[(4′-Chloro[1,1′-biphenyl]-2-yl)methyl]-1-piperazinyl]- N -[[4-[[(1 R )-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide (ABT-737), a BH3-mimetic that neutralizes Bcl-2 and Bcl-xL. In this study, we used ABT-737 to explore the dynamic regulation of Bcl-2 proteins in living cells of different origins. Using ABT-737 as well as RNA interference or the application of growth factors, we examined the impact of the functional availability of the antiapoptotic proteins Bcl-2 and Bcl-2-extra large (Bcl-xL) on the Bcl-2 network. We report that ABT-737 increases the expression of Bcl-2-associated death promoter (Bad), a proapoptotic partner of the proteins Bcl-2 and Bcl-xL. Our observations indicate that Bad overexpression induced by ABT-737 results from the control of its normally rapid protein turnover, leading to the stabilization of this protein. We demonstrate the relevance of Bad post-translational regulation by Bcl-xL to the physiological setting using RNA interference against Bcl-xL as well as the application of epidermal growth factor, a growth factor that promotes the dissociation of Bad from Bcl-xL. Our results highlight a new facet of the mode of action of the antiapoptotic proteins Bcl-2 and Bcl-xL consisting of the regulation of the stability of the protein Bad. Finally, our results shed light on the mode of action of ABT-737, currently the best characterized inhibitor of the antiapoptotic proteins of the Bcl-2 family, and bear important implications regarding its use as an anticancer drug.

The calcium-sensing receptor (CaR) is responsive to changes in the extracellular Ca(2+) (Ca(2+)(o)) concentration. It is a member of the largest family of cell surface receptors, the G protein-coupled receptors, and it has been shown to be involved in Ca(2+)(o) homeostasis. Apart from its primary role in Ca(2+)(o) homeostasis, the CaR may be involved in phenomena that allow for the development of many types of benign or malignant tumors, from parathyroid adenomas to breast, prostate, and colon cancers. For example, whereas the CaR is expressed in both normal and malignant breast tissue, increased CaR levels have been reported in highly metastatic primary breast cancer cells and breast cancer cell lines, possibly contributing to their malignancy and associated alterations in their biological properties. In these settings the CaR exhibits oncogenic properties. Enhanced CaR expression and altered proliferation of prostate cancer cells in response to increased Ca(2+)(o) have also been described. In contrast, colon and parathyroid cancers often present with reduced or absent CaR expression, and activation of this receptor decreases cell proliferation, suggesting a role for the CaR as a tumor suppressor gene. Thus, the CaR may play an important role in the development of many types of neoplasia. Herein, we review the role of the CaR in various benign and malignant tumors in further detail, describing its contribution to parathyroid tumors, breast, prostate, and colon cancers, and we evaluate how pharmacological manipulations of this receptor may be of interest for the treatment of certain cancers in the future.

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC). A decrease in the serum levels of Alpha-fetoprotein (AFP) is reported to be the biological parameter that is best associated with disease control by sorafenib. In order to provide a biological rationale for the variations of AFP, we analyzed the various steps of AFP production in human HCC cell lines exposed to sorafenib. Sorafenib dramatically reduced the levels of AFP produced by HCC cells independently of its effect on cell viability. The mRNA levels of AFP decreased upon sorafenib treatment, while the AFP protein remained localized in the Golgi apparatus. Sorafenib activated the Regulated Inositol-Requiring Enzyme-1α (IRE-1α) and the PKR-like ER Kinase (PERK)-dependent arms of the Unfolded Protein Response (UPR). The inhibition of IRE-1α partially restored the mRNA levels of AFP upon treatment with sorafenib. The inhibition of both pathways partially prevented the drop in the production of AFP induced by sorafenib. The findings provide new insights on the regulation of AFP, and identify it as a biomarker suitable for the exploration of HCC cell proteostasis in the context of therapeutic targeting.

Sorafenib is the medical reference for treatment of hepatocellular carcinoma (HCC). Multiple forms of cytotoxicity are induced by sorafenib in HCC cells in vitro but it is unclear what extent of apoptosis and necrosis is induced in HCC patients receiving sorafenib.The M30 and M65 biomarkers, which reflect the release of cytokeratin-18 and its apoptotic cleavage fragments, were measured in patients with HCC (n=36) and matched patients with cirrhosis (n=47). A serum sample was collected from 20 patients with HCC four weeks after the onset of treatment with sorafenib.Basal serum levels of M30 and M65 were increased in patients with HCC compared to those with uncomplicated cirrhosis. No statistically significant increase in the level of M30 or M65 was found in the sera of patients with HCC after sorafenib.The findings indicate that sorafenib is not a potent inducer of HCC cell death in most patients.

The α5β1 integrin is a key fibronectin (FN) receptor that binds to RGD-containing peptides to mediate cell adhesion. We previously reported that α5β1 integrin promotes osteogenic differentiation in mesenchymal skeletal cells (MSCs), but the underlying mechanisms are not fully understood. In this study, we determined the signaling mechanisms induced by α5β1 integrin interaction with its high-affinity ligand CRRETAWAC in murine and human MSCs and in vivo. We show that cyclized CRRETAWAC fully displaced MSC adhesion to FN, whereas related peptides lacking the full RRET sequence produced a partial displacement, indicating that RRET acts as an RGD-like sequence that is required to antagonize FN-mediated cell adhesion. However, all peptides increased focal adhesion kinase phosphorylation, OSE2 transcriptional activity, osteoblast gene expression, and matrix mineralization in MSCs, indicating that peptide-induced α5β1 integrin priming can promote osteogenic differentiation independently of the RRET sequence. Biochemical analyses showed that peptide-induced α5β1 integrin priming transiently increased PI3K/Akt phosphorylation and promoted Wnt/β-catenin transcriptional activity independently of RRET. Consistently, pharmacological inhibition of PI3K activity reduced osteoblast differentiation and abolished Wnt regulatory gene expression induced by α5β1 integrin priming. In vivo, systemic delivery of cyclized GACRETAWACGA linked to (DSS)6 to allow delivery to bone-forming sites for 6 weeks increased serum osteocalcin levels and improved long bone mass and microarchitecture in SAMP-6 senescent osteopenic mice. The results support a mechanism whereby α5β1 integrin priming by high-affinity ligands integrates Wnt/β-catenin signaling to promote osteoblast differentiation independently of cell adhesion, which could be used to improve bone mass and microarchitecture in the aging skeleton.

We investigated the ability of the activated mu-opioid receptor (MOR) to differentiate between myristoylated G(alphai1) and G(alphaoA) type G(alpha) proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each G(alpha) protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The G(alpha) subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified G(alpha) protein by CB(1) cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[(35)S]GTP(gamma)S exchange was then compared for G(alphai1) and G(alphaoA). Activation of MOR by DAMGO produced a high-affinity saturable interaction for G(alphaoA) (K(m)=20+/-1 nM) but a low-affinity interaction with G(alphai1) (K(m)=116+/-12 nM). DAMGO, met-enkephalin and leucine-enkephalin displayed maximal G(alpha) activation among the agonists evaluated. Endomorphins 1 and 2, methadone and beta-endorphin activated both G(alpha) to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between G(alphai1) and G(alphaoA). Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two G(alpha). Differences in maximal activity and potency, for G(alphai1) versus G(alphaoA), are both indicative of agonist selective activation of G-proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects.

Ferroptosis is a recently identified form of regulated necrosis that can be experimentally induced in cancer cells with the chemical inducer erastin. Recently, we identified sorafenib, an inhibitor of oncogenic kinases, as an inducer of ferroptosis in hepatocellular carcinoma cells. Whether sorafenib is able to exert its ferroptotic activity in cancer cells originating from other tissues is presently unclear.We compared the levels of ferroptosis induced by sorafenib with those induced by the reference compound erastin in a panel of ten human cell lines originating from various tissues.Sorafenib induced ferroptosis in different cancer cell lines. We found a positive correlation between the ferroptotic potency of sorafenib and erastin. Compared to other kinase inhibitors, sorafenib is the only drug that displays ferroptotic efficacy.The findings establish sorafenib as the first clinically-approved anticancer drug that can induce ferroptosis.