Distinct Regions of the Large Extracellular Domain of Tetraspanin CD9 Are Involved in the Control of Human Multinucleated Giant Cell Formation

Multinucleated giant cells, formed by the fusion of monocytes/macrophages, are features of chronic granulomatous inflammation associated with infections or the persistent presence of foreign material. The tetraspanins CD9 and CD81 regulate multinucleated giant cell formation: soluble recombinant proteins corresponding to the large extracellular domain (EC2) of human but not mouse CD9 can inhibit multinucleated giant cell formation, whereas human CD81 EC2 can antagonise this effect. Tetraspanin EC2 are all likely to have a conserved three helix sub-domain and a much less well-conserved or hypervariable sub-domain formed by short helices and interconnecting loops stabilised by two or more disulfide bridges. Using CD9/CD81 EC2 chimeras and point mutants we have mapped the specific regions of the CD9 EC2 involved in multinucleated giant cell formation. These were primarily located in two helices, one in each sub-domain. The cysteine residues involved in the formation of the disulfide bridges in CD9 EC2 were all essential for inhibitory activity but a conserved glycine residue in the tetraspanin-defining ‘CCG’ motif was not. A tyrosine residue in one of the active regions that is not conserved between human and mouse CD9 EC2, predicted to be solvent-exposed, was found to be only peripherally involved in this activity. We have defined two spatially-distinct sites on the CD9 EC2 that are required for inhibitory activity. Agents that target these sites could have therapeutic applications in diseases in which multinucleated giant cells play a pathogenic role

Retroviral matrix and lipids, the intimate interaction

Retroviruses are enveloped viruses that assemble on the inner leaflet of cellular membranes. Improving biophysical techniques has recently unveiled many molecular aspects of the interaction between the retroviral structural protein Gag and the cellular membrane lipids. This interaction is driven by the N-terminal matrix domain of the protein, which probably undergoes important structural modifications during this process, and could induce membrane lipid distribution changes as well. This review aims at describing the molecular events occurring during MA-membrane interaction, and pointing out their consequences in terms of viral assembly. The striking conservation of the matrix membrane binding mode among retroviruses indicates that this particular step is most probably a relevant target for antiviral research

Physiopathologie et modulation pharmacologique de l’entérite radique

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Ciblage de la famille HER dans les cancers ORL : efficacité biologique de l’association de cétuximab et de pertuzumab combinée à l’irradiation photonique

International audienceObjectif de l’étudeLes cancers épidermoïdes ORL sont associés à un fort taux de récidive loco-régionale et métastatique. Les cellules souches cancéreuses, sous-population hautement migratoire, semblent être une hypothèse majeure à l’origine de la résistance aux traitements. L’objectif du travail était de comparer l’efficacité du blocage pan-HER par une association cétuximab-pertuzumab associé à l’irradiation photonique dans les processus d’invasion et migration de la lignée SQ20B et sa sous-population souche.Matériel et méthodeLa sous-population de CSCs de la lignée SQ20B a été isolée par double tri selon les critères SQ20B/SP/CD44High. La prolifération des cellules SQ20B et SQ20B/CSCs a été étudiée après traitement par cétuximab 5 nM et/ou pertuzumab 20 μg/mL avec ou sans irradiation photonique à 10 Gy. L’analyse de la migration et de l’invasion a été réalisée par test de blessure avec et sans matrigel (IncuCyte). L’activation de récepteur de l’epidermal growth factor (EGFR) (Tyr1068) et des voies de signalisation intracellulaires (phospho-AKT et phospho-MEK1/2) a été étudiée en réponse aux traitements par western blot (WES).RésultatsLe cétuximab inhibe la prolifération cellulaire des cellules SQ20B et non celle de la sous-population souche. L’association cétuximab-pertuzumab inhibe significativement la prolifération des cellules SQ20B et SQ20B/CSCs. La double association cétuximab-pertuzumab associée à une irradiation de 10 Gy inhibe significativement la migration et l’invasion des deux populations cellulaires. Le double traitement inhibe la phosphorylation d’EGFR dans les deux populations. Les cellules SQ20B expriment fortement phospho-AKT à l’inverse des SQ20B/CSCs qui expriment phospho-MEK1/2. Enfin, l’association cétuximab-pertuzumab-10 Gy inhibe fortement l’expression de phospho-AKT et phospho-MEK1/2.ConclusionLe double traitement par cétuximab-pertuzumab associé à l’irradiation photonique inhibe significativement la prolifération, la migration et l’invasion de la lignée SQ20B et sa sous-population souche

Targeting Cancer Stem Cells in HNSCC: Synergic Effect of Cetuximab and ABT-199 in Combination with Photon Radiation

International audiencePurpose/Objective(s)Concurrent cetuximab based radio-chemotherapy is a validated scheme in head and neck squamous cell carcinoma (HNSCC). Cancer Stem Cells (CSCs) are highly resistant to treatment, have large migratory abilities, and are hypothesized to be responsible for a significant part of recurrences. Apoptotic signaling in CSCs is a major way of treatment escape, through Bcl-2 proteins family. The aim of the present study was to explore the synergic effect between cetuximab and an anti-Bcl-2 antibody (ABT-199), when combined or not with photon radiation.Materials/MethodsHNSCC chemo and radio resistant human cell line (SQ20B) and its corresponding stem cell line (SQ20B/CSCs) were used to test the treatment combinations. HaCaT cell line was used to assess toxicity on healthy cell population. SQ20B/CSCs subpopulation was isolated through double cell sorting: side population (SP) (Hoechst exclusion) and CD44 staining: SP/CD44High. SQ20B and SQ20B/CSCs proliferation, invasion/migration, and apoptosis were studied after an exposition to cetuximab 5nM + ABT-199 10mM treatment +/- 10Gy photon irradiation. Invasion and migration were assessed based on scratch wound assay with or without matrigel. Apoptosis was measured using caspases 3/7. 3D spheroid assay was performed to validate the results in a 3D culture approach. EGFR, phospho-EGFR (Tyr1068), Bcl-2 and Bcl-xl protein expression were studied.ResultsCetuximab strongly inhibited SQ20B proliferation, migration and invasion whereas it had little effect on SQ20B/CSCs. Conversely, ABT-199 significantly inhibited these properties on SQ20B/CSCs, without showing any effect on SQ20B parental cell line. Cetuximab-ABT-199 combined with radiation had a significant inhibitory effect on both SQ20B and SQ20B/CSCs proliferation, migration and invasion. Although exclusive cetuximab had no pro-apoptotic effect, activation of caspases 3/7 was induced by ABT-199 and enhanced by the cetuximab+ABT-199 combination in both populations. Cetuximab was a strong inhibitor of 3D-spheroid proliferation in SQ20B, whereas ABT-199 strongly decreased spheroid size in CSCs. EGFR was overexpressed in SQ20B, and under-expressed in SQ20B/CSCs. Bcl2 was overexpressed in SQ20B/CSCs. Although cetuximab moderately inhibited HaCaT cell proliferation, the drug combination did not significantly enhanced toxicity.ConclusionCetuximab+ABT-199 combined with photon radiation significantly inhibited proliferation, invasion and migration of SQ20B HNSCC cell line and its CSCs subpopulation, with an acceptable toxicity profile on healthy cell lines. Apoptotic cell death was enhanced by this drug combination

Outcome and Prognosis Factors of Stage IV Cervical Cancer Patients: A Decade Experience

International audiencePurpose/Objective(s)The aim of this study was to identify and compare prognostic factors, management strategies, and outcomes of very locally advanced cervical cancer (CC) (i.e., stages IVA) and metastatic CC (i.e., stages IV).Materials/MethodsA retrospective review was conducted, based on all consecutive patients treated for stage IV CC in a comprehensive cancer care center between 2004 and 2017.ResultsSixty-eight patients were included. Performance status (PS) was ≥ 2 for 35.9%. Median age at diagnosis was 60.5. There were 24 stage IVA CC (35.3%) and 44 stage IVB CC (64.7%). Seventeen patients with stage IVB CC had only para-aortic lymph nodes metastases (38.6%), 13 had only distant metastases (29.5%) and 14 had both (31.8%). Patients with stage IVA CC experienced a radiotherapy with curative intent (n=14, 58.3%) +/- a concomitant chemotherapy, or a palliative treatment (n=10, 41.7%). Twenty-three patients with stage IVB CC received a prior chemotherapy (52.3%), eleven a primary concomitant chemoradiation (25%), and ten a palliative treatment (22.7%). The mean follow-up was 18.0 months. The 5-year overall survival was 5.1% for stages IVA (95%CI=0.7-33.9), and 10.5% for stages IVB (95%CI=3.7-29.7). In multivariate analysis, PS>1 was identified as a poor prognostic factor of disease-specific survival for stage IVA CC. PS>1 and pelvic lymph nodes involvement were identified as poor prognostic factors of overall survival and disease-specific survival for stage IVB CC.ConclusionIn daily clinical practice, outcomes of stages IV CC are poor. Treatment of advanced and metastatic CC remains challenging. New management strategies are needed, as well as efficient preventive strategies

HER Family Blockade in Head and Neck Squamous Cell Carcinoma: Couple Therapy Efficacy of Cetuximab and Pertuzumab Combined With Photon Irradiation

International audiencePurpose/Objective(s)Head and neck squamous cell carcinoma (HNSCC) is a malignancy still associated with severe mortality, due to loco-regional recurrences or distant metastasis. Head and neck cancer stem cells (CSCs) are highly resistant to treatment and have large migratory abilities. These particular properties could explain treatment resistances in this location. If EGFR is strongly overexpressed in 80-100% of HNSCC, HER2 and HER3 seem to be also expressed in these lines. The aim of the present study was to explore the efficacy of HER1-2-3 blockade through cetuximab-pertuzumab association with or without photon irradiation on the proliferation and migration/invasion capabilities of a HNSCC chemo and radio resistant human cell line (SQ20B) and its corresponding stem cell line (SQ20B/CSCs).Materials/MethodsSQ20B/CSCs subpopulation was isolated through double cell sorting: side population (SP) (Hoechst exclusion) and CD44 staining: SP/CD44High. SQ20B and SQ20B/CSCs proliferation was studied after treatment with cetuximab 5nM + pertuzumab 20mg/mL treatment +/- 10Gy photon irradiation. Invasion and migration were assessed with scratch wound assay with or without matrigel. EGFR, phospho-EGFR (Tyr1068), HER2 and HER3 basal protein expression was studied. Activation or inhibition of RAS/MAPK and AKT-mTOR downstream signaling cascade was studied through phospho-AKT (Ser473), phospho-MEK1/2(Ser217/221) expression exposed to combined treatments.ResultsCetuximab strongly inhibits SQ20B proliferation, migration and invasion when it has a small effect on SQ20B/CSCs. Cetuximab-pertuzumab treatment combined with radiation has a potent significant inhibitory effect on SQ20B and SQ20B/CSCs proliferation, migration and invasion. EGFR is overexpressed in SQ20B, and under-expressed in SQ20B/CSCs, while HER2 and HER3 are expressed equivalently in both populations in basal conditions. Phospho-AKT is strongly expressed in SQ20B, at the opposite of SQ20B/CSCs which express phospho-MEK1/2. Cetuximab-pertuzumab treatment combination with 10Gy photon irradiation switches off both phospho-AKT and phospho-MEK1/2 expression in the two populations.ConclusionCetuximab-pertuzumab couple pan-HER treatment combined with photon irradiation significantly inhibits proliferation, invasion and migration of SQ20B HNSCC cell line and its CSCs subpopulation, through both AKT-mTOR and Ras-MAPK downstream signaling blockade. HER family seems to be a promising therapeutic target in HNSCC

Navigating the highlights of phase III trials: a watchful eye on evidence-based radiotherapy

International audienceBackground: Phase III randomized controlled trials (RCTs) are the cornerstone of evidence-based oncology. However, there is no exhaustive review describing the radiotherapy RTCs characteristics. The objective of the present study was to describe features of all phase III RCTs including at least a radiation therapy. Methods and materials: Requests were performed in the Medline database (via PubMed). The latest update was performed in April 2016, using the following MESH terms: 'clinical trials: phase III as topic', 'radiotherapy', 'brachytherapy', as keywords. Results: A total of 454 phase III RCTs were identified. Studies were mainly based on open (92.1%) multicenter (77.5%) designs, analyzed in intend to treat (67.6%), aiming at proving superiority (91.6%) through overall survival assessment (46.5%). Most frequently studied malignancies were head and neck (21.8%), lung (14.3%) and prostate cancers (9.9%). Patients were mainly recruited with a locally advanced disease (73.7%). Median age was 59 years old. Out of 977 treatment arms, 889 arms experienced radiotherapy, mainly using 3D-conformal radiotherapy (288 arms, 32.4%). Intensity-modulated techniques were tested in 12 arms (1.3%). The intervention was a non-cytotoxic agent addition in 89 studies (19.6%), a radiation dose/ fractionation modification in 74 studies (16.3%), a modification of chemotherapy regimen in 63 studies (13.9%), a chemotherapy addition in 63 studies (13.9%) and a radiotherapy addition in 53 trials (11.7%). With a median follow-up of 50 months, acute all-grade and grade 3–5 toxicities were reported in 49.6% and 69.4% of studies, respectively. Radiotherapy technique, follow-up and late toxicities were reported in 60.1%, 74%, and 31.1% of studies, respectively. Conclusion: Phase III randomized controlled trials featured severe limitations, since a third did not report radiotherapy technique, follow-up or late toxicities. The fast-paced technological evolution creates a discrepancy between literature and radiotherapy techniques performed in daily-routine, suggesting that phase III methodology needs to be reinvented