Extracapsular extension of neck nodes and absence of human papillomavirus 16-DNA are predictors of impaired survival in p16-positive oropharyngeal squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinomas (OPSCCs) demonstrate superior outcome compared with HPV-negative OPSCCs. The eighth edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) tumor, lymph node, metastasis (TNM) classification (TNM 2017) modifies OPSCC staging based on p16 positivity as a surrogate for HPV-driven disease. In p16-negative OPSCCs, lymph node (N) categories include extracapsular/extranodal extension (ECE); and, in p16-positive OPSCCs, N categories are based on the number of positive neck lymph nodes omitting ECE status. The objective of the current study was to assess the prognostic impact of positive ECE status and the detection of HPV16 DNA in patients with p16-positive OPSCC. METHODS: In a cohort of 92 patients with p16-positive, lymph node (N)-positive (stage III-IVB) OPSCC who underwent surgery and neck dissection, allowing for a pathologic examination of positive lymph nodes, 66 of 92 patients (71.4%) were p16-positive/HPV16 DNA-positive, 62 of 92 (67%) were ECE-positive, and 45 of 62 (72.6%) were ECE-positive, p16-positive, and HPV16 DNA-positive. Differences in outcome were assessed using Kaplan-Meier plots and Cox proportional hazard regression (CoxR) for tumor-specific survival and overall survival (OS). RESULTS: The mean numbers of positive lymph nodes in ECE-positive patients (5.0 positive lymph nodes; 95% CI, 3.8-6.4 positive lymph nodes) and ECE-negative patients (2.4 positive lymph nodes; 95% CI, 1.8-2.9 positive lymph nodes) were different (P = .0007). ECE affected OS and tumor-specific survival in p16-positive patients (P = .007 and P = .047, respectively) and in p16-positive/HPV16 DNA-positive patients (P = .013 and P = .026, respectively). Related to the unequal distributions of ECE-positive/HPV16 DNA-negative tumors, the TNM 2017 failed to discriminate OS in patients with UICC stage I, II, and III disease (mean OS, 54.5, 73.4, and 45 months, respectively; median OS, 64.7 months, not reached, and 41.1 months, respectively). According to a univariate CoxR, the presence of ECE predicted impaired OS in patients with p16-positive OPSCC (hazard ratio, 3.40; 95% CI, 1.17-9.89; P = .025) and even greater impaired OS in those with p16-positive/HPV16 DNA-positive OPSCC (HR, 8.64; 95% CI, 1.12-66.40; P = .038). Multivariate CoxR confirmed ECE and HPV16 DNA detection as independent predictors. CONCLUSIONS: ECE and HPV16 DNA status should be included in the prognostic staging of patients with p16-positive OPSCC because several lines of evidence demonstrate their impact on survival.

High-Risk Human Papillomavirus (HR-HPV) DNA Detection in Mouthwashes for Diagnosis of HPV-Driven Oropharynx Cancer and Its Curative Therapy-A Feasibility Study.

Detection of p16 through immunohistochemistry (IHC) is the standard for determining the HPV status of the tumor according the TNM eighth edition released in 2017 and has become crucial for determining the HPV status of oropharyngeal squamous cell carcinomas (OPSCC) with direct impact on staging and prognostication. In recent years, detection of HPV DNA in mouthwashes has been proposed as a noninvasive alternative, both for OPSCCs and for other head and neck squamous cell carcinomas (HNSCCs). However, the prospect of using the mouthwashes to monitor the response to therapy is unclear. To evaluate the effect of curative therapy on the detection of HPV DNA, we performed a prospective study comparing the detection frequency of high-risk HPV DNA (HR-HPV-DNA) in pre- and post-therapy mouthwashes. We collected 137 mouthwashes from 88 pathologically confirmed HNSCC patients for DNA isolation and HPV genotyping with the Inno-LiPA assay. We show that HPV DNA in pretherapeutic mouthwashes can detect HPV-driven HNSCCs with a sensitivity of 50.0% and specificity of 85.4%, alongside a high negative predictive value of 79.5% and an accuracy of 74.5%. Furthermore, we observed a notable decrease in the detection frequency of HR-HPV-DNA after successful treatment (pre-therapy 50.0% (9/18) versus post-therapy 9.7% (3/28)). However, the comparatively low sensitivity regarding detection of HPV-driven OPSCC argues against its use in clinical routine.

Micronucleus Formation in Primary Oropharyngeal Epithelial Cells Reveals Mutagenicity of Cement Dusts.

BACKGROUND/AIM: Epidemiological studies showed an increased risk of developing laryngeal head and neck squamous cell carcinoma (HNSCC) for employees working in the construction business. This suggested a causal link between exposure to cement particles and development of HNSCC but data were missing. MATERIALS AND METHODS: We established an Organisation for Economic Co-operation and Development (OECD) guideline 487-conform micronucleus assay (MNA) using oropharyngeal mucosa-derived primary epithelial cells (OPCs) ex vivo. OPCs from healthy mucosa of 52 donors were cultured in vitro and incubated with serial concentrations of two common cement particles. Mitomycin C was used as a soluble positive control, and TiO2 and DQ12 were used as negative and positive particle controls. Bi-nucleated cells were counted and the mitotic index (MI) was determined. Subsequently, micronuclei-containing bi-nucleated cells (MN+) were counted. RESULTS: Cement particles, in concentrations not significantly reducing ex vivo proliferation according to mitotic index, dose-dependently increased micronuclei formation. CONCLUSION: Through the establishment of an OECD guideline 487 conform MNA, we demonstrate the mutagenic effects of cement on human OPCs.

Pre-Therapeutic VEGF Level in Plasma Is a Prognostic Bio-Marker in Head and Neck Squamous Cell Carcinoma (HNSCC).

Vascular endothelial growth factor (VEGF) is centrally involved in cancer angiogenesis. We hypothesized that pre-therapeutic VEGF levels in serum and plasma differ in their potential as biomarkers for outcomes in head and neck squamous cell carcinoma (HNSCC) patients. As prospectively defined in the study protocols of TRANSCAN-DietINT and NICEI-CIH, we measured VEGF in pretreatment serum and plasma of 75 HNSCC test cohort (TC) patients. We analyzed the prognostic value of VEGF concentrations in serum (VEGFSerum) and plasma (VEGFPlasma) for event-free survival (EFS) utilizing receiver-operating characteristics (ROC). Mean VEGF concentrations in plasma (34.6, 95% CI 26.0-43.3 ng/L) were significantly lower (p = 3.35 × 10-18) than in serum (214.8, 95% CI 179.6-250.0 ng/L) but, based on ROC (area under the curve, AUCPlasma = 0.707, 95% CI 0.573-0.840; p = 0.006 versus AUCSerum = 0.665, 95% CI 0.528-0.801; p = 0.030), superiorly correlated with event-free survival (EFS) of TC patients. Youden indices revealed optimum binary classification with VEGFPlasma 26 ng/L and VEGFSerum 264 ng/L. Kaplan-Meier plots demonstrated superiority of VEGFPlasma in discriminating patients regarding outcome. Patients with VEGFPlasma < 26 ng/L had superior nodal (NC), local (LC) and loco-regional control (LRC) leading to significant prolonged progression-free survival (PFS) and EFS. We successfully validated VEGFPlasma according the cut-off <26 ng/L as predictive for superior outcome in an independent validation cohort (iVC) of 104 HNSCC patients from the studies DeLOS-II and LIFE and found better outcomes including prolonged tumor-specific (TSS) and overall survival (OS). Outcomes in TC and iVC combined again was related to VEGFPlasma, and multivariate Cox regression revealed that VEGFPlasma was an independent outcome predictor. In HNSCC, pre-therapeutic VEGFPlasma is prognostic for outcomes.

Leptin decreases circulating inflammatory IL-6 and MCP-1 in mice.

Leptin influences inflammation and immune response. Dose-dependent effects of leptin on biomarkers of inflammation have not been studied in vivo, so far. Leptin-deficient low-density lipoprotein receptor (LDLR) knockout (LDLR-/- ;ob/ob) female mice were treated with three different leptin doses or saline for 12 weeks. The effect of leptin on plasma interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 concentrations and Il-6 and Mcp-1 mRNA expression in vivo were assessed. Macrophage infiltration in epididymal adipose tissue (epiAT) after leptin treatment was determined by quantitative immunohistochemical analysis. Aortic root atherosclerotic lesions were analyzed by oil red O staining. Mean plasma IL-6 and MCP-1 decreased significantly in the 3.0 mg/kg BW/day group as compared to control mice (both P < 0.01). Messenger RNA expression of Il-6 and Mcp-1 was significantly down-regulated by leptin treatment in different adipose tissues in vivo. Characteristic crown-like structures formed by adipose tissue macrophages were significantly reduced by leptin treatment in epiAT. Recombinant leptin dose-dependently diminished plaque area in the aortic root. Leptin administration within the subphysiological to physiological range diminishes circulating pro-inflammatory IL-6 and MCP-1. Reduction of Il-6 and Mcp-1 gene expression in adipose tissue, as well as decreased adipose tissue macrophage infiltration might contribute. © 2018 BioFactors, 45(1):43-48, 2019.

Discrimination of Head and Neck Squamous Cell Carcinoma Patients and Healthy Adults by 10-Color Flow Cytometry: Development of a Score Based on Leukocyte Subsets.

BACKGROUND: Leukocytes in peripheral blood (PB) are prognostic biomarkers in head and neck squamous cell carcinoma cancer patients (HNSCC-CPs), but differences between HNSCC-CPs and healthy adults (HAs) are insufficiently described. METHODS: 10-color flow cytometry (FCM) was used for in-depth immunophenotyping of PB samples of 963 HAs and 101 therapy-naïve HNSCC-CPs. Absolute (AbsCC) and relative cell counts (RelCC) of leukocyte subsets were determined. A training cohort (TC) of 43 HNSCC-CPs and 43 HAs, propensity score (PS)-matched according to age, sex, alcohol, and smoking, was used to develop a score consecutively approved in a validation cohort (VC). RESULTS: Differences in AbsCC were detected in leukocyte subsets (p < 0.001), but had low power in discriminating HNSCC-CPs and HAs. Consequently, RelCC of nine leukocyte subsets in the TC were used to calculate 36 ratios; receiver operating characteristic (ROC) curves defined optimum cut-off values. Binary classified data were combined in a score based on four ratios: monocytes-to-granulocytes (MGR), classical monocytes-to-monocytes (clMMR), monocytes-to-lymphocytes (MLR), and monocytes-to-T-lymphocytes (MTLR); ≥3 points accurately discriminate HNSCC-CPs and HAs in the PS-matched TC (p = 2.97 × 10-17), the VC (p = 4.404 × 10-178), and both combined (p = 7.74 × 10-199). CONCLUSIONS: RelCC of leukocyte subsets in PB of HNSCC-CPs differ significantly from those of HAs. A score based on MGR, clMMR, MLR, and MTLR allows for accurate discrimination.

Development of a Human Leukocyte Antigen Score to Predict Progression-Free Survival in Head and Neck Squamous Cell Carcinoma Patients.

BACKGROUND: In personalized medicine and treatment stratification of head and neck squamous cell carcinoma (HNSCC), the heterogeneous genetic background of patients is not considered. Human leukocyte antigen (HLA) alleles and HLA haplotypes (HLA traits) are linked to development of HNSCC and affect progression-free survival (PFS) of HNSCC patients but most head and neck oncologists are not familiar with HLA typing. Hence, we developed an HLA-score abstracting from complexity of HLA-typing results to facilitate potential use of HLA-associated hazard ratios (HR) for prognostic stratification. METHODS: The HR for PFS of 8 HLA traits shown to be independent predictors (Pi) of PFS in a test cohort (TC) of 90 HNSCC patients were used to build the HLA-score based on the natural logarithm (ln) of the Pi-associated HR. Crude ln-transformed HR of the eight Pi, alleles B*13 (2), B*35 (1), B*51 (2), DQB1*06 (1), homozygous Cw (1), homozygous DRB4 (2), and haplotypes A*01/B*08 (-6) and B*08/C*07 (4), were summed up to yield the individual patient's HLA-score. Receiver operating characteristic (ROC) and Kaplan-Meier curves were used to proof the suitability of the HLA-score as prognostic marker for PFS. An independent validation cohort (iVC) of 32 patients treated in the larynx-organ preservation trial DeLOS-II was utilized for validation. RESULTS: The individual HLA-scores (range -2 to 6) in TC classified HNSCC patients regarding PFS. ROC analysis (area under the curve = 0.750, 95% CI 0.665-0.836; P = 0.0000034) demonstrated an optimum cutoff for the HLA-score at 0.5 (97.9% sensitivity, 34.7% specificity), and 70/90 patients in TC with HLA-score > 0 had significant reduced PFS (P = 0.001). Applying the same classifier (HLA-score > 0) confirmed these findings in the iVC revealing reduced PFS of 25/32 patients (P = 0.040). CONCLUSION: HLA traits constitute critical Pi. Considering the HLA-score may potentially facilitate the use of genetic information from HLA typing for prognostic stratification, e.g., within clinical trials.

Correction: The anti-tumorigenic activity of A2M-A lesson from the naked mole-rat.

[This corrects the article DOI: 10.1371/journal.pone.0189514.].

Reduced Cytokine Release in Ex Vivo Response to Cilengitide and Cetuximab Is a Marker for Improved Survival of Head and Neck Cancer Patients.

Targeting of αVß3 and αVß5 integrins by cilengitide may reduce growth of solid tumors including head and neck squamous cell carcinoma (HNSCC). Preclinical investigations suggest increased activity of cilengitide in combination with other treatment modalities. The only published trial in HNSCC (ADVANTAGE) investigated cisplatin, 5-fluorouracil, and cetuximab (PFE) without or with once (PFE+CIL1W) or twice weekly cilengitide (PFE+CIL2W) in recurrent/metastatic HNSCC. ADVANTAGE showed good tolerability of the cilengitide arms and even lower adverse events (AEs) compared to PFE but not the benefit in overall survival expected based on preclinical data. As we found in the FLAVINO assay, a short-time ex vivo assay for prediction of chemosensitivity, only a subgroup of HNSCC had an increased suppressive effect of cilengitide containing combination therapies on colony formation of epithelial cells (CFec) and release of pro-angiogenetic and pro-inflammatory cytokines, whereas other HNSCC failed to respond. Response to αVß3 and αVß5 integrin targeting by cilengitide classifies HNSCC regarding outcome. We present FLAVINO data arguing for further development of cilengitide plus cetuximab in treatment of a subgroup of HNSCC potentially identified by the FLAVINO assay using a set of biomarkers for response evaluation.

Verification and characterization of an alternative low density lipoprotein receptor-related protein 1 splice variant.

BACKGROUND: Low density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a ubiquitously expressed multi-ligand endocytosis receptor implicated in a wide range of signalling, among others in tumour biology. Tumour-associated genomic mutations of the LRP1 gene are described, but nothing is known about cancer-associated expression of LRP1 splice variants Therefore, the focus of this study was on an annotated truncated LRP1 splice variant (BC072015.1; NCBI GenBank), referred to as smLRP1, which was initially identified in prostate and lung carcinoma. METHODS: Using PCR and quantitative PCR, the expression of LRP1 and smLRP1 in different human tissues and tumour cell lines was screened and compared on tumour biopsies of head and neck squamous cell carcinoma (HNSCC). Using a recently developed anti-smLRP1 antibody, the expression of the putative LRP1 protein isoform in tumour cell lines in Western blot and immunofluorescence staining was further investigated. RESULTS: The alternative transcript smLRP1 is ubiquitously expressed in 12 human cell lines of different origin and 22 tissues which is similar to LRP1. A shift in expression of smLRP1 relative to LRP1 towards smLRP1 was observed in most tumour cell lines compared to healthy tissue. The expression of LRP1 as well as smLRP1 is decreased in HNSCC cell lines in comparison to healthy mucosa. In vitro results were checked using primary HNSCC. Furthermore, the expression of the protein isoform smLRP1 (32 kDa) was confirmed in human tumour cell lines. CONCLUSIONS: Similar to LRP1, the truncated splice variant smLRP1 is ubiquitously expressed in healthy human tissues, but altered in tumours pointing to a potential role of smLRP1 in cancer. Comparative results suggest a shift in expression in favour of smLRP1 in tumour cells that warrant further evaluation. The protein isoform is suggested to be secreted.

HLA traits linked to development of head and neck squamous cell carcinoma affect the progression-free survival of patients.

BACKGROUND: Personalized medicine and treatment stratification of patients with head and neck squamous cell carcinoma (HNSCC) today mostly ignore genetic heterogeneity in HNSCC but especially the patient's genetic background. We hypothesized that particular human leukocyte antigen (HLA) class I (HLA-A, B, Cw) and II proteins (DR, DQ) confer susceptibility for and influence development of HNSCC and may be prognostic factors for progression-free survival (PFS). METHODS: 90 consecutive HNSCC patients of the prospective observational cohort study LIFE treated between 08/2010 and 05/2011 at the University Leipzig underwent low resolution typing of HLA-A, B, Cw, DR, and DQ. Antigen and haplotype frequencies were compared to those in German blood donors. Effects on PFS were analyzed using Kaplan-Meier curves and Cox models. RESULTS: HNSCC patients had overall altered HLA-B frequencies (P<0.05); frequencies of B∗44 were lower, those of B∗13, B∗52, and B∗57 increased (P<0.05). Almost all other antigen frequencies showed no deviation. Homozygous HLA-Cw and DRB4 were frequent and associated with reduced PFS (P<0.05). Altered haplotype frequencies were common and particular haplotypes accompanied by differing PFS. B∗13/Cw∗06 carriers had poorest outcome (P=0.011). However, multivariate Cox proportional hazard models revealed 3 clinical covariates (localization oropharynx, loco-regional metastasis, and T4 category), HPV16-DNA positivity, and 10 HLA traits as independent predictors for PFS. CONCLUSIONS: The relevance of the genetic background of HNSCC patients calls for future research to clarify the role of HLA traits in HNSCC and if PFS depends on HLA.

Cilengitide and Cetuximab Reduce Cytokine Production and Colony Formation of Head and Neck Squamous Cell Carcinoma Cells Ex Vivo.

BACKGROUND/AIM: To analyze ex vivo effects of combined targeting of the epidermal growth factor-receptor (EGFR) by cetuximab (E) plus αVß3 and αVß5 integrins by cilengitide (Cil) on colony formation of epithelial cells (CFec) and release of pro-angiogenetic and pro-inflammatory cytokines in head and neck squamous cell carcinoma (HNSCC) cells. MATERIALS AND METHODS: Collagenase-IV digests of 43 histopathological confirmed HNSCC cases were seeded into laminin-coated 96-well plates containing E, Cil, or Cil+E in final concentrations of 66.7 µg/ml, 10 µM, and 10 µM+66.7 µg/ml, respectively. Following the FLAVINO-assay protocol, supernatants were harvested after 3 days and adherent cells fixed in ethanol. Counting of CFec was facilitated by FITC-labeled pan-cytokeratin antibodies. Out of 43 HNSCC cases, 39 had adherent growth (mean CFec≥4/well in triplicate controls). Cytokines in supernatants were measured using ELISA were interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1) and vascular endothelial growth factor A (VEGFA). RESULTS: CFec on laminin was significantly reduced by Cil, E, and Cil+E. Cytokine measurements also revealed significant suppression of MCP-1, IL-6 and VEGFA. The strongest suppression of CFec, MCP-1 and VEGFA release was exerted by Cil and E combined. Efficacy of Cil+E exceeded those of the solely applied pharmaceutics but failed regarding significant synergism of both treatments as E was unable to significantly boost the effects of Cil. In contrast, IL-6 release was significantly suppressed by E but not by Cil, while their combination strongly reduced it. CONCLUSION: Combined targeting of EGFR and integrins with E and Cil heightens their suppressive effects regarding CFec as well as release of pro-angiogenetic and pro-inflammatory cytokines.

Reduced proliferation and colony formation of head and neck squamous cell carcinoma (HNSCC) after dual targeting of EGFR and hedgehog pathways.

PURPOSE: The hedgehog signalling pathway (Hh) is frequently active in head and neck squamous cell carcinoma (HNSCC). Overexpressed Hh associates with poor prognosis. The Hh inhibitor vismodegib targets smoothened, and based on molecular data, may prevent resistance to EGFR targeting. METHODS: To elucidate potential roles of vismodegib in HNSCC therapy, its sole effects and those combined with cisplatin, docetaxel, and cetuximab on HNSCC cell lines were assessed by MTT metabolisation and BrdU incorporation. Colony formation (CF) of primary HNSCC cells was studied utilizing the FLAVINO-protocol. Combinatory effects were analysed regarding antagonism, additivity or synergism. Associations between the ex vivo detected mode of action of vismodegib with other treatments related to patient characteristics were assessed and progression-free survival (PFS) in patient groups compared using Kaplan-Meier curves. RESULTS: Vismodegib suppressed BrdU incorporation significantly stronger than MTT turnover; CF was significantly inhibited at ≥20 µM vismodegib while concentrations <20 µM acted hormetic. Combining 20 µM vismodegib plus docetaxel (T), cisplatin (P), and cetuximab (E), additively enhanced anti-tumoral activity in HNSCC samples from patients with superior PFS highlighting a potential role for ex-vivo testing of this combination for use as a prognostic classifier. CONCLUSION: We provide ex-vivo evidence for vismodegib's potential in HNSCC therapies, especially if combined with cetuximab, cisplatin and docetaxel.

Induction chemotherapy followed by radiotherapy for larynx preservation in advanced laryngeal and hypopharyngeal cancer: Outcome prediction after one cycle induction chemotherapy by a score based on clinical evaluation, computed tomography-based volumetry and 18F-FDG-PET/CT.

BACKGROUND: Long-term laryngectomy-free (LFS), tumour-specific (TSS) and overall survival (OS) is achieved by non-surgical larynx preservation (LP) only in a proportion of patients with locally advanced laryngeal or hypopharyngeal cancer. A score facilitating decision-making after 1 cycle induction chemotherapy (IC-1) may improve LFS and TSS. METHODS: Early response to IC-1 with TPF ± cetuximab was assessed in 52 patients using endoscopic tumour staging for selecting total laryngectomy for non-responders with endoscopic tumour surface shrinkage <30% versus induction chemotherapy plus radiotherapy (IC + RT) for responders. Computed tomography (CT)-based volumetry was used to assess volumes of primary tumour, neck nodes and their sum; maximum and mean standardised uptake value (SUVmax, SUVmean) were measured by 18F-FDG-PET/CT. Baseline and residual values after IC-1 were calculated and correlated with LFS, TSS and OS. RESULTS: After IC-1, 39/52 patients (75%) were early responders. Early response predicted complete response to IC + RT (p = 8.48 × 10-9). Early laryngectomised non-responders and responders with endoscopic tumour surface shrinkage > 70% had best OS. Significant independent predictors for LFS in responders are number of CT-staged suspect positive neck nodes (N+), residual primary tumour volume, residual total tumour volume and the ratio of residual SUVmax and SUVmean (resSUVmax/resSUVmean). Our LFS-score combines >2N+, residual primary tumour volume > 20%, residual total tumour volume > 5.6 mL and resSUVmax/resSUVmean > 1.51 weighted by their hazard ratio (12, 6, 5 and 4); LFS-score ≤ 16 predicts increased LFS, OS and TSS (p < 0.05). CONCLUSION: LFS-score ≤ 16 identifies in responders to IC-1 the patients with maximum benefit of non-surgical LP achieving long-term LFS. Even more importantly, a LFS-score > 16 defines patients unsuitable for LP applying the TPF/TP IC + RT protocol.

The anti-tumorigenic activity of A2M-A lesson from the naked mole-rat.

Cancer resistance is a major cause for longevity of the naked mole-rat. Recent liver transcriptome analysis in this animal compared to wild-derived mice revealed higher expression of alpha2-macroglobulin (A2M) and cell adhesion molecules, which contribute to the naked mole-rat's cancer resistance. Notably, A2M is known to dramatically decrease with age in humans. We hypothesize that this might facilitate tumour development. Here we found that A2M modulates tumour cell adhesion, migration and growth by inhibition of tumour promoting signalling pathways, e.g. PI3K / AKT, SMAD and up-regulated PTEN via down-regulation of miR-21, in vitro and in tumour xenografts. A2M increases the expression of CD29 and CD44 but did not evoke EMT. Transcriptome analysis of A2M-treated tumour cells, xenografts and mouse liver demonstrated a multifaceted regulation of tumour promoting signalling pathways indicating a less tumorigenic environment mediated by A2M. By virtue of these multiple actions the naturally occurring A2M has strong potential as a novel therapeutic agent.

Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma.

BACKGROUND: The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. RESULTS: Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma. CONCLUSION: We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti-aging mechanisms in the NMR.

Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression.

The Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-ß-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration.