Tumor microtubes (TMs) are actin- and tubulin-based protrusions interconnecting glioma cells into multicellular networks linked to tumor aggressiveness, but the molecular signals exchanged through them and the impact of IDH1 mutation on TM function remain poorly defined. Using isogenic U87 glioma cells expressing wild-type IDH1 (U87IDH-WT) or the IDH1-R132H mutant (U87IDH-R132H), we combined quantitative immunofluorescence, RT-qPCR, fluorescent microRNA transfection, vertical co-culture with primary normal human astrocytes, and live-cell time-lapse imaging to analyze TM architecture, connexin-43 (Cx43) enrichment, and intercellular trafficking of the oncogenic miR-21 and tumor-suppressive miR-340. Cell viability, apoptosis, and proliferation were assessed by XTT, caspase-3/7 activity, and Ki-67 immunostaining. The IDH1-R132H mutation was associated with significant reductions in TM number, length, diameter, and Cx43 enrichment, indicating profound network remodeling. Despite these structural changes, TMs mediated intercellular microRNA transfer. miR-21 was overexpressed in glioma cells compared with astrocytes and promoted glioma cell survival while inducing apoptosis in astrocytes; TM-mediated miR-21 transfer was visualized between glioma cells, from glioma cells to astrocytes, and between astrocytes themselves. In contrast, miR-340 was downregulated in glioma cells, and its direct transfection markedly inhibited proliferation without astrocyte toxicity; fluorescent miR-340 was also detected within TMs and recipient glioma cells. Together, these findings identify TMs as structural features shaped by IDH1 mutation and as conduits for intercellular exchange of both oncogenic and tumor-suppressive microRNAs, providing a framework for understanding TM-mediated communication and its potential therapeutic exploitation in glioma.

The Hippo kinase, NDR2, plays a key role in the natural history of several human cancers, particularly lung cancer, by regulating processes such as proliferation, apoptosis, migration, invasion, vesicular trafficking, autophagy, ciliogenesis and immune response. To examine the specificity of NDR2's action, interaction and function in physiological or tumoral contexts, we first focus on the structural differences in the amino-acid sequence between NDR1 and NDR2. We then establish a correlation between these NDR1/2 differences and specific post-translational regulation, as well as the distinct action, interactions, and functions of NDR2 in physiological or tumoral paradigms, such as lung cancer. Furthermore, the full set of NDR2 partners and/or substrates remains to be identified. Given that it is hypothesized that NDR2 and its partners may offer new perspectives for anticancer therapies, we emphasize potential clustering or functional enrichment networks among the NDR2-specific interactants. Additionally, we provide an unpublished proteomic comparison of the NDR1 versus NDR2 interactome, focusing on human bronchial epithelial cells (HBEC-3), lung adenocarcinoma cells (H2030), and their brain metastasis-derived counterparts (H2030-BrM3). In conclusion, this study underscores the pivotal role of NDR2 in cancer progression, particularly lung cancer, and helps to better understand their specific functions and interactions in both normal and tumor contexts. The identification of NDR2 partners and substrates remains essential, with the potential to open new avenues for anticancer therapies.

Les gliomes de haut grade, en particulier les glioblastomes (GBM), figurent parmi les tumeurs cérébrales primitives les plus agressives, caractérisées par une récidive inévitable et un pronostic sombre malgré les stratégies thérapeutiques multimodales actuelles. Des études récentes ont mis en évidence le rôle crucial de protrusions membranaires spécialisées appelées microtubes tumoraux (MT), qui interconnectent les cellules gliales tumorales en réseaux multicellulaires robustes. Ces MT permettent une communication intercellulaire directe via des jonctions communicantes, facilitant l’échange d’ions et de petites molécules, et contribuant significativement à la progression tumorale, à la résistance thérapeutique, à l’invasion et à la récidive. Alors que les tunneling nanotubes (TNT), structures plus courtes et transitoires également observées dans les gliomes, ont été clairement démontrés capables de transférer des organites et des microARNs (miRs), l’implication potentielle des MT dans le transfert de miRs reste à confirmer expérimentalement. Les miRs sont de petits ARN non codants qui régulent l’expression génique post-transcriptionnelle et jouent un rôle clé dans l’oncogenèse des gliomes, influençant la prolifération, l’invasion et la résistance thérapeutique. Cette revue synthétise les avancées récentes concernant la structure, les fonctions et les implications cliniques des MT dans les gliomes, en mettant particulièrement l’accent sur leur rôle potentiel dans le transfert intercellulaire de miRs. Nous discutons des lacunes actuelles dans les connaissances et soulignons la nécessité d’études expérimentales rigoureuses sur le transfert de miRs médié par les MT. Une meilleure compréhension de ces mécanismes pourrait ouvrir de nouvelles pistes thérapeutiques, incluant des stratégies visant à perturber les réseaux basés sur les MT ou à exploiter les MT comme vecteurs innovants pour la délivrance ciblée de miRs thérapeutiques dans la niche tumorale.

Non-small cell lung cancer (NSCLC) is characterized by the deregulation of the Hippo kinase NDR2 and high basal autophagic activity. NDR2 promotes autophagy-driven tumor growth in some cancers, but evidence in lung cancer is lacking. Human bronchial epithelial tumor cell (HBEC) lines H2030, H2030-BrM3, and H1299, with or without NDR2 depletion via siRNA or shRNA, were cultured for up to 24 h in the presence or absence of serum, and with or without the autophagosome–lysosome fusion inhibitor chloroquine (CQ). Autophagosome biogenesis, migration and Golgi apparatus functionality were analyzed. Serum deprivation of HBECs silences the expression of NDR1 but not NDR2. As shown by the increased expression of the autophagosome marker LC3-II, NDR2 participates to the formation and distribution of phagophores/autophagosomes in HBECs in an ATG9A-dependent manner. NDR2 is required for cargos degradation since its depletion disrupts lysosomal trafficking and/or fusion with autophagosomes. Finally, NDR2 silencing inhibits filopodia formation and cell polarization during HBEC migration under serum deprivation by disrupting Golgi repositioning to the leading edge, a process essential for cell migration. These data highlight NDR2’s role in Golgi- and autophagy-regulated migration during starvation. Unlike NDR1, NDR2 is stabilized under starvation and promotes autophagy by regulating LC3 and ATG9A, thereby supporting NSCLC cell proliferation and migration. Routine staining for NDR2 and/or ATG9 could aid in diagnosing NSCLC with high migratory potential.

BACKGROUND:The Hippo kinase Nuclear Dbf2-related kinase 2 (NDR2) promotes brain metastasis (BM) in non-small cell lung cancer (NSCLC) by the disrupting Yes-associated protein 1 (YAP-1), suggesting a role in circulating tumor cells and/or brain colonization. The underlying mechanism remains to be clarified. METHODS:Human bronchial epithelial tumor cells (A549, H1975, H2030 and the brain-tropic H2030-BrM3 line) with or without NDR2 depletion (via siRNA or shRNA), were exposed to shear stress (up to 40 dyn/cm² for 3 h) using an Ibidi® pump system, in the presence or absence of exogenous Amphiregulin (AREG), and subsequently reseeded or not. Viability, apoptosis, proliferation, and YAP-1-dependent gene expression were analyzed. H2030-BrM3 cells (shControl or shNDR2) were injected intracardially into nude athymic mice (n = 10/group) to evaluate plasma AREG levels correlation with BM. AREG expression was assessed in human NSCLC tumor samples from primary and/or brain metastatic sites. RESULTS:NSCLC cells tolerated shear stress and showed reduced apoptosis after reseeding when expressing NDR2. Shear stress induced a dedifferentiation (via Sox2/9 variation) and increased AREG expression in most NSCLC cell lines. AREG enhanced NSCLC cells survival and proliferation under shear stress. In mice, plasma AREG levels correlated with BM volume. In human NSCLC samples, AREG-positive tumors displayed elevated Programmed Death-Ligand 1 (PD-L1), suggesting immune escape. Exogenous AREG treatment in H2030-BrM3 cells induced PD-L1 expression in vitro. CONCLUSION:AREG supports tumor adaptation to mechanical stress and may drive immune tolerance via PD-L1. Its correlation with BM burden highlights its potential as a biomarker and therapeutic target in NSCLC.

PDF file - 59K, IHC score according probability of response to chemo

PDF file - 352K, TUBB3 mRNA and protein expression according to K-RAs Si-RNA

PDF file - 252K, TUBB3 protein expression in HBEC cells is reduced by hypoxia cells culture conditions

PDF file - 144K, Representative SiRNA K-Ras depletion in HBEC and A549 (K-Ras mutated) cells

The molecular mechanisms induced by hypoxia are misunderstood in non-small cell lung cancer (NSCLC), and above all the hypoxia and RASSF1A/Hippo signaling relationship. We confirmed that human NSCLC ( n = 45) as their brain metastases (BM) counterpart are hypoxic since positive with CAIX-antibody (target gene of Hypoxia-inducible factor (HIF)). A severe and prolonged hypoxia (0.2% O2, 48 h) activated YAP (but not TAZ) in Human Bronchial Epithelial Cells (HBEC) lines by downregulating RASSF1A/kinases Hippo (except for NDR2) regardless their promoter methylation status. Subsequently, the NDR2-overactived HBEC cells exacerbated a HIF-1A, YAP and C-Jun-dependent-amoeboid migration, and mainly, support BM formation. Indeed, NDR2 is more expressed in human tumor of metastatic NSCLC than in human localized NSCLC while NDR2 silencing in HBEC lines (by shRNA) prevented the xenograft formation and growth in a lung cancer-derived BM model in mice. Collectively, our results indicated that NDR2 kinase is over-active in NSCLC by hypoxia and supports BM formation. NDR2 expression is thus a useful biomarker to predict the metastases risk in patients with NSCLC, easily measurable routinely by immunohistochemistry on tumor specimens.

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.

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.

Lung cancer patients frequently develop brain metastases (BM). Despite aggressive treatment including neurosurgery and external-radiotherapy, overall survival remains poor. There is a pressing need to further characterize factors in the microenvironment of BM that may confer resistance to radiotherapy (RT), such as hypoxia. Here, hypoxia was first evaluated in 28 biopsies from patients with non‑small cell lung cancer (NSCLC) BM, using CA-IX immunostaining. Hypoxia characterization (pimonidazole, CA-IX and HIF-1α) was also performed in different preclinical NSCLC BM models induced either by intracerebral injection of tumor cells (H2030-Br3M, H1915) into the cortex and striatum, or intracardial injection of tumor cells (H2030-Br3M). Additionally, [18F]-FMISO-PET and oxygen-saturation-mapping-MRI (SatO2-MRI) were carried out in the intracerebral BM models to further characterize tumor hypoxia and evaluate the potential of Hypoxia-image-guided-RT (HIGRT). The effect of RT on proliferation of BM ([18F]-FLT-PET), tumor volume and overall survival was determined. We showed that hypoxia is a major yet heterogeneous feature of BM from lung cancer both preclinically and clinically. HIGRT, based on hypoxia heterogeneity observed between cortical and striatal metastases in the intracerebrally induced models, showed significant potential for tumor control and animal survival. These results collectively highlight hypoxia as a hallmark of BM from lung cancer and the value of HIGRT in better controlling tumor growth.

Trente à trente-cinq pourcent des patients atteints de cancer bronchique non à petites cellules (CBNPC) développeront des métastases cérébrales au cours de leur maladie [1]. L'activation inappropriée de l'effecteur terminal de la voie Hippo, YAP, pourrait être impliquée dans la formation de ces métastases [2] de même qu'un environnement hypoxique [3]. Nous avons voulu déterminer quelle(s) kinase(s) de la voie Hippo n'assurai(en) t plus son rôle inactivateur de Yap dans les cellules de CBNPC. Des cellules de lignées humaines de CBNPC (A549, H1229) ou immortalisées (HBEC-3, BEAS-2B) ont été transfectée avec un siARN ou shARN ciblant l'une des kinases de la voie Hippo (MST1/2, NDR1/2, LATS1/2) puis cultivées en normoxie ou hypoxie (0,2 % O2, 48 h). La capacité des cellules à se déplacer (migration 2D, invasion), à former des xénogreffes chez des souris SCID beige (autorisation de projet utilisant des animaux à des fins scientifiques numéro : 2018030814474695 (# 13256) et l'état d'activation des kinases de la voie Hippo et de YAP a été évaluée. Nous rapportons que l'extinction de la kinase MST2, LATS2, NDR1 comme de NDR2 diminue la vitesse de migration 2D et l'invasion des cellules de CBNPC ou bronchiques immortalisées, mais que seules les kinases NDR1 et NDR2 contrôlent la motilité de ces cellules de façon YAP-dépendante. Enfin, seule NDR2 est stabilisée en hypoxie et seule l'extinction de la kinase NDR2 ralentit la prise de xénogreffes de cellules de lignées humaines de CBNPC injectées en sous-cutanée chez la souris SCID Beige ainsi que leur formation de métastases. Ce travail décrit pour la première fois à notre connaissance l'implication de la kinase NDR2 dans la capacité des cellules de CBNPC à se mouvoir, former des xénogreffes puis des métastases lorsqu'implantées sur des souris SCID. Ce rôle la désigne comme une cible thérapeutique de choix chez les patientes atteints de CBNPC.

By allowing insured communication between cancer cells themselves and with the neighboring stromal cells, tunneling nanotubes (TNTs) are involved in the multistep process of cancer development from tumorigenesis to the treatment resistance. However, despite their critical role in the biology of cancer, the study of the TNTs has been announced challenging due to not only the absence of a specific biomarker but also the fragile and transitory nature of their structure and the fact that they are hovering freely above the substratum. Here, we proposed to review guidelines to follow for studying the structure and functionality of TNTs in tumoral neuroendocrine cells (PC12) and nontumorigenic human bronchial epithelial cells (HBEC-3, H28). In particular, we reported how crucial is it (i) to consider the culture conditions (culture surface, cell density), (ii) to visualize the formation of TNTs in living cells (mechanisms of formation, 3D representation), and (iii) to identify the cytoskeleton components and the associated elements (categories, origin, tip, and formation/transport) in the TNTs. We also focused on the input of high-resolution cell imaging approaches including Stimulated Emission Depletion (STED) nanoscopy, Transmitted and Scanning Electron Microscopies (TEM and SEM). In addition, we underlined the important role of the organelles in the mechanisms of TNT formation and transfer between the cancer cells. Finally, new biological models for the identification of the TNTs between cancer cells and stromal cells (liquid air interface, ex vivo , in vivo ) and the clinical considerations will also be discussed.

Bien que l’activité oncogènique de l’amphiréguline (AREG) ait déjà été décrite dans de nombreux cancers humains [1], son rôle dans l’histoire naturelle du mésothéliome pleural malin (MPM), un cancer rare mais agressif et de mauvais pronostic, principalement causé par une exposition professionnelle à l’amiante [2], reste controversée. L’objectif de ce travail était d’étudier le possible rôle de l’AREG dans le développement du MPM. L’AREG plasmatique et l’expression tumorale de l’AREG ont été quantifiées par dosage ELISA (Amphiregulin DuoSet, R&D system) et immunohistochimie (H-Score) chez respectivement 373 et 288 patients atteints de MPM et enrôlés dans l’essai de phase 3 MAPS (IFCT-GFPC-0701). L’AREG plasmatique détectée chez seulement 42/373 patients testés (score : 48,24 ± 455,1 pg/mL, médiane : 0) n’influence pas significativement la survie globale ou sans progression des patients atteints de MPM. En revanche, la plupart des prélèvements de MPM analysés avaient un marquage AREG fortement positif (64,3 % des cas ; score : 59,87 ± 26,92, médiane : 70). La médiane de survie globale des patients exprimant une AREG cytosolique est de 24,8 mois, contre 15,9 mois lorsque l’AREG se trouve dans un autre compartiment cellulaire (HR ajusté : 0,62, IC95 % [0,44–0,86], p = 0,005). La médiane de survie sans progression des patients exprimant un AREG cytosolique est de 10,4 mois contre 8,2 mois pour les autres localisations subcellulaires (HR ajusté : 0,61, IC95 % [0,45–0,84], p = 0,0007). Aucune corrélation entre les quantités plasmatiques et tissulaires d’AREG n’a été trouvée (coefficient de corrélation = 0,007, p = 0,91). L’expression cytoplasmique d’AREG s’avère donc étonnamment être un facteur de bon pronostic chez les patients atteints de MPM. N’étant pas retrouvée dans le plasma de ces patients, nous supposons qu’il existe un défaut de relargage de l’AREG par les cellules de MPM, l’empêchant ainsi d’agir sur son récepteur membranaire.

Breast cancer (BC) is the primary cause of cancer-related mortality among women. Patients who express the estrogen receptor (ER), which mediates the tumorigenic effects of estrogens, respond to antihormonal therapy. Loss of ER expression or acquired resistance to E2 is associated with aggressive malignant phenotypes, which lead to relapse. These BC subtypes overexpress syndecan-1 (SDC1), a transmembrane heparan sulfate proteoglycan that mediates angiogenesis as well as the proliferation and invasiveness of cancer cells. We showed here that the activation of ER-alpha (ERα) by estrogens induces downregulation of SDC1 expression in ER(+) MCF7 cells but not in T47D cells. Loss of ERα expression, induced by RNA interference or a selective ER downregulator, led to subsequent SDC1 overexpression. E2-dependent downregulation of SDC1 expression required de novo protein synthesis and was antagonized by treatment with BAY 11-7085, an irreversible inhibitor of IκBα phosphorylation, which inhibits the activation of NFκB. Downregulation of SDC1 expression required ERα and activation of IKK, but was independent to downstream transcriptional regulators of NFκB. BAY 11-7085 prevented E2-mediated phosphorylation of ERα on Ser118, increasing its proteasomal degradation, suggesting that IKK stabilized E2-activated ERα, leading to subsequent downregulation of SDC1 expression. Our results showed that sustained ER signaling inhibits SDC1 expression. Such antagonism elucidates the inverse correlation between SDC1 and ER expression in ER(+) BC as well as the overexpression of SDC1 in hormone receptor-negative BC subtypes with the most aggressive phenotypes. These results identify SDC1 as an attractive therapeutic target for BC as well as for other endocrine-associated cancers.