Proteomic Study of HPV-Positive Head and Neck Cancers: Preliminary Results

Human papillomavirus (HPV) was recently recognized as a new risk factor for head and neck squamous cell carcinoma. For oropharyngeal cancers, an HPV+ status is associated with better prognosis in a subgroup of nonsmokers and nondrinkers. However, HPV infection is also involved in the biology of head and neck carcinoma (HNC) in patients with a history of tobacco use and/or alcohol consumption. Thus, the involvement of HPV infection in HN carcinogenesis remains unclear, and further studies are needed to identify and analyze HPV-specific pathways that are involved in this process. Using a quantitative proteomics-based approach, we compared the protein expression profiles of two HPV+ HNC cell lines and one HPV− HNC cell line. We identified 155 proteins that are differentially expressed (P<0.01) in these three lines. Among the identified proteins, prostate stem cell antigen (PSCA) was upregulated and eukaryotic elongation factor 1 alpha (EEF1α) was downregulated in the HPV+ cell lines. Immunofluorescence and western blotting analyses confirmed these results. Moreover, PSCA and EEF1α were differentially expressed in two clinical series of 50 HPV+ and 50 HPV− oral cavity carcinomas. Thus, our study reveals for the first time that PSCA and EEF1α are associated with the HPV-status, suggesting that these proteins could be involved in HPV-associated carcinogenesis

Immune Cell Density Evaluation Improves the Prognostic Values of Staging and p16 in Oropharyngeal Cancer

The incidence of oropharyngeal cancers (OPSCCs) has continued to rise over the years, mainly due to human papillomavirus (HPV) infection. Although they were newly reclassified in the last TNM staging system, some groups still relapse and have poor prognoses. Based on their implication in oncogenesis, we investigated the density of cytotoxic and regulatory T cells, macrophages, and Langerhans cells in relation to p16 status, staging and survival of patients. Biopsies from 194 OPSCCs were analyzed for HPV by RT-qPCR and for p16 by immunohistochemistry, while CD8, FoxP3, CD68 and CD1a immunolabeling was performed in stromal (ST) and intratumoral (IT) compartments to establish optimal cutoff values for overall survival (OS). High levels of FoxP3 IT and CD1a ST positively correlated with OS and were observed in p16-positive and low-stage patients, respectively. Then, their associations with p16 and TNM were more efficient than the clinical parameters alone in describing patient survival. Using multivariate analyses, we demonstrated that the respective combination of FoxP3 or CD1a with p16 status or staging was an independent prognostic marker improving the outcome of OPSCC patients. These two combinations are significant prognostic signatures that may eventually be included in the staging stratification system to develop personalized treatment approaches

Immune Cell Density Evaluation Improves the Prognostic Values of Staging and p16 in Oropharyngeal Cancer

The incidence of oropharyngeal cancers (OPSCCs) has continued to rise over the years, mainly due to human papillomavirus (HPV) infection. Although they were newly reclassified in the last TNM staging system, some groups still relapse and have poor prognoses. Based on their implication in oncogenesis, we investigated the density of cytotoxic and regulatory T cells, macrophages, and Langerhans cells in relation to p16 status, staging and survival of patients. Biopsies from 194 OPSCCs were analyzed for HPV by RT-qPCR and for p16 by immunohistochemistry, while CD8, FoxP3, CD68 and CD1a immunolabeling was performed in stromal (ST) and intratumoral (IT) compartments to establish optimal cutoff values for overall survival (OS). High levels of FoxP3 IT and CD1a ST positively correlated with OS and were observed in p16-positive and low-stage patients, respectively. Then, their associations with p16 and TNM were more efficient than the clinical parameters alone in describing patient survival. Using multivariate analyses, we demonstrated that the respective combination of FoxP3 or CD1a with p16 status or staging was an independent prognostic marker improving the outcome of OPSCC patients. These two combinations are significant prognostic signatures that may eventually be included in the staging stratification system to develop personalized treatment approaches.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Heat shock factor 1 is a potent therapeutic target for enhancing the efficacy of treatments for multiple myeloma with adverse prognosis

International audienceAbstractDeregulated expression of heat shock proteins (HSPs) encoding genes is frequent in multiple myeloma. HSPs, which are molecular chaperones involved in protein homeostasis pathways, have emerged recently as promising therapeutic targets. Using human myeloma cell lines and primary myeloma cells belonging to various molecular groups, we tested the efficacy of HSP90, HSP70, and heat shock factor 1 (HSF1) inhibitors alone or associated with current antimyeloma drugs. We report here that KNK-437 (an inhibitor of HSF1) and bortezomib have additive effects on apoptosis induction in cells belonging to groups with bad prognosis

Targeting Oxidative Stress With Auranofin or Prima-1Met to Circumvent p53 or Bax/Bak Deficiency in Myeloma Cells

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Immunoscore Combining CD8, FoxP3, and CD68-Positive Cells Density and Distribution Predicts the Prognosis of Head and Neck Cancer Patients

We assessed immune cell infiltrates to develop an immunoscore for prognosis and to investigate its correlation with the clinical data of patients with head and neck cancer. CD8, FoxP3, and CD68 markers were evaluated by immunohistochemistry in 258 carcinoma samples and positive cells were counted in stromal and intra-tumoral compartments. The RStudio software was used to assess optimal cut-offs to divide the population according to survival while the prognostic value was established by using Kaplan–Meier curves and Cox regression models for each immune marker alone and in combination. We found with univariate analysis that the infiltration of immune cells in both compartments was predictive for recurrence-free survival and overall survival. Multivariate analysis revealed that CD8+ density was an independent prognostic marker. Additionally, the combination of CD8, FoxP3, and CD68 in an immunoscore provided a significant association with overall survival (p = 0.002, HR = 9.87). Such an immunoscore stayed significant (p = 0.018, HR = 11.17) in a multivariate analysis in comparison to tumor stage and histological grade, which had lower prognostic values. Altogether, our analysis indicated that CD8, FoxP3, and CD68 immunoscore was a strong, independent, and significant prognostic marker that could be introduced into the landscape of current tools to improve the clinical management of head and neck cancer patients.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Myeloma Cell Self-Renewal Depends on JAG2 Expression and Is Mediated by IGF1 or SCF Loop

International audienceThe purpose of this study was to identify the pathways associated with the ability of human myeloma cells (HMCLs) to spontaneous self-renew in a serum-free semi-solid human collagen-based assay. Among 32 HMCLs analyzed, 8 were able to grow spontaneously (from 5% to 35% of seeded cells) without any addition of cytokines or growth factors and this capacity to grow correlated with the presence of RAS mutations (p=0.04). Gene expression profile analysis of HMCLs identified one gene, JAG2, overexpressed in HMCLs that are able to self-renew. Interestingly, flow cytometry analysis of JAG2 expression showed that the level of membrane JAG2 expression positively correlated (r=0.87) to the percentage of colony formation (p=0.004). Blocking Jag-Notch interactions with Notch-Fc chimeric molecules impaired self-colony formation underlying a role for Jag-Notch pathway in colony formation. Furthermore, direct JAG2 silencing in two independent HMCLs (KMM1 and JJN3) prevented colony formation. Moreover, xenografts in SCID mice showed that JAG2 silencing fully impaired tumor growth of both KMM1 and JJN3. RT-PCR evaluation of JAG2 expression showed that 20 of 30 CD138+ purified primary myeloma cells expressed JAG2 and a Jag2+ subpopulation was identified by flow cytometry within primary CD138+ MM cells of patients at diagnosis or relapse.We further identified the growth factors involved in the self-renewal. By using blocking anti-IGF1R Ab or C-KIT inhibitor (imatinib mesylate), we showed that self-renewal of HMCLs was dependent on IGF1/IGF1R (5 of 8) or on C-KIT/SCF (1) or on both loops (2 of 8). Of note, C-KIT+ HMCLs expressed high JAG2 level at the cell membrane that was decreased by imatinib mesylate. Interestingly, none HMCL self-renewal was dependent on IL6/IL6R loop despite the high efficiency of paracrine IL6 to induce colony formation in most HMCLs. To address expression of C-KIT/SCF and IGF1R/IGF1 in primary myeloma cells, we used public data from patients at diagnosis published by Arkansas University. Expression of C-KIT and IGF1R was found in 56% and 50% of patients at diagnosis, respectively: 53% of patients express one or the other receptor, 27% express both and 20% express none. Expression of receptors is not similar with regard to the molecular classification of patients as previously shown by cytometry: indeed, MS patients underexpress C-KIT (p<0.001) but overexpress IGF1R (p<0.001), in full contrast to HY patients who overexpress C-KIT (p<0.001) but underexpress IGF1R (p<0.001). Moreover, IGF1R expression is lower on C-KIT+ patients as compared with C-KIT− ones (p=0.049). Of note, CD-1 and CD-2 patients underexpress both C-KIT (p=0.039) and IGF1R (p<0.001). IGF1 and SCF are produced by the microenvironment although IGF1 (but not SCF) mRNA was found in myeloma cells too. Altogether, these data suggest that IGF1 and SCF could be the main growth factors for 80% of the patients. Blocking these two tyrosine kinase receptors (in good agreement with their expression in patients) as well as Jag2/Notch interactions could decrease myeloma progression and/or relapse and thus increase survival

Dealing with Macrophage Plasticity to Address Therapeutic Challenges in Head and Neck Cancers

The head and neck tumor microenvironment (TME) is highly infiltrated with macro-phages. More specifically, tumor‐associated macrophages (TAM/M2‐like) are one of the most critical components associated with poor overall survival in head and neck cancers (HNC). Two extreme states of macrophage phenotypes are described as conducting pro‐inflammatory/anti‐tumoral (M1) or anti‐inflammatory/pro‐tumoral (M2) activities. Moreover, specific metabolic pathways as well as oxidative stress responses are tightly associated with their phenotypes and functions. Hence, due to their plasticity, targeting M2 macrophages to repolarize in the M1 phenotype would be a promising cancer treatment. In this context, we evaluated macrophage infiltration in 60 HNC patients and demonstrated the high infiltration of CD68+ cells that were mainly related to CD163+ M2 macro-phages. We then optimized a polarization protocol from THP1 monocytes, validated by specific gene and protein expression levels. In addition, specific actors of glutamine pathway and oxidative stress were quantified to indicate the use of glutaminolysis by M2 and the production of reactive oxygen species by M1. Finally, we evaluated and confirmed the plasticity of our model using M1 activators to repolarize M2 in M1. Overall, our study provides a complete reversible polarization protocol allowing us to further evaluate various reprogramming effectors targeting glutaminolysis and/or oxidative stress in macrophages.SCOPUS: ar.jinfo:eu-repo/semantics/publishe