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Aim: Malignant pleural mesothelioma is a chemoresistant tumor, and biphasic and sarcomatoid histologies portend the worst prognosis for malignant pleural mesothelioma (MPM) patients. We obtained the microRNA expression profile of three biphasic-sarcomatoid MPM cell lines to identify commonly expressed microRNAs and evaluate the effect of butein, a chemo-sensitizing compound, on this microRNA subset. Methods: Nanostring-based microRNA profiling and analysis through the ROSALIND platform were employed to identify the commonly modulated microRNAs and their targets. MicroRNA-mimic transfection, Luciferase assay, and Western blotting were employed to show specific perturbation of TWIST1 levels by miR-186-5p. Sphere-forming assays, invasion assay, and metabolic profiling were used to assess the biological consequences of the butein-instigated miR-186-5p-mediated perturbation of TWIST1 levels. TGCA analysis was used to search for the correlation between TWIST1 and miR-186-5p levels in biphasic and epithelioid MPM specimens. Results: We identified a set of perturbed microRNAs, common to three biphasic/sarcomatoid MPM cell lines, after butein treatment. When focusing on miR-186-5p, we unraveled a butein-ignited and miR-186-5p-mediated modulation of TWIST1 levels which affected the 3D anchorage-independent growth, cisplatin resistance, invasion, and bioenergetics of the MPM cell lines tested. We showed that miR-186-5p and TWIST1 levels are anti-correlated in biphasic MPM specimens from TCGA. Conclusion: We unraveled a novel mechanism of action of butein, which attenuated the pro-tumorigenic features of MPM at least through a miR-186-5p-TWIST1 axis. We suggest that those activities converge into the chemo-sensitizing effect of this compound and may be of translational relevance.

Chemoresistance is a hallmark of malignant pleural mesothelioma (MPM) management and the expression of ALDH1A3 is responsible for the survival and activity of MPM chemoresistant cell subpopulations (ALDHbright cells). We enriched mesothelioma ALDHbright cells to near homogeneity by FACS sorting and an Aldefluor assay and performed unbiased Affymetrix gene expression profiling. Viability and ELISA assays were used to rule out significant apoptosis in the sorted cell subpopulations and to assess target engagement by butein. Statistical analysis of the results, pathway enrichment and promoter enrichment were employed for the generation of the data. Q-RTPCR was used to validate a subset of the identified, modulated mRNAs In this work, we started from the observation that the mRNA levels of the ALDH1A3 isoform could prognostically stratify MPM patients. Thus, we purified MPM ALDHbright cells from NCI-H2595 cells and interrogated their gene expression (GES) profile. We analyzed the GES of the purified cells at both a steady state and upon treatment with butein (a multifunctional tetrahydroxy-chalcone), which abates the ALDHbright cell number, thereby exerting chemo-sensitizing effects in vitro and in vivo. We identified 924 genes modulated in a statistically significant manner as a function of ALDH status and of the response to the inhibitor. Pathway and promoter enrichment identified the molecular determinant of high ALDH status and how butein treatment altered the molecular portrait of those chemoresistant cell subpopulations. Further, we unraveled an eighteen-gene signature with high prognostic significance for MPM patients, and showed that most of the identified prognostic contributors escaped the analysis of unfractionated samples. This work proves that digging into the unexplored field of intra-tumor heterogeneity (ITH) by working at the cell subpopulation level may provide findings of prognostic relevance, in addition to mechanistic insights into tumor resistance to therapy.

Simple Summary The Ephrin receptors and their ligands play important roles in organ formation and tissue repair, by orchestrating complex programs of cell adhesion and repulsion, however, this same system plays a role in cancer development In fact, EphA2 levels are higher in tumors vs normal tissue and further increased upon treatment, in vivo and in vitro. Changes in the molecular status of EphA2, of its subcellular localization, the absence of ligand and signals derived from the tumor context unleash the oncogenic role of EphA2 and its broad ability to promote resistance to radiotherapy, chemotherapy and targeted agents, including inhibitors of Epidermal-Growth-Factor-Receptor (EGFR). High levels of EphA2 may reduce response to cetuximab even in RAS wt CRC patients. In this work, we aim to review the current knowledge of the EphA2 function which is crucial for achieving a more effective therapeutic management of tumors resistant to EGFR inhibitors and to many other agents. Abstract The Eph receptors represent the largest group among Receptor Tyrosine kinase (RTK) families. The Eph/ephrin signaling axis plays center stage during development, and the deep perturbation of signaling consequent to its dysregulation in cancer reveals the multiplicity and complexity underlying its function. In the last decades, they have emerged as key players in solid tumors, including colorectal cancer (CRC); however, what causes EphA2 to switch between tumor-suppressive and tumor-promoting function is still an active theater of investigation. This review summarizes the recent advances in understanding EphA2 function in cancer, with detail on the molecular determinants of the oncogene-tumor suppressor switch function of EphA2. We describe tumor context-specific examples of EphA2 signaling and the emerging role EphA2 plays in supporting cancer—stem—cell-like populations and overcoming therapy-induced stress. In such a frame, we detail the interaction of the EphA2 and EGFR pathway in solid tumors, including colorectal cancer. We discuss the contribution of the EphA2 oncogenic signaling to the resistance to EGFR blocking agents, including cetuximab and TKIs.

Tumor heterogeneity impinges on all the aspects of tumor history, from onset to metastasis and relapse. It is growingly recognized as a propelling force for tumor adaptation to environmental and micro-environmental cues. Metabolic heterogeneity perfectly falls into this process. It strongly contributes to the metabolic plasticity which characterizes cancer cell subpopulations—capable of adaptive switching under stress conditions, between aerobic glycolysis and oxidative phosphorylation—in both a convergent and divergent modality. The mitochondria appear at center-stage in this adaptive process and thus, targeting mitochondria in cancer may prove of therapeutic value. Metformin is the oldest and most used anti-diabetic medication and its relationship with cancer has witnessed rises and falls in the last 30 years. We believe it is useful to revisit the main mechanisms of action of metformin in light of the emerging views on tumor heterogeneity. We first analyze the most consolidated view of its mitochondrial mechanism of action and then we frame the latter in the context of tumor adaptive strategies, cancer stem cell selection, metabolic zonation of tumors and the tumor microenvironment. This may provide a more critical point of view and, to some extent, may help to shed light on some of the controversial evidence for metformin’s anticancer action.

Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of “active chromatin” by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.

Alteration in microRNAs (miRNAs) expression is a frequent finding in human cancers. In particular, widespread miRNAs down-regulation is a hallmark of malignant transformation. In the present report, we showed that the miR-128-3p, which is up-regulated in lung cancer tissues, has Drosha and Dicer, two key enzymes of miRNAs processing, as the main modulation targets leading to the widespread down-regulation of miRNA expression. We observed that the miRNAs downregulation induced by miR-128-3p contributed to the tumorigenic properties of lung cancer cells. In particular, miR-128-3p-mediated miRNAs down-regulation contributed to aberrant SNAIL and ZEB1 expression thereby promoting the epithelial-to-mesenchymal transition (EMT) program. Drosha also resulted to be implicated in the control of migratory phenotype as its expression counteracted miR-128-3p functional effects. Our study provides mechanistic insights into the function of miR-128-3p as a key regulator of the malignant phenotype of lung cancer cells. This also enforces the remarkable impact of Drosha and Dicer alteration in cancer, and in particular it highlights a role for Drosha in non-small-cell lung cancer cells migration.

// Harvey I. Pass 1 , Carmencita Lavilla 2 , Claudia Canino 1, 5 , Chandra Goparaju 1 , Jordan Preiss 1 , Samrah Noreen 2 , Giovanni Blandino 3, 4 , Mario Cioce 1, 3 1 Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Langone Medical Center, New York University, New York, USA 2 New York University Langone Medical Center, New York University, New York, USA 3 Translational Oncogenomics Unit, Italian National Cancer Institute 'Regina Elena', Rome, Italy 4 Department of Oncology, Juravinski Cancer Center-McMaster University, Hamilton, Ontario, Canada 5 University Campus Biomedico, Rome, Italy Correspondence to: Mario Cioce, email: mario.cioce@ifo.gov.it Keywords: lung cancer, chemoresistance, CSF-1, CSF-1R, ALDH Received: May 10, 2016 Accepted: June 30, 2016 Published: July 28, 2016 ABSTRACT In the present work we show that multiple lung cancer cell lines contain cisplatin resistant cell subpopulations expressing the Colony-Stimulating-Factor-Receptor-1 (CSF-1R) and surviving chemotherapy-induced stress. By exploiting siRNA-mediated knock down in vitro and the use of an investigational CSF-1R TKI (JNJ-40346527) in vitro and in vivo , we show that expression and function of the receptor are required for the clonogenicity and chemoresistance of the cell lines. Thus, inhibition of the kinase activity of the receptor reduced the levels of EMT-associated genes, stem cell markers and chemoresistance genes. Additionally, the number of high aldehyde dehydrogenase (ALDH) expressing cells was reduced, consequent to the lack of cisplatin-induced increase of ALDH isoforms. This affected the collective chemoresistance of the treated cultures. Treatment of tumor bearing mice with JNJ-40346527, at pharmacologically relevant doses, produced strong chemo-sensitizing effects in vivo . These anticancer effects correlated with a reduced number of CSF-1R pos cells, in tumors excised from the treated mice. Depletion of the CD45 pos cells within the treated tumors did not, apparently, play a major role in mediating the therapeutic response to the TKI. Thus, lung cancer cells express a functional CSF-1 and CSF-1R duo which mediates pro-tumorigenic effects in vivo and in vitro and can be targeted in a therapeutically relevant way. These observations complement the already known role for the CSF-1R at mediating the pro-tumorigenic properties of tumor-infiltrating immune components.

Emerging evidence point to a crucial role for non-coding RNAs in modulating homeostatic signaling under physiological and pathological conditions. MicroRNAs, the best-characterized non-coding RNAs to date, can exquisitely integrate spatial and temporal signals in complex networks, thereby confer specificity and sensitivity to tissue response to changes in the microenvironment. MicroRNAs appear as preferential partners for Receptor Tyrosine Kinases (RTKs) in mediating signaling under stress conditions. Stress signaling can be especially relevant to disease. Here we focus on the ability of microRNAs to mediate RTK signaling in cancer, by acting as both tumor suppressors and oncogenes. We will provide a few general examples of microRNAs modulating specific tumorigenic functions downstream of RTK signaling and integrate oncogenic signals from multiple RTKs. A special focus will be devoted to epidermal growth factor receptor (EGFR) signaling, a system offering relatively rich information. We will explore the role of selected microRNAs as bidirectional modulators of EGFR functions in cancer cells. In addition, we will present the emerging evidence for microRNAs being specifically modulated by oncogenic EGFR mutants and we will discuss how this impinges on EGFRmut driven chemoresistance, which fits into the tumor heterogeneity-driven cancer progression. Finally, we discuss how other non-coding RNA species are emerging as important modulators of cancer progression and why the scenario depicted herein is destined to become increasingly complex in the future.

Adaptive resistance to therapy is a hallmark of cancer progression. To date, it is not entirely clear how microenvironmental stimuli would mediate emergence of therapy-resistant cell subpopulations, although a rearrangement of the cancer cell secretome following therapy-induced stress can be pivotal for such a process. Here, by using the highly chemoresistant malignant pleural mesothelioma (MPM) as an experimental model, we unveiled a key contribution of the chaperone HSP90 at assisting a chemotherapy-instigated Senescence-Associated-Secretory-Phenotype (SASP). Thus, administration of a clinical trial grade, HSP90, inhibitor blunted the release of several cytokines by the chemotherapy-treated MPM cells, including interleukin (IL)-8. Reduction of IL-8 levels hampered the FAK-AKT signaling and inhibited 3D growth and migration. This correlated with downregulation of key EMT and chemoresistance genes and affected the survival of chemoresistant ALDHbright cell subpopulations. Altogether, inhibition of HSP90 provoked a switch from a pro-tumorigenic SASP to a pro-apoptotic senescence status, thus resulting in chemosensitizing effects. In mouse xenografts treated with first-line agents, inhibiting HSP90 blunted FAK activation and reduced the expression of ALDH1A3 and the levels of circulating human IL-8, these latter strongly correlating with the effect on tumor growth. We validated the above findings in primary mesothelioma cultures, a more clinically relevant model. We unveiled here a key contribution of the chaperone HSP90 at assisting the secretory stress in chemotherapy-treated cells, which may warrant further investigation in combinatorial therapeutic settings.

// Claudia Canino 1 , YuYing Luo 2 , Paola Marcato 3 , Giovanni Blandino 4,5 , Harvey I. Pass 1 and Mario Cioce 1 1 Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Langone Medical Center, New York University, New York, USA 2 New York University School of Medicine, New York, USA 3 Department of Pathology and Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia , Canada 4 Translational Oncogenomics Unit, Italian National Cancer Institute ‘Regina Elena’, Rome, Italy 5 Department of Oncology, Juravinski Cancer Center-McMaster University, Hamilton, Ontario, Canada Correspondence to: Mario Cioce, email: // Keywords : ALDH, STAT3, NFkB, DDIT3, CEBPβ Received : November 26, 2014 Accepted : March 02, 2015 Published : March 30, 2015 Abstract Here we studied the relevance and modulation of aldehyde dehydrogenase (ALDH) expression in malignant pleural mesothelioma (MPM) chemoresistant cell subpopulations (ALDH bright cells), which survive pemetrexed + cisplatin treatment in vitro and in vivo. Expression of the ALDH1A3 isoform was invariably enriched in purified ALDH bright cells from multiple MPM cell lines and accounted for the enzymatic activity of those cells. RNAi mediated downregulation of ALDH1A3 reduced the survival of the ALDH bright cells at steady state and, much more, after pemetrexed + cisplatin treatment. We demonstrated, for the first time, that a pSTAT3(tyr705)-NFkB(p65) complex is required for the repression of DDIT3 mRNA and this ensures high levels of CEBPβ-dependent ALDH1A3 promoter activity. Inhibition of STAT3-NFkB activity allowed high levels of DDIT3 expression with increased formation of a DDIT3-CEBPβ complex. This reduced the occupancy of the ALDH1A3 promoter by CEBPβ, thus largely reducing the ALDH1A3 expression. Consequently, survival of ALDH bright cells in pemetrexed + cisplatin-treated cultures was impaired, following increased apoptosis. We show that such a mechanism is relevant in vivo and underlies the action of butein, a dual STAT3-NFkB inhibitor capable of abating the chemoresistance of mesothelioma cells in vivo . The possible broad translational relevance of the described mechanism is discussed.

Metformin is a commonly prescribed type II diabetes medication that exhibits promising anticancer effects. Recently, these effects were found to be associated, at least in part, with a modulation of microRNA expression. However, the mechanisms by which single modulated microRNAs mediate the anticancer effects of metformin are not entirely clear and knowledge of such a process could be vital to maximize the potential therapeutic benefits of this safe and well-tolerated therapy. Our analysis here revealed that the expression of miR-21-5p was downregulated in multiple breast cancer cell lines treated with pharmacologically relevant doses of metformin. Interestingly, the inhibition of miR-21-5p following metformin treatment was also observed in mouse breast cancer xenografts and in sera from 96 breast cancer patients. This modulation occurred at the levels of both pri-miR-21 and pre-miR-21, suggesting transcriptional modulation. Antagomir-mediated ablation of miR-21-5p phenocopied the effects of metformin on both the clonogenicity and migration of the treated cells, while ectopic expression of miR-21-5p had the opposite effect. Mechanistically, this reduction in miR-21-5p enhanced the expression of critical upstream activators of the AMP-activated protein kinase, calcium-binding protein 39-like and Sestrin-1, leading to AMP-activated protein kinase activation and inhibition of mammalian target of rapamycin signaling. Importantly, these effects of metformin were synergistic with those of everolimus, a clinically relevant mammalian target of rapamycin inhibitor, and were independent of the phosphatase and tensin homolog status. This highlights the potential relevance of metformin in combinatorial settings for the treatment of breast cancer.

// Valeria Canu 1,* , Andrea Sacconi 1,* , Laura Lorenzon 2 , Francesca Biagioni 1 , Federica Lo Sardo 1 , Maria Grazia Diodoro 4 , Paola Muti 5 , Alfredo Garofalo 3 , Sabrina Strano 5,6 , Antonietta D’Errico 7 , Gian Luca Grazi 3 , Mario Cioce 1 and Giovanni Blandino 1,5 1 Oncogenomic and Epigenetic Unit, Italian National Cancer Institute ‘Regina Elena’, Rome, Italy 2 Faculty of Medicine and Psychology, Surgical and Medical Department of Clinical Sciences, Biomedical Technologies and Translational Medicine, University of Rome ‘La Sapienza’, Sant’Andrea Hospital, Rome, Italy 3 HepatoBiliary Pancreatic Surgery, ‘Regina Elena’ National Cancer Institute, Rome, Italy 4 Department of Research, Advanced Diagnostic, and Technological Innovation, Regina Elena National Cancer Institute, Rome, Italy 5 Department of Oncology, Juravinski Cancer Center, McMaster University Hamilton, Hamilton, Ontario, Canada 6 Molecular Chemoprevention Group, Italian National Cancer Institute ‘Regina Elena’, Rome, Italy 7 Department of Medical and Surgical Sciences, Pathology Unit, S. Orsola-Malpighi Hospital, Alma Mater Studiorum, University of Bologna, Bologna, Italy * These authors have equally contributed to this work Correspondence to: Giovanni Blandino, email: // Keywords : gastric cancer, cholangiocarcinoma, micro-RNA, miR-204, prognostic Received : September 18, 2016 Accepted : January 27, 2017 Published : February 11, 2017 Abstract Background & Aims: There is high need of novel diagnostic and prognostic tools for tumors of the digestive system, such as gastric cancer and cholangiocarcinoma. We recently found that miR-204 was deeply downregulated in gastric cancer tissues. Here we investigated whether this was common to other tumors of the digestive system and whether this elicited a miR-204-dependent gene target signature, diagnostically and therapeutically relevant. Finally, we assessed the contribution of the identified target genes to the cell cycle progression and clonogenicity of gastric cancer and cholangiocarcinoma cell lines. Methods: We employed quantitative PCR and Affymetrix profiling for gene expression studies. In silico analysis aided us to identifying a miR-204 target signature in publicly available databases (TGCA). We employed transient transfection experiments, clonogenic assays and cell cycle profiling to evaluate the biological consequences of miR-204 perturbation. Results: We identified a novel miR-204 gene target signature perturbed in gastric cancer and in cholangiocarcinoma specimens. We validated its prognostic relevance and mechanistically addressed its biological relevance in GC and CC cell lines. Conclusions: We suggest that restoring the physiological levels of miR-204 in some gastrointestinal cancers might be exploited therapeutically.

// Mario Cioce 1* , MariaCristina Valerio 2* , Luca Casadei 2 , Claudio Pulito 3 , Andrea Sacconi 4 , Federica Mori 3 , Francesca Biagioni 4 , Cesare Manetti 2 , Paola Muti 5 , Sabrina Strano 3 , and Giovanni Blandino 4 . 1 Department of Cardiothoracic Surgery, NYU Langone Medical Center, New York, NY USA 2 Department of Chemistry, University of Rome ‘La Sapienza’, 00185 Rome, Italy 3 Molecular Chemoprevention Group, Italian National Cancer Institute “Regina Elena”, Rome, Italy 4 Translational Oncogenomic Unit, Italian National Cancer Institute “Regina Elena”, Rome, Italy 5 Department of Oncology, McMaster University, Hamilton, Ontario, L8V 5C2, Canada * These two authors contributed equally Correspondence: Giovanni Blandino , email: // Keywords : Metformin, metabolism, chemoresistance, ALDH, metabolic reprogramming, cancer Received : January 22, 2014 Accepted : March 24, 2014 Published : March 26, 2014 Abstract Metabolic remodeling is a hallmark of cancer progression and may affect tumor chemoresistance. Here we investigated by 1H-NMR/PCA analysis the metabolic profile of chemoresistant breast cancer cell subpopulations (ALDH bright cells) and their response to metformin, a promising anticancer metabolic modulator. The purified ALDH bright cells exhibited a different metabolic profile as compared to their chemosensitive ALDH low counterparts. Metformin treatment strongly affected the metabolism of the ALDHbright cells thereby affecting, among the others, the glutathione metabolism, whose upregulation is a feature of progenitor-like, chemoresistant cell subpopulations. Globally, metformin treatment reduced the differences between ALDH bright and ALDH low cells, making the former more similar to the latter. Metformin broadly modulated microRNAs in the ALDH bright cells, with a large fraction of them predicted to target the same metabolic pathways experimentally identified by 1H-NMR. Additionally, metformin modulated the levels of c-MYC and IRS-2, and this correlated with changes of the microRNA-33a levels. In summary, we observed, both by 1H-NMR and microRNA expression studies, that metformin treatment reduced the differences between the chemoresistant ALDH bright cells and the chemosensitive ALDH low cells. This works adds on the potential therapeutic relevance of metformin and shows the potential for metabolic reprogramming to modulate cancer chemoresistance.

Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.

Chemoresistance is a major challenge for cancer therapy and drives tumor relapse. The emergence, within the treated tumor mass, of specific cancer cell subpopulations endowed with high tolerance to the microenvironment stress induced by therapy is being growingly recognized as a mechanism of tumor progression. To obtain detailed information with regard to the pathways underlying survival, expansion, and microenvironmental cross talk of such chemoresistant cell subpopulations may be instrumental for cancer chemoprevention. Additionally, the obtained cell subpopulations may be used for direct screening of cancer chemopreventive compounds, in appropriate experimental settings. Here we report detailed experimental procedures that we and others have setup in order to obtain cell cultures enriched for chemoresistant cells from both malignant pleural mesothelioma specimens and primary cell cultures. We provide indications for the purification and characterization of those chemoresistant cell populations and to generally validate the obtained enriched cell populations for their chemoresistance.

Chemoresistance is a major challenge for cancer therapy and drives tumor relapse. The emergence, within the treated tumor mass, of specific cancer cell subpopulations endowed with high tolerance to the microenvironment stress induced by therapy is being growingly recognized as a mechanism of tumor progression. To obtain detailed information with regard to the pathways underlying survival, expansion, and microenvironmental cross talk of such chemoresistant cell subpopulations may be instrumental for cancer chemoprevention. Additionally, the obtained cell subpopulations may be used for direct screening of cancer chemopreventive compounds, in appropriate experimental settings. Here we report detailed experimental procedures that we and others have setup in order to obtain cell cultures enriched for chemoresistant cells from both malignant pleural mesothelioma specimens and primary cell cultures. We provide indications for the purification and characterization of those chemoresistant cell populations and to generally validate the obtained enriched cell populations for their chemoresistance.

Tumor transformation is the result of genetic and epigenetic modifications that alter gene transcription, consequently producing a specific oncogenic program. Multiple myeloma (MM) is a neoplasia characterized by the accumulation of proliferating antibodies producing plasma cells in the bone marrow. This disease is the second most frequent haematological malignancy in the US and Europe. In this study we demonstrate that Che-1 plays a crucial role in the control of transcription and cellular proliferation by regulating the state of the chromatin and by increasing its accessibility, highlighting Che-1 as an essential component of the transcription machinery in MM. Che-1 loss induces a global transcription shut-off, by reducing RNA Pol II recruitment onto the DNA. Most importantly, transcriptional inhibition in Che-1 depleted cells could already be detected at pre-mRNA levels, leading to the hypothesis that Che-1 could regulate RNA pol II activity directly. Our study shows that Che-1 is required for the maintenance of open chromatin in MM cells, and is involved in general histone acetylation. Morever, we found that Che-1 downregulation further sensitizes MM cells to bromodomain and extra-terminal (BET) inhibitors. Indeed Che-1 silencing increased JQ1 sensitivity of MM cells. In summary, our findings identify Che-1 as a key player for maintaining the open chromatin structure required for sustaining MM growth. These findings support Che-1 as a possible target for MM therapy, alone or in combination with BET inhibitors. Citation Format: Tiziana Bruno, Francesca De Nicola, Frauke Goeman, Matteo Pallocca, Cristina Sorino, Valeria Catena, Gianluca Bossi, Bruno Amadio, Giovanni Cigliana, Enrico Spugnini, Maria Rosaria Ricciardi, Maria Teresa Petrucci, Alfonso Baldi, Mario Cioce, Giancarlo Cortese, Elisabetta Mattei, Roberta Merola, Umberto Gianelli, Francesco Pisani, Svitlana Gumenyuk, Andrea Mengarelli, Katja Hopker, Thomas Benzing, Aristide Floridi, Claudio Passananti, Giovanni Blandino, Simona Iezzi, Maurizio Fanciulli. Che-1/aatf-induced transcriptionally active chromatin promotes cell growth in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 350.

TPS2606Background: MEN1309/OBT076 is a DM4-loaded Antibody Drug Conjugate (ADC), directed against CD205/Ly75, a type I transmembrane surface protein belonging to the macrophage mannose receptor fam...

The morphology and composition of subnuclear organelles, such as Cajal bodies (CBs), nucleoli, and other nuclear bodies, is dynamic and can change in response to a variety of cell stimuli, including stress. We show that UV-C irradiation disrupts CBs and alters the distribution of a specific subset of CB components. The effect of UV-C on CBs differs from previously reported effects of transcription inhibitors. We demonstrate that the mechanism underlying the response of CBs to UV-C is mediated, at least in part, by PA28gamma (proteasome activator subunit gamma). The presence of PA28gamma in coilin-containing complexes is increased by UV-C. Overexpression of PA28gamma, in the absence of UV-C treatment, provokes a similar redistribution of the same subset of CB components that respond to UV-C. RNA interference-mediated knockdown of PA28gamma attenuates the nuclear disruption caused by UV-C. These data demonstrate that CBs are specific nuclear targets of cellular stress-response pathways and identify PA28gamma as a novel regulator of CB integrity.

This review surveys what is known about the structure and function of the subnuclear domains called Cajal bodies (CBs). The major focus is on CBs in mammalian cells but we provide an overview of homologous CB structures in other organisms. We discuss the protein and RNA components of CBs, including factors recently found to associate in a cell cycle-dependent fashion or under specific metabolic or stress conditions. We also consider the dynamic properties of both CBs and their molecular components, based largely on recent data obtained thanks to the advent of improved in vivo detection and imaging methods. We discuss how these data contribute to an understanding of CB functions and highlight major questions that remain to be answered. Finally, we consider the interesting links that have emerged between CBs and alterations in nuclear structure apparent in a range of human pathologies, including cancer and inherited neurodegenerative diseases. We speculate on the relationship between CB function and molecular disease.

A general strategy for inactivation of target proteins is presented, which we have termed “oligomerization chain reaction.” This technique is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins that are able to self-associate naturally. Oligomerization through the CC region of promyelocytic leukemia, and through the natural self-associating domain, triggers the oligomerization chain reaction, leading to formation of large molecular weight complexes and functional inactivation of the target. As a test case, we have chosen the oncosuppressor p53, naturally occurring as a tetramer. Fusion of the CC to p53 leads to formation of stable high molecular weight complexes—as shown by size exclusion chromatography—to which wild-type p53 is recruited with high efficiency. CC-p53 chimeras delocalize wild-type p53 to the cytoplasm and inhibit its transcriptional regulatory properties, resulting in a loss of p53 function. We propose that this strategy may be of general application to self-associating factors and represent a complementary approach to currently used functional inactivation-based strategies.

We identified a discrete number of microRNAs differentially expressed in benign or malignant mesothelial tissues. We focused on mir-145 whose levels were significantly downregulated in malignant mesothelial tissues and malignant pleural mesothelioma (MPM) cell lines as compared to benign tissues (pleura, peritoneum or cysts). We show that promoter hyper-methylation caused very low levels in MPM cell lines and specimens. Treatment of MPM cell lines with mir-145 agonists negatively modulated some protumorigenic properties of MPM cells, such as clonogenicity, cell migration and resistance to pemetrexed treatment. The main effector mechanism of the clonogenic death induced by mir-145 was that of accelerated senescence. We found that mir-145 targeted OCT4 via specific binding to its 3'-UTR. Increased intracellular levels of mir-145 decreased the levels of OCT4 and its target gene ZEB1, thereby counteracting the increase of OCT4 induced by pemetrexed treatment which is known to favor the development of chemoresistant cells. In line with this, reintroduction of OCT4 into mimic-145 treated cells counteracted the effects on clonogenicity and replicative senescence. This further supports the relevance of the mir-145-OCT4 interaction for the survival of MPM cells. The potential use of mir-145 expression levels to classify benign vs malignant mesothelial tissues and the differences between pemetrexed-induced senescence and that induced by the re-expression of mir-145 are discussed.

Clinical management of malignant pleural mesothelioma (MPM) is very challenging because of the uncommon resistance of this tumor to chemotherapy. We report here increased expression of macrophage colony-stimulating-factor-1-receptor (M-CSF/CSF-1R) mRNA in mesothelioma versus normal tissue specimens and demonstrate that CSF-1R expression identifies chemoresistant cells of mesothelial nature in both primary cultures and mesothelioma cell lines. By using RNAi or ligand trapping, we demonstrate that the chemoresistance properties of those cells depend on autocrine CSF-1R signaling. At the single-cell level, the isolated CSF-1Rpos cells exhibit a complex repertoire of pluripotency, epithelial–mesenchymal transition and detoxifying factors, which define a clonogenic, chemoresistant, precursor-like cell sub-population. The simple activation of CSF-1R in untransformed mesothelial cells is sufficient to confer clonogenicity and resistance to pemetrexed, hallmarks of mesothelioma. In addition, this induced a gene expression profile highly mimicking that observed in the MPM cells endogenously expressing the receptor and the ligands, suggesting that CSF-1R expression is mainly responsible for the phenotype of the identified cell sub-populations. The survival of CSF1Rpos cells requires active AKT (v-akt murine thymoma viral oncogene homolog 1) signaling, which contributed to increased levels of nuclear, transcriptionally competent β-catenin. Inhibition of AKT reduced the transcriptional activity of β-catenin-dependent reporters and sensitized the cells to senescence-induced clonogenic death after pemetrexed treatment. This work expands what is known on the non-macrophage functions of CSF-1R and its role in solid tumors, and suggests that CSF-1R signaling may have a critical pathogenic role in a prototypical, inflammation-related cancer such as MPM and therefore may represent a promising target for therapeutic intervention.

Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony-stimulating factor-1 receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane followed by mass spectrometric analysis, we identified receptor-type protein-tyrosine phosphatase ζ (PTP-ζ), a cell surface chondroitin sulfate (CS) proteoglycan, as a novel IL-34 receptor. PTP-ζ is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-ζ in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the proliferation, clonogenicity, and motility of U251 cells in a PTP-ζ-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-ζ downstream effectors focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and CS competed with IL-34 for binding to the extracellular domain of PTP-ζ and to the cells, indicating a dependence of binding on PTP-ζ CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets. Background: IL-34 and the known IL-34 receptor, CSF-1R, are differentially expressed in mouse brain; thus, IL-34 may signal via an additional receptor(s). Results: IL-34 binds to PTP-ζ on U251 human glioblastoma cells to stimulate intracellular signaling and responses. Conclusion: PTP-ζ is an IL-34 receptor. Significance: CSF-1R-independent actions of IL-34 via PTP-ζ should be considered in evaluating IL-34 roles in development and disease.

[Abstract] This is a general protocol for the isolation and maintenance of primary MPM cultures as a tool for the identification of Tumor Initiating Cells and early progenitor-targeting drugs (Cioce et al., 2010). Primary cultures can be propagated efficiently for 8-12 weeks and xenotransplanted in NOD/SCID mice while retaining the histofeatures of the originating tumor (Canino et al., 2012). The protocol is suitable for both MPM solid specimens and pleural effusion. For increased clarity, initially two separate sections addressing the isolation of MPM cells from solid tumors and pleural effusions are here provided.

Chronic inflammation appears to be a driving force behind cancer progression. NFκB and STAT3 activation plays a pertinent role in this process by mediating chemoresistance and the acquisition of mesenchymal features of protumorigenic cells. Epidemiological data and experimental observations suggest that Malignant Pleural Mesothelioma (MPM) is a prototype of chronic inflammation-driven cancer. Chemoresistance is a major feature of MPM. Thus, this paper explores the effect of butein (3,4,2',4'-tetrahydroxychalcone), a naturally occurring NFκB and STAT3 inhibitor, on the tumorigenic properties of MPM cells. MPM cells harbor high nuclear levels of NFκB and pSTAT3(Y(705)). Butein inhibits pSTAT3(Y(705)) phosphorylation, nuclear localization of NFκB and the physical interaction of NFκB and pSTAT3. This correlates with a downregulation of several genes involved in cancer progression (such as ICAM1, Vimentin, MMP9, Twist) of proangiogenic cytokines (VEGF) and of IL-6 and IL-8, key growth factors for MPM. Hence, butein inhibits the migration of MPM cells and strongly affects the clonogenicity of MPM cells in vitro. Finally, we show that the in vitro actions of butein translate into anticancer effects in vivo. In fact, butein treatment severely affects tumor engraftment and potentiates the anticancer effects of pemetrexed in mouse xenograft models. Butein does not significantly affect the viability of human, untransformed mesothelial cells in vitro, nor does it affect survival of tumor-free mice in vivo. The possibility of using butein as an additional treatment to current MPM therapies is discussed here.

Diabetic patients treated with metformin have a reduced incidence of cancer and cancer-related mortality. Here we show that metformin affects engraftment and growth of breast cancer tumours in mice. This correlates with the induction of metabolic changes compatible with clear anticancer effects. We demonstrate that microRNA modulation underlies the anticancer metabolic actions of metformin. In fact, metformin induces DICER expression and its effects are severely impaired in DICER knocked down cells. Conversely, ectopic expression of DICER recapitulates the effects of metformin in vivo and in vitro. The microRNAs upregulated by metformin belong mainly to energy metabolism pathways. Among the messenger RNAs downregulated by metformin, we found c-MYC, IRS-2 and HIF1alpha. Downregulation of c-MYC requires AMP-activated protein kinase-signalling and mir33a upregulation by metformin. Ectopic expression of c-MYC attenuates the anticancer metabolic effects of metformin. We suggest that DICER modulation, mir33a upregulation and c-MYC targeting have an important role in the anticancer metabolic effects of metformin.

Here we show that pemetrexed-treated mesothelioma cells undergo accelerated senescence. This is characterized by the secretion of proinflammatory and mitogenic cytokines, reminiscent of an SASP (senescence-associated secretory phenotype). Conditioned media from senescent MPM (malignant pleural mesothelioma) cells trigger the emergence of EMT (epithelial-to-mesenchymal)-like, clonogenic and chemoresistant cell subpopulations, expressing high levels of ALDH (aldehyde dehydrogenase) activity (ALDH(bright) cells). We show by fluorescence-activated cell sorting of purified ALDH(bright) and ALDH(low) cells, that both cell-autonomous and cell-non-autonomous mechanisms converge to maintain the SASP-induced, EMT-like cell subpopulations. Chemoresistant ALDH(bright) cells exist within primary MPM specimens and enrichment for ALDH(bright) cells correlates with an earlier tumor onset into NOD/SCID mice. We show that RAS(v12) expression induces SASP-like changes in untransformed human mesothelial cells, and that p53 ablation increases the effect of RAS(v12) expression. We identify STAT3 activation as a crucial event downstream to SASP signaling. In fact, small hairpin RNA-mediated ablation of STAT3 deeply attenuates the induction of EMT genes and the increase of ALDH(bright) cells induced by SASP-cytokines. This strongly affects the chemoresistance of MPM cells in vitro and leads to anticancer effects in vivo.

Here we show that a subpopulation of MCF-7 breast cancer cells which stains pale to Toluidine Blue (Light Cells- LCs), is endowed with features of CSCs. LCs give rise to self-renewing mammospheres and express typical CSC markers; moreover this subpopulation is chemoresistant and highly tumorigenic in vivo. LCs can be identified in several other breast cancer cell lines, irrespectively of their histological origin (luminal vs. mesenchymal vs. basal) and represent an heterogeneous cell population composed mainly of CSC-like and early progenitor cells. By a limited in vitro drug screening assay, we identify compounds which can specifically interfere with the viability of LCs from multiple breast cancer cell lines. Analysis of the Sphere-Forming Efficiency (SFE) and of the distribution of ALDH(bright) cells within the treated cell lines suggest that one of the identified compounds acts in vitro by modulating the CSC phenotype. Interestingly, a subset of the identified compounds is known to affect directly or indirectly the NFkappaB pathway which is emerging as an important modulator of CSC proliferation and chemoresistance.

Clinical management of Malignant Pleural Mesothelioma (MPM) is very challenging because of the uncommon resistance of the tumor to chemotherapy. We started from the observation that 7/7 mesothelioma cell lines tested expressed both the Colony Stimulating Factor Receptor-1 and its -ligands CSF-1 and IL-34, a signaling module mainly characterized in hematopoietic cell subpopulations. Pemetrexed and cisplatin treatment did not affect the number of mesothelioma CSF1R-expressing cells. Additionally, interference with CSF1R expression or function significantly abrogated the resistance of these cells to pemetrexed, suggesting the involvement of CSF-1R signaling in mediating chemoresistance of mesothelioma. This prompted us to isolate and characterize the CSF-1Rpos cells and we found that the purified cells expressed a complex repertoire of pluripotency markers, EMT factors and chemoresistance genes. The functions of the CSF-1R expressing MPM cells relied on the autocrine production of CSF-1 and IL-34, whose downregulation affected the viability of the purified cells. We identified AKT, NFkB and STAT3 activation as distinct features of the CSF-1Rpos cells. We showed that all three pathways contributed to the survival of the CSF1Rpos cells, with AKT exerting the most relevant effects. The forced activation of CSF-1R in untransformed mesothelial cells was sufficient to confer clonogenicity and resistance to pemetrexed, typical feature of transformed mesothelial cells. Furthermore, this induced a gene expression profile very similar to that observed in the FACS sorted CSF-1Rpos cells, suggesting a causal relationship between CSF-1R activation and cell transformation. Active AKT signaling contributed to increased levels of nuclear β-catenin in the CSF-1R expressing cells. Inhibition of AKT reduced the transcriptional activity of β-catenin dependent reporters and sensitized the CSF-1Rpos cells to pemetrexed-induced senescence, thereby reversing the resistance of the CSF1Rpos cells to pemetrexed. This correlated with downregulation of c-MYC, a functional CSF-1R target, and upregulation of p21. Finally, we recapitulated the main findings in primary mesothelioma cultures, which exhibited long-term surviving, chemoresistant CSF-1Rpos cells of mesothelial nature and sensitive to CSF-1R inhibition. This work expands what is known on the non-macrophage functions of CSF-1R and its role in solid tumors, suggests that CSF-1R signaling may have a critical pathogenic role in malignant pleural mesothelioma and therefore may represent a promising target for therapeutic intervention. Citation Format: Mario Cioce, Claudia Canino, Chandra Goparaju, Haining Yang, Michele Carbone, Harvey I. Pass. CSF-1R signaling drives mesothelioma stemness and chemoresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2014-4404

In this issue of Molecular Cell , Sen et al. (2011) report the involvement of PGC1α in modulating the transcriptional activity of p53 in metabolically challenged cells. They provide important insights into the mechanisms linking length and strength of the metabolic stress stimuli to the specific activation of p53 target genes.

7577 Background: Clinically silent progression and extraordinary resistance to therapy dictate poor survival for malignant pleural mesothelioma (MPM) patients. Current standard therapies such as an...

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

The transforming proteins of acute promyelocytic leukaemias (APL) are fusions of the promyelocytic leukaemia (PML) and the promyelocytic leukaemia zinc-finger (PLZF) proteins with retinoic acid receptor-alpha (RARalpha). These proteins retain the RARalpha DNA- and retinoic acid (RA)-binding domains, and their ability to block haematopoietic differentiation depends on the RARalpha DNA-binding domain. Thus RA-target genes are downstream effectors. However, treatment with RA induces differentiation of leukaemic blast cells and disease remission in PML-RARalpha APLs, whereas PLZF-RARa APLs are resistant to RA. Transcriptional regulation by RARs involves modifications of chromatin by histone deacetylases, which are recruited to RA-target genes by nuclear co-repressors. Here we show that both PML-RARalpha and PLZF-RARalpha fusion proteins recruit the nuclear co-repressor (N-CoR)-histone deacetylase complex through the RARalpha CoR box. PLZF-RARalpha contains a second, RA-resistant binding site in the PLZF amino-terminal region. High doses of RA release histone deacetylase activity from PML-RARalpha, but not from PLZF-RARalpha. Mutation of the N-CoR binding site abolishes the ability of PML-RARalpha to block differentiation, whereas inhibition of histone deacetylase activity switches the transcriptional and biological effects of PLZF-RARalpha from being an inhibitor to an activator of the RA signalling pathway. Therefore, recruitment of histone deacetylase is crucial to the transforming potential of APL fusion proteins, and the different effects of RA on the stability of the PML-RARalpha and PLZF-RARalpha co-repressor complexes determines the differential response of APLs to RA.

In the last years, two independent concepts have improved our understanding of cancer recurrence and spread: (1) the cancer stem cell (CSC) hypothesis and (2) the occurrence of epithelial-to-mesenchymal transition (EMT). Recent evidences, such as the one presented in a recent issue by Borgna et al.,1 point to a line of convergence of the two concepts. EMT is a physiological cell reprogramming event utilized in tissue remodeling during embryonic development and activated in normal adult tissues during regeneration.2 The presence of EMT-like cells in tumors has been linked to increased invasive and metastatic properties. The CSC hypothesis postulates the existence of hierarchically high-positioned, chemoresistant cells, which are responsible for disease relapse after treatment with debulking agents. These cells are endowed with the ability to reconstitute the histological heterogeneity of the originating tumor upon transplantation in immunodeficient hosts.3 These properties of CSCs have strict resemblance to tissue remodeling and repair, which are typical features of mesenchymal tissues. Indeed, it has been shown that cultured breast cells that have undergone EMT in vitro also possess cancer stem cell signatures and properties.4 Growth of breast cancer cells as non-adherent spheroids in relatively non-differentiating conditions is regarded as a useful tool to enrich cells endowed with CSC-like features, such as chemoresistance and tumor-repopulating ability.5 In the November 2012 issue of Cell Cycle, Silvia Borgna and colleagues provide evidence, at a molecular level and by using a large panel of cell lines corresponding to different breast cancer subtypes, that mammosphere-inducing growth conditions enrich for EMT-like cell subpopulations as well.1 This is especially true for Claudin-low breast cancer cell lines, which are highly enriched for CSC-like, CD44high/CD24low cells. Their work once more suggests that acquisition of EMT and CSC features are highly interconnected processes, possibly relevant for the organization of mammospheres. Indeed, recent evidence has been provided that the interaction of cell subpopulations with distinct mesenchymal and epithelial traits is instrumental for the maintenance of CSC-like cells6 and relies upon cytokine-mediated signaling7 (Fig. 1). In light of this, it may be worth noticing that mammospheres are heterogeneous in composition and represent an ideal place for paracrine signaling to occur between different cell subpopulations. One may thus predict that compounds interfering with this crosstalk can block mammosphere formation (Fig. 1). Indeed Butein, a naturally occurring STAT3 and NFκB inhibitor, impairs mammosphere formation from multiple breast cancer cell lines,5 possibly by blocking IL-6 signaling.6 Figure 1. Schematic hypothetical working model. Mammospheres contain both CSC and EMT-like cells. When grown as mammospheres in non-adherent and no-serum conditions, breast cancer cell cultures are progressively enriched in EMT-like cells. Inset: ... Interestingly, Borgna et al. found at least one of the known EMT-promoting transcription factors to be dynamically modulated in most of their cultures in time (mainly SNAI2 and TWIST1) when shifting from adherent to mammosphere culture conditions.1 This underscores the relevance of such a process and its activation by distinct, converging and interconnected pathways. Indeed, forced expression of individual EMT-inducing transcription factors in stabilized cell lines has led Weinberg and collaborators to postulate the existence of an EMT interactome of transcription factors which are capable of reciprocally influencing each other. It will be interesting to evaluate the levels of EMT-promoting factors upon chemotherapy treatment of spheroids in vitro and to establish whether the enrichment for EMT-like cells is relevant to chemoresistance of mammospheres. Culturing cancer cells as 3D spheroids may represent, therefore, a simplified albeit very useful tool for reproducing in vitro transient dynamic states of the tumor growth. It may also stimulate a shift in the way we envision hunting for novel therapeutic tools. Finally, this methodology is of general value, as it can be applied to cancers from other histotypes. For example, cells derived from biopsies or from malignant pleural effusions of patients with NSCLC give rise efficiently to propagating tumor spheroids in culture,8 which are, again, enriched in CSC markers.8,9 In conclusion, in vitro cultures of tumor spheroids from stabilized cell lines and from fresh tumor specimens may therefore be considered a useful in vitro model to screen for new agents capable of co-targeting both CSCs or EMT malignant features of cancer cells.

RON belongs to the c-MET family of receptor tyrosine kinases. As its well-known family member MET, RON and its ligand macrophage-stimulating protein have been implicated in the progression and metastasis of tumors and have been shown to be overexpressed in cancer. We generated and tested a large number of human monoclonal antibodies (mAbs) against human RON. Our screening yielded three high-affinity antibodies that efficiently block ligand-dependent intracellular AKT and MAPK signaling. This effect correlates with the strong reduction of ligand-activated migration of T47D breast cancer cell line. By cross-competition experiments, we showed that the antagonistic antibodies fall into three distinct epitope regions of the RON extracellular Sema domain. Notably, no inhibition of tumor growth was observed in different epithelial tumor xenografts in nude mice with any of the antibodies. These results suggest that distinct properties beside ligand antagonism are required for anti-RON mAbs to exert antitumor effects in vivo.