Use of LoopDeelab during the COVID-19 Pandemic: An Innovative Device for Field Diagnosis.

Rapid and accurate diagnosis of SARS-CoV-2 infection is essential for the management of the COVID-19 outbreak. RT-LAMP LoopDeetect COVID-19 (LoopDeescience, France) is a rapid molecular diagnostic tool which operates with the LoopDeelab (LoopDeescience, France) device. RAPID COVID is a prospective double-blind research protocol which was conducted to evaluate the concordance between Loopdeetect COVID-19 and RT-PCR Allplex 2019 n-Cov (Seegene, Korea). Between 11 May 2020 and 14 June 2021, a total of 1122 nasopharyngeal swab specimens were collected, of which 741 were finally analysed. There were 32 "positive" and "indeterminate" RT-PCR results. The intrinsic performances of Loopdeetect COVID-19 are equivalent to other commercial RT-LAMP PCR COVID-19 kits, with a sensitivity and specificity of 69.23% [CI 95%: 48.21-85.67] and 100% [CI 95%: 99.58-100.00], respectively. To the best of our knowledge, LoopDeelab is the only LAMP PCR diagnostic device allowing such a fast and reliable analysis with low-cost equipment; this makes it a new and innovative technology, designed for field use. This device being portable, the development of other detection kits will be useful for the management of epidemics with a high attack rate and would facilitate the rapid application of health measures.

Molecular Alterations in Malignant Pleural Mesothelioma: A Hope for Effective Treatment by Targeting YAP.

Malignant pleural mesothelioma is a rare and aggressive neoplasm, which has primarily been attributed to the exposure to asbestos fibers (83% of cases); yet, despite a ban of using asbestos in many countries, the incidence of malignant pleural mesothelioma failed to decline worldwide. While little progress has been made in malignant pleural mesothelioma diagnosis, bevacizumab at first, then followed by double immunotherapy (nivolumab plus ipilumumab), were all shown to improve survival in large phase III randomized trials. The morphological analysis of the histological subtyping remains the primary indicator for therapeutic decision making at an advanced disease stage, while a platinum-based chemotherapy regimen combined with pemetrexed, either with or without bevacizumab, is still the main treatment option. Consequently, malignant pleural mesothelioma still represents a significant health concern owing to poor median survival (12-18 months). Given this context, both diagnosis and therapy improvements require better knowledge of the molecular mechanisms underlying malignant pleural mesothelioma's carcinogenesis and progression. Hence, the Hippo pathway in malignant pleural mesothelioma initiation and progression has recently received increasing attention, as the aberrant expression of its core components may be closely related to patient prognosis. The purpose of this review was to provide a critical analysis of our current knowledge on these topics, the main focus being on the available evidence concerning the role of each Hippo pathway's member as a promising biomarker, enabling detection of the disease at earlier stages and thus improving prognosis.

A defect of amphiregulin release predicted longer survival independently of YAP expression in patients with pleural mesothelioma in the IFCT-0701 MAPS phase 3 trial.

The Hippo pathway effector YAP is dysregulated in malignant pleural mesothelioma (MPM). YAP's target genes include the secreted growth factor amphiregulin (AREG), which is overexpressed in a wide range of epithelial cancers and plays an elusive role in MPM. We assayed the expression of YAP and AREG in MPM pathology samples and that of AREG additionally in plasma samples of patients from the randomized phase 3 IFCT-0701 Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS) using immunohistochemistry and ELISA assays, respectively. MPM patients frequently presented high levels of tumor AREG (64.3%), a high cytosolic AREG expression being predictive of a better prognosis with longer median overall and progression-free survival. Surprisingly, tumor AREG cytosolic expression was not correlated with secreted plasma AREG. By investigating the AREG metabolism and function in MPM cell lines H2452, H2052, MSTO-211H and H28, in comparison with the T47D ER+ breast cancer cell line used as a positive control, we confirm that AREG is important for cell invasion, growth without anchorage, proliferation and apoptosis in mesothelioma cells. Yet, most of these MPM cell lines failed to correctly execute AREG posttranslational processing by metalloprotease ADAM17/tumor necrosis factor-alpha-converting enzyme (TACE) and extracell secretion. The favorable prognostic value of high cytosolic AREG expression in MPM patients could therefore be sustained by default AREG posttranslational processing and release. Thus, the determination of mesothelioma cell AREG content could be further investigated as a prognostic marker for MPM patients and used as a stratification factor in future clinical trials.

Role of the YAP-1 Transcriptional Target cIAP2 in the Differential Susceptibility to Chemotherapy of Non-Small-Cell Lung Cancer (NSCLC) Patients with Tumor RASSF1A Gene Methylation from the Phase 3 IFCT-0002 Trial.

RASSF1 gene methylation predicts longer disease-free survival (DFS) and overall survival (OS) in patients with early-stage non-small-cell lung cancer treated using paclitaxel-based neo-adjuvant chemotherapy compared to patients receiving a gemcitabine-based regimen, according to the randomized Phase 3 IFCT (Intergroupe Francophone de Cancérologie Thoracique)-0002 trial. To better understand these results, this study used four human bronchial epithelial cell (HBEC) models (HBEC-3, HBEC-3-RasV12, A549, and H1299) and modulated the expression of RASSF1A or YAP-1. Wound-healing, invasion, proliferation and apoptosis assays were then carried out and the expression of YAP-1 transcriptional targets was quantified using a quantitative polymerase chain reaction. This study reports herein that gemcitabine synergizes with RASSF1A, silencing to increase the IAP-2 expression, which in turn not only interferes with cell proliferation but also promotes cell migration. This contributes to the aggressive behavior of RASSF1A-depleted cells, as confirmed by a combined knockdown of IAP-2 and RASSF1A. Conversely, paclitaxel does not increase the IAP-2 expression but limits the invasiveness of RASSF1A-depleted cells, presumably by rescuing microtubule stabilization. Overall, these data provide a functional insight that supports the prognostic value of RASSF1 gene methylation on survival of early-stage lung cancer patients receiving perioperative paclitaxel-based treatment compared to gemcitabine-based treatment, identifying IAP-2 as a novel biomarker indicative of YAP-1-mediated modulation of chemo-sensitivity in lung cancer.

NDR2 kinase contributes to cell invasion and cytokinesis defects induced by the inactivation of RASSF1A tumor-suppressor gene in lung cancer cells.

BACKGROUND: RASSF1A, a tumor suppressor gene, is frequently inactivated in lung cancer leading to a YAP-dependent epithelial-mesenchymal transition (EMT). Such effects are partly due to the inactivation of the anti-migratory RhoB GTPase via the inhibitory phosphorylation of GEF-H1, the GDP/GTP exchange factor for RhoB. However, the kinase responsible for RhoB/GEF-H1 inactivation in RASSF1A-depleted cells remained unknown. METHODS: NDR1/2 inactivation by siRNA or shRNA effects on epithelial-mesenchymal transition, invasion, xenograft formation and growth in SCID-/- Beige mice, apoptosis, proliferation, cytokinesis, YAP/TAZ activation were investigated upon RASSF1A loss in human bronchial epithelial cells (HBEC). RESULTS: We demonstrate here that depletion of the YAP-kinases NDR1/2 reverts migration and metastatic properties upon RASSF1A loss in HBEC. We show that NDR2 interacts directly with GEF-H1 (which contains the NDR phosphorylation consensus motif HXRXXS/T), leading to GEF-H1 phosphorylation. We further report that the RASSF1A/NDR2/GEF-H1/RhoB/YAP axis is involved in proper cytokinesis in human bronchial cells, since chromosome proper segregation are NDR-dependent upon RASSF1A or GEF-H1 loss in HBEC. CONCLUSION: To summarize, our data support a model in which, upon RASSF1A silencing, NDR2 gets activated, phosphorylates and inactivates GEF-H1, leading to RhoB inactivation. This cascade induced by RASSF1A loss in bronchial cells is responsible for metastasis properties, YAP activation and cytokinesis defects.

MST1/Hippo promoter gene methylation predicts poor survival in patients with malignant pleural mesothelioma in the IFCT-GFPC-0701 MAPS Phase 3 trial.

BACKGROUND: The Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS/NCT00651456) phase 3 trial demonstrated the superiority of bevacizumab plus pemetrexed-cisplatin triplet over chemotherapy alone in 448 malignant pleural mesothelioma (MPM) patients. Here, we evaluated the prognostic role of Hippo pathway gene promoter methylation. METHODS: Promoter methylations were assayed using methylation-specific polymerase chain reaction in samples from 223 MAPS patients, evaluating their prognostic value for overall survival (OS) and disease-free survival in univariate and multivariate analyses. MST1 inactivation effects on invasion, soft agar growth, apoptosis, proliferation, and YAP/TAZ activation were investigated in human mesothelial cell lines. RESULTS: STK4 (MST1) gene promoter methylation was detected in 19/223 patients tested (8.5%), predicting poorer OS in univariate and multivariate analyses (adjusted HR: 1.78, 95% CI (1.09-2.93), p = 0.022). Internal validation by bootstrap resampling supported this prognostic impact. MST1 inactivation reduced cellular basal apoptotic activity while increasing proliferation, invasion, and soft agar or in suspension growth, resulting in nuclear YAP accumulation, yet TAZ cytoplasmic retention in mesothelial cell lines. YAP silencing decreased invasion of MST1-depleted mesothelial cell lines. CONCLUSIONS: MST1/hippo kinase expression loss is predictive of poor prognosis in MPM patients, leading to nuclear YAP accumulation and electing YAP as a putative target for therapeutic intervention in human MPM.

A role for RASSF1A in tunneling nanotube formation between cells through GEFH1/Rab11 pathway control.

BACKGROUND: By allowing intercellular communication between cells, tunneling nanotubes (TNTs) could play critical role in cancer progression. If TNT formation is known to require cytoskeleton remodeling, key mechanism controlling their formation remains poorly understood. METHODS: The cells of human bronchial (HBEC-3, A549) or mesothelial (H2452, H28) lines are transfected with different siRNAs (inactive, anti-RASSF1A, anti-GEFH1 and / or anti-Rab11). At 48 h post-transfection, i) the number and length of the nanotubes per cell are quantified, ii) the organelles, previously labeled with specific tracers, exchanged via these structures are monitored in real time between cells cultured in 2D or 3D and in normoxia, hypoxia or in serum deprivation condition. RESULTS: We report that RASSF1A, a key-regulator of cytoskeleton encoded by a tumor-suppressor gene on 3p chromosome, is involved in TNTs formation in bronchial and pleural cells since controlling proper activity of RhoB guanine nucleotide exchange factor, GEF-H1. Indeed, the GEF-H1 inactivation induced by RASSF1A silencing, leads to Rab11 accumulation and subsequent exosome releasing, which in turn contribute to TNTs formation. Finally, we provide evidence involving TNT formation in bronchial carcinogenesis, by reporting that hypoxia or nutriment privation, two almost universal conditions in human cancers, fail to prevent TNTs induced by the oncogenic RASSF1A loss of expression. CONCLUSIONS: This finding suggests for the first time that loss of RASSF1A expression could be a potential biomarker for TNTs formation, such TNTs facilitating intercellular communication favoring multistep progression of bronchial epithelial cells toward overt malignancy.