B-cell receptor signaling activity identifies patients with mantle cell lymphoma at higher risk of progression.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

MEK1/2 regulate normal BCR and ABL1 tumor-suppressor functions to dictate ATO response in TKI-resistant Ph+ leukemia.

Resistance to tyrosine kinase inhibitors (TKIs) remains a clinical challenge in Ph-positive variants of chronic myeloid leukemia. We provide mechanistic insights into a previously undisclosed MEK1/2/BCR::ABL1/BCR/ABL1-driven signaling loop that may determine the efficacy of arsenic trioxide (ATO) in TKI-resistant leukemic patients. We find that activated MEK1/2 assemble into a pentameric complex with BCR::ABL1, BCR and ABL1 to induce phosphorylation of BCR and BCR::ABL1 at Tyr360 and Tyr177, and ABL1, at Thr735 and Tyr412 residues thus provoking loss of BCR's tumor-suppression functions, enhanced oncogenic activity of BCR::ABL1, cytoplasmic retention of ABL1 and consequently drug resistance. Coherently, pharmacological blockade of MEK1/2 induces dissociation of the pentameric MEK1/2/BCR::ABL1/BCR/ABL1 complex and causes a concurrent BCRY360/Y177, BCR::ABL1Y360/Y177 and cytoplasmic ABL1Y412/T735 dephosphorylation thereby provoking the rescue of the BCR's anti-oncogenic activities, nuclear accumulation of ABL1 with tumor-suppressive functions and consequently, growth inhibition of the leukemic cells and an ATO sensitization via BCR-MYC and ABL1-p73 signaling axes activation. Additionally, the allosteric activation of nuclear ABL1 was consistently found to enhance the anti-leukemic effects of the MEK1/2 inhibitor Mirdametinib, which when combined with ATO, significantly prolonged the survival of mice bearing BCR::ABL1-T315I-induced leukemia. These findings highlight the therapeutic potential of MEK1/2-inhibitors/ATO combination for the treatment of TKI-resistant leukemia.

Tumor Suppressor Role of Wild-Type P53-Dependent Secretome and Its Proteomic Identification in PDAC.

The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space. Thus, loss of p53 has a profound impact on the secretome composition of cancer cells and marks the transition to invasiveness. Here, we demonstrate the tumor suppressive role of wild-type p53 on cancer cell secretome, showing the anti-proliferative, apoptotic and chemosensitivity effects of wild-type p53 driven conditioned medium. By using high-resolution SWATH-MS technology, we characterized the secretomes of p53-deficient and p53-expressing PDAC cells. We found a great number of secreted proteins that have known roles in cancer-related processes, 30 of which showed enhanced and 17 reduced secretion in response to p53 silencing. These results are important to advance our understanding on the link between wt-p53 and cancer microenvironment. In conclusion, this approach may detect a secreted signature specifically driven by wild-type p53 in PDAC.

Hyaluronated and PEGylated Liposomes as a Potential Drug-Delivery Strategy to Specifically Target Liver Cancer and Inflammatory Cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and is characterized by poor clinical outcomes, with the majority of patients not being eligible for curative therapy and treatments only being applicable for early-stage tumors. CD44 is a receptor for hyaluronic acid (HA) and is involved in HCC progression. The aim of this work is to propose HA- and PEGylated-liposomes as promising approaches for the treatment of HCC. It has been found, in this work, that CD44 transcripts are up-regulated in HCC patients, as well as in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Cell culture experiments indicate that HA-liposomes are more rapidly and significantly internalized by Huh7 cells that over-express CD44, compared with HepG2 cells that express low levels of the receptor, in which the uptake seems due to endocytic events. By contrast, human and murine macrophage cell lines (THP-1, RAW264.7) show improved and rapid uptake of PEG-modified liposomes without the involvement of the CD44. Moreover, the internalization of PEG-modified liposomes seems to induce polarization of THP1 towards the M1 phenotype. In conclusion, data reported in this study indicate that this strategy can be proposed as an alternative for drug delivery and one that dually and specifically targets liver cancer cells and infiltrating tumor macrophages in order to counteract two crucial aspect of HCC progression.

Browsing the oldest antioxidant enzyme: catalase and its multiple regulation in cancer.

Aerobic organisms possess numerous antioxidant enzymatic families, including catalases, superoxide dismutases (SODs), peroxiredoxins (PRDXs), and glutathione peroxidases (GPXs), which work cooperatively to protect cells from an excess of reactive oxygen species (ROS) derived from endogenous metabolism or external microenvironment. Catalase, as well as other antioxidant enzymes, plays an important dichotomous role in cancer. Therefore, therapies aimed at either reverting the increased or further escalating catalase levels could be effective, depending on the metabolic landscape and on the redox status of cancer cells. This dichotomous role of catalase in cancers highlights the importance to deepen comprehensively the role and the regulation of this crucial antioxidant enzyme. The present review highlights the role of catalase in cancer and provides a comprehensive description of the molecular mechanisms associated with the multiple levels of catalase regulation.

Progressively De-Differentiated Pancreatic Cancer Cells Shift from Glycolysis to Oxidative Metabolism and Gain a Quiescent Stem State.

Pancreatic ductal adenocarcinoma (PDAC) is typically characterized by high chemoresistance and metastatic spread, features mainly attributable to cancer stem cells (CSCs). It is of central interest the characterization of CSCs and, in particular, the study of their metabolic features in order to selectively identify their peculiarities for an efficient therapeutic approach. In this study, CSCs have been obtained by culturing different PDAC cell lines with a specific growth medium. Cells were characterized for the typical stem/mesenchymal properties at short-, medium-, and long-term culture. Metabolomics, proteomics, analysis of oxygen consumption rate in live cells, and the effect of the inhibition of lactate transporter on cell proliferation have been performed to delineate the metabolism of CSCs. We show that gradually de-differentiated pancreatic cancer cells progressively increase the expression of both stem and epithelial-to-mesenchymal transition markers, shift their metabolism from a glycolytic to an oxidative one, and lastly gain a quiescent state. These quiescent stem cells are characterized by high chemo-resistance, clonogenic ability, and metastatic potential. Re-differentiation reverts these features, re-activating their proliferative capacity and glycolytic metabolism, which generally correlates with high aggressiveness. These observations add an important piece of knowledge to the comprehension of the biology of CSCs, whose metabolic plasticity could be exploited for the generation of promising and selective therapeutic approaches for PDAC patients.

The Mutant p53-Driven Secretome Has Oncogenic Functions in Pancreatic Ductal Adenocarcinoma Cells.

The cancer secretome is a rich repository of useful information for both cancer biology and clinical oncology. A better understanding of cancer secretome is particularly relevant for pancreatic ductal adenocarcinoma (PDAC), whose extremely high mortality rate is mainly due to early metastasis, resistance to conventional treatments, lack of recognizable symptoms, and assays for early detection. TP53 gene is a master transcriptional regulator controlling several key cellular pathways and it is mutated in ~75% of PDACs. We report the functional effect of the hot-spot p53 mutant isoforms R175H and R273H on cancer cell secretome, showing their influence on proliferation, chemoresistance, apoptosis, and autophagy, as well as cell migration and epithelial-mesenchymal transition. We compared the secretome of p53-null AsPC-1 PDAC cells after ectopic over-expression of R175H-mutp53 or R273H-mutp53 to identify the differentially secreted proteins by mutant p53. By using high-resolution SWATH-MS technology, we found a great number of differentially secreted proteins by the two p53 mutants, 15 of which are common to both mutants. Most of these secreted proteins are reported to promote cancer progression and epithelial-mesenchymal transition and might constitute a biomarker secreted signature that is driven by the hot-spot p53 mutants in PDAC.

Regulation of succinate dehydrogenase and role of succinate in cancer.

Succinate dehydrogenase (SDH) has been classically considered a mitochondrial enzyme with the unique property to participate in both the citric acid cycle and the electron transport chain. However, in recent years, several studies have highlighted the role of the SDH substrate, i.e. succinate, in biological processes other than metabolism, tumorigenesis being the most remarkable. For this reason, SDH has now been defined a tumor suppressor and succinate an oncometabolite. In this review, we discuss recent findings regarding alterations in SDH activity leading to succinate accumulation, which include SDH mutations, regulation of mRNA expression, post-translational modifications and endogenous SDH inhibitors. Further, we report an extensive examination of the role of succinate in cancer development through the induction of epigenetic and metabolic alterations and the effects on epithelial to mesenchymal transition, cell migration and invasion, and angiogenesis. Finally, we have focused on succinate and SDH as diagnostic markers for cancers having altered SDH expression/activity.

Hyaluronic Acid-Decorated Liposomes as Innovative Targeted Delivery System for Lung Fibrotic Cells.

Collagen Tissue Disease-associated Interstitial Lung Fibrosis (CTD-ILDs) and Bronchiolitis Obliterans Syndrome (BOS) represent severe lung fibrogenic disorders, characterized by fibro-proliferation with uncontrolled extracellular matrix deposition. Hyaluronic acid (HA) plays a key role in fibrosis with its specific receptor, CD44, overexpressed by CTD-ILD and BOS cells. The aim is to use HA-liposomes to develop an inhalatory treatment for these diseases. Liposomes with HA of two molecular weights were prepared and characterized. Targeting efficiency was assessed toward CTD-ILD and BOS cells by flow cytometry and confocal microscopy and immune modulation by RT-PCR and ELISA techniques. HA-liposomes were internalized by CTD-ILD and BOS cells expressing CD44, and this effect increased with higher HA MW. In THP-1 cells, HA-liposomes decreased pro-inflammatory cytokines IL-1ß, IL-12, and anti-fibrotic VEGF transcripts but increased TGF-ß mRNA. However, upon analyzing TGF-ß release from healthy donors-derived monocytes, we found liposomes did not alter the release of active pro-fibrotic cytokine. All liposomes induced mild activation of neutrophils regardless of the presence of HA. HA liposomes could be also applied for lung fibrotic diseases, being endowed with low pro-inflammatory activity, and results confirmed that higher MW HA are associated to an increased targeting efficiency for CD44 expressing LFs-derived from BOS and CTD-ILD patients.

Regulation of Autophagy by Nuclear GAPDH and Its Aggregates in Cancer and Neurodegenerative Disorders.

Several studies indicate that the cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has pleiotropic functions independent of its canonical role in glycolysis. The GAPDH functional diversity is mainly due to post-translational modifications in different amino acid residues or due to protein-protein interactions altering its localization from cytosol to nucleus, mitochondria or extracellular microenvironment. Non-glycolytic functions of GAPDH include the regulation of cell death, autophagy, DNA repair and RNA export, and they are observed in physiological and pathological conditions as cancer and neurodegenerative disorders. In disease, the knowledge of the mechanisms regarding GAPDH-mediated cell death is becoming fundamental for the identification of novel therapies. Here, we elucidate the correlation between autophagy and GAPDH in cancer, describing the molecular mechanisms involved and its impact in cancer development. Since autophagy is a degradative pathway associated with the regulation of cell death, we discuss recent evidence supporting GAPDH as a therapeutic target for autophagy regulation in cancer therapy. Furthermore, we summarize the molecular mechanisms and the cellular effects of GAPDH aggregates, which are correlated with mitochondrial malfunctions and can be considered a potential therapeutic target for various diseases, including cancer and neurodegenerative disorders.

Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O2-· production in cancer cells.

BACKGROUND: The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 gain-of-function isoforms actively promote cancer malignancy. METHODS: A panel of wild-type and mutant p53 cancer cell lines of different tissues, including pancreas, breast, skin, and lung were used, as well as chronic lymphocytic leukemia (CLL) patients with different TP53 gene status. The effects of mutant p53 were evaluated by confocal microscopy, reactive oxygen species production assay, immunoblotting, and quantitative reverse transcription polymerase chain reaction after cellular transfection. RESULTS: We demonstrate that oncogenic mutant p53 isoforms are able to inhibit SESN1 expression and consequently the amount of SESN1/AMPK complex, resulting in the downregulation of the AMPK/PGC-1α/UCP2 axis and mitochondrial O2-· production. We also show a correlation between the decrease of reduced thiols with a poorer clinical outcome of CLL patients bearing mutant TP53 gene. The restoration of the mitochondrial uncoupling protein 2 (UCP2) expression, as well as the addition of the radical scavenger N-acetyl-L-cysteine, reversed the oncogenic effects of mutant p53 as cellular hyper-proliferation, antiapoptotic effect, and resistance to drugs. CONCLUSIONS: The inhibition of the SESN1/AMPK/PGC-1α/UCP2 axis contributes to the pro-oxidant and oncogenic effects of mutant p53, suggesting pro-oxidant drugs as a therapeutic approach for cancer patients bearing mutant TP53 gene.

Regenerative potential of the Bichat fat pad determined by the quantification of multilineage differentiating stress enduring cells.

Published studies regarding Bichat fat pad focused, quite exclusively, on the implant of this adipose depot for different facial portions reconstruction. The regenerative components of Bichat fat pad were poorly investigated. The present study aimed to describe by an ultrastructural approach the Bichat fat pad, providing novel data at the ultrastructural and cellular level. This data sets improve the knowledge about the usefulness of the Bichat fat pad in regenerative and reconstructive surgery. Bichat fat pads were harvested form eight patients subjected to maxillofacial, dental and aesthetic surgeries. Biopsies were used for the isolation of mesenchymal cell compartment and for ultrastructural analysis. Respectively, Bichat fat pads were either digested and placed in culture for the characterization of mesenchymal stem cells (MSCs) or, were fixed in glutaraldehyde 2% and processed for transmission or scanning electron microscopy. Collected data showed very interesting features regarding the cellular composition of the Bichat fat pad and, in particular, experiments aimed to characterized the MSCs showed the presence of a sub-population of MSCs characterized by the expression of specific markers that allow to classify them as multilineage differentiating stress enduring cells.  This data set allows to collect novel information about regenerative potential of Bichat fat pad that could explain the success of its employment in reconstructive and regenerative medicine.

MYC-related microRNAs signatures in non-Hodgkin B-cell lymphomas and their relationships with core cellular pathways.

In order to investigate the role of microRNAs in the pathogenesis of different B-cell lymhoma subtypes, we have applied an array-based assay to a series of 76 mixed non-Hodgkin B-cell lymphomas, including Burkitt's lymphoma (BL), diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma (MCL) and follicular lymphoma. Lymphomas clustered according to histological subtypes, driven by two miRNA clusters (the miR-29 family and the miR-17-92 cluster). Since the two miRNA clusters are known to be MYC-regulated, we investigated whether this would be supported in MYC-driven experimental models, and found that this signature separated BL cell lines and a MYC-translocated MCL cell lines from normal germinal center B-cells and other B-cell populations. Similar results were also reproduced in tissue samples comparing BL and reactive lymph node samples. The same series was then quantitatively analyzed for MYC expression by immunohistochemistry and MYC protein levels were compared with corresponding miRNA signatures. A specific metric was developed to summarize the levels of MYC-related microRNAs and the corresponding protein levels. We found that MYC-related signatures are directly related to MYC protein expression across the whole spectrum of B-cells and B-cell lymphoma, suggesting that the MYC-responsive machinery shows predominantly quantitative, rather than qualitative, modifications in B-cell lymphoma. Novel MYC-related miRNAs were also discovered by this approach. Finally, network analysis found that in BL MYC-related differentially expressed miRNAs could control, either positively or negatively, a limited number of hub proteins, including BCL2, CDK6, MYB, ZEB1, CTNNB1, BAX and XBP1.

MicroRNA signatures and Foxp3+ cell count correlate with relapse occurrence in follicular lymphoma.

First line drug treatment of follicular lymphoma (FL) patients is followed by a highly variable disease-free time before relapse in about one third of patients. No molecular marker is able to predict efficiently the risk of relapse. We investigated the expression profile of microRNAs (miRNAs) by microarrays and of the tumor microenvironment by immunohistochemistry in 26 FLs and 12 reactive lymph nodes (rLN) as reference. Twenty-nine miRNAs were differentially expressed in FLs compared to rLNs and some of them discriminated grade 1 from 3a FLs. Both FLs and rLNs displayed molecular heterogeneity. FLs grouped into two clusters mostly driven by the tumor T-cell content. Among 21 drug-treated FL patients with an average follow-up of 13.5 years, eight cases relapsed. Twenty-six miRNAs discriminated between relapsed and non-relapsed FLs. Ten miRNAs also correlated with Foxp3+ cells number. Notably, Foxp3+ cells were significantly less in relapsed patients and lower Foxp3+ cell number associated with shorter time-to-relapse. Foxp3+ cells did not co-expressed follicular helper T-cell markers and were therefore classified as regulatory T cells rather than follicular regulatory T-cells. These findings introduce new knowledge about the relationship between miRNA alterations and infiltrating immune cells and show that Foxp3+ cells might be predictive of disease relapse.

Corrigendum: HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity.

This corrects the article DOI: 10.1038/srep40037.

Low catalase expression confers redox hypersensitivity and identifies an indolent clinical behavior in CLL.

B-cell receptor (BCR) signaling is a key determinant of variable clinical behavior and a target for therapeutic interventions in chronic lymphocytic leukemia (CLL). Endogenously produced H2O2 is thought to fine-tune the BCR signaling by reversibly inhibiting phosphatases. However, little is known about how CLL cells sense and respond to such redox cues and what effect they have on CLL. We characterized the response of BCR signaling proteins to exogenous H2O2 in cells from patients with CLL, using phosphospecific flow cytometry. Exogenous H2O2 in the absence of BCR engagement induced a signaling response of BCR proteins that was higher in CLL with favorable prognostic parameters and an indolent clinical course. We identified low catalase expression as a possible mechanism accounting for redox signaling hypersensitivity. Decreased catalase could cause an escalated accumulation of exogenous H2O2 in leukemic cells with a consequent greater inhibition of phosphatases and an increase of redox signaling sensitivity. Moreover, lower levels of catalase were significantly associated with a slower progression of the disease. In leukemic cells characterized by redox hypersensitivity, we also documented an elevated accumulation of ROS and an increased mitochondrial amount. Taken together, our data identified redox sensitivity and metabolic profiles that are linked to differential clinical behavior in CLL. This study advances our understanding of the redox and signaling heterogeneity of CLL and provides the rationale for the development of therapies targeting redox pathways in CLL.

Hyaluronated mesoporous silica nanoparticles for active targeting: influence of conjugation method and hyaluronic acid molecular weight on the nanovector properties.

We have prepared and evaluated the physico-chemical and biological properties of four different hyaluronated mesoporous silica nanoparticles (MSNs) samples (MSN/HA). Hyaluronic acid (HA) with two different molecular weights (200 and 6.4 kDa) was used for the conjugation of aminopropyl-functionalized MSN (NH2-MSN), following two different procedures. Namely, samples HA200A and HA6.4A were prepared by reacting activated HA with NH2-MSN (method A), while samples HA200B and HA6.4B were obtained carrying out HA activation in the presence of the nanoparticles (method B). The four samples showed similar hydrophilicity, but clear differences in the HA loading, textural properties, surface charge and stability of the suspensions. More in detail, conjugation using low molecular weight HA with method A resulted in low HA loading, with consequent scarce effects on dispersity and stability in physiological media. The highest yield and corresponding best performances were obtained with method B using high molecular weight HA. HA loading and molecular weight also influenced in a concerted way the biological response towards the MSNs of CD44 target cancer cells (CD44+) and control cells (CD44-): MDA-MB-231 and A2780, respectively. The absence of cytotoxicity was assessed. Moreover, the targeting ability of the best performing MSN/HA was confirmed by cellular uptake studies.

Stability and Expression Levels of HLA-C on the Cell Membrane Modulate HIV-1 Infectivity.

HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/ß2 microglobulin [ß2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to ß2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to ß2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication.IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes ß2 microglobulin/peptide; the other conformation is not bound to ß2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from ß2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

Endothelin-1 receptor blockade as new possible therapeutic approach in multiple myeloma.

New effective treatments are needed to improve outcomes for multiple myeloma (MM) patients. Receptors with restricted expression on plasma cells (PCs) represent attractive new therapeutic targets. The endothelin-1 (EDN1) axis, consisting of EDN1 acting through EDN-receptor A (EDNRA) and B (EDNRB), was previously shown to be overexpressed in several tumours, including MM. However, there is incomplete understanding of how EDN1 axis regulates MM growth and response to therapy. Besides EDNRA, the majority of MM cell lines and primary malignant PCs express high levels of EDNRB and release EDN1. Similarly, bone-marrow microenvironment cells also secrete EDN1. Investigating the extent of epigenetic dysregulation of EDNRB gene in MM, we found that hypermethylation of EDNRB promoter and subsequent down-regulation of EDNRB gene was observed in PCs or B lymphocytes from healthy donors compared to EDNRB-expressing malignant PCs. Pharmacological blockade with the dual EDN1 receptor antagonist bosentan decreased cell viability and MAPK activation of U266 and RPMI-8226 cells. Interestingly, the combination of bosentan and the proteasome inhibitor bortezomib, currently approved for MM treatment, resulted in synergistic cytotoxic effects. Overall, our data has uncovered EDN1-mediated autocrine and paracrine mechanisms that regulate malignant PCs growth and drug response, and support EDN1 receptors as new therapeutic targets in MM.