Etienne Moussay

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.

MicroRNA as biomarkers and regulators in B-cell chronic lymphocytic leukemia

Early cancer detection and disease stratification or classification are critical to successful treatment. Accessible, reliable, and informative cancer biomarkers can be medically valuable and can provide some relevant insights into cancer biology. Recent studies have suggested improvements in detecting malignancies by the use of specific extracellular microRNAs (miRNAs) in plasma. In chronic lymphocytic leukemia (CLL), an incurable hematologic disorder, sensitive, early, and noninvasive diagnosis and better disease classification would be very useful for more effective therapies. We show here that circulating miRNAs can be sensitive biomarkers for CLL, because certain extracellular miRNAs are present in CLL patient plasma at levels significantly different from healthy controls and from patients affected by other hematologic malignancies. The levels of several of these circulating miRNAs also displayed significant differences between zeta-associated protein 70 (ZAP-70)+ and ZAP-70− CLL. We also determined that the level of circulating miR-20a correlates reliably with diagnosis-to-treatment time. Network analysis of our data, suggests a regulatory network associated with BCL2 and ZAP-70 expression in CLL. This hypothesis suggests the possibility of using the levels of specific miRNAs in plasma to detect CLL and to determine the ZAP-70 status.

Hypoxic tumor-derived microvesicles negatively regulate NK cell function by a mechanism involving TGF-β and miR23a transfer

ABSTRACT Tumor-derived microvesicles (TD-MVs) are key mediators which are shed by cancer cells and can sensitize neighboring cells in the tumor microenvironment. TD-MVs are extracellular vesicles composed of exosomes and MVs and promote cancer invasion and metastasis. Intratumoral hypoxia is an integral component of all solid tumors. The relationship between hypoxic tumor-shed MVs and NK-mediated cytotoxicity remains unknown. In this paper, we reported that MVs derived from hypoxic tumor cells qualitatively differ from those derived from normoxic tumor cells. Using multiple tumor models, we showed that hypoxic MVs inhibit more NK cell function as compared to normoxic MVs. Hypoxic TD-MVs package two immunosuppressive factors involved in the impairment of natural killer (NK) cell cytotoxicity against different tumor cells in vitro and in vivo. We showed that following their uptake by NK cells, hypoxic TD-MVs transfer TGF-β1 to NK cells, decreasing the cell surface expression of the activating receptor NKG2D, thereby inhibiting NK cell function. MicroRNA profiling revealed the presence of high levels of miR-210 and miR-23a in hypoxic TD-MVs. We demonstrated that miR-23a in hypoxic TD-MVs operates as an additional immunomosuppressive factor, since it directly targets the expression of CD107a in NK cells. To our knowledge, this is the first study to show that hypoxic tumor cells by secreting MVs can educate NK cells and decrease their antitumor immune response. This study highlights the existence of a novel mechanism of immune suppression mediated by hypoxic TD-MVs and further improves our understanding of the immunosuppressive mechanisms prevailing in the hypoxic tumor microenvironment.

The critical role of the tumor microenvironment in shaping natural killer cell-mediated anti-tumor immunity.

Considerable evidence has been gathered over the last 10 years showing that the tumor microenvironment (TME) is not simply a passive recipient of immune cells, but an active participant in the establishment of immunosuppressive conditions. It is now well documented that hypoxia, within the TME, affects the functions of immune effectors including natural killer (NK) cells by multiple overlapping mechanisms. Indeed, each cell in the TME, irrespective of its transformation status, has the capacity to adapt to the hostile TME and produce immune modulatory signals or mediators affecting the function of immune cells either directly or through the stimulation of other cells present in the tumor site. This observation has led to intense research efforts focused mainly on tumor-derived factors. Notably, it has become increasingly clear that tumor cells secrete a number of environmental factors such as cytokines, growth factors, exosomes, and microRNAs impacting the immune cell response. Moreover, tumor cells in hostile microenvironments may activate their own intrinsic resistance mechanisms, such as autophagy, to escape the effective immune response. Such adaptive mechanisms may also include the ability of tumor cells to modify their metabolism and release several metabolites to impair the function of immune cells. In this review, we summarize the different mechanisms involved in the TME that affect the anti-tumor immune function of NK cells.

The acquisition of resistance to TNFα in breast cancer cells is associated with constitutive activation of autophagy as revealed by a transcriptome analysis using a custom microarray

While the autophagic process is mainly regulated at the post-translational level, a growing body of evidence suggests that autophagy might also be regulated at the transcriptional level. The identification of transcription factors involved in the regulation of autophagy genes has provided compelling evidence for such regulation. In this context, a powerful high throughput analysis tool to simultaneously monitor the expression level of autophagy genes is urgently needed. Here we describe setting up the first comprehensive human autophagy database (HADb, available at www.autophagy.lu) and the development of a companion Human Autophagy-dedicated cDNA Microarray which comprises 234 genes involved in or related to autophagy. The autophagy microarray tool used on breast adenocarcinoma MCF-7 cell line allowed the identification of 47 differentially expressed autophagy genes associated with the acquisition of resistance to the cytotoxic effect of TNFα. The autophagy-core machinery genes DRAM (Damage-Regulated Autophagy Modulator), BNIP3L (BCL2/adenovirus E1B 19 kDa interacting protein 3-like), BECN1 (Beclin 1), GABARAP (Gamma-AminoButyric Acid Receptor-Associated Protein) and UVRAG (UV radiation resistance associated gene) were found upregulated in TNF-resistant cells, suggesting a constitutive activation of the autophagy machinery in these cells. More interestingly, we identified NPC1 as the most upregulated genes in TNF-resistant compared to TNF-sensitive MCF-7 cells, suggesting a relation between the intracellular transport of cholesterol, the regulation of autophagy and NPC1 expression in TNF-resistant tumor cells. In conclusion, we describe here new tools that may help investigating autophagy gene regulation in various cellular models and diseases.

The Histone Deacetylase Inhibitor MGCD0103 Induces Apoptosis in B-Cell Chronic Lymphocytic Leukemia Cells through a Mitochondria-Mediated Caspase Activation Cascade

Clinical trials have shown activity of the isotype-selective histone deacetylase (HDAC) inhibitor MGCD0103 in different hematologic malignancies. There are data to support the use of HDAC inhibitors in association with other cancer therapies. To propose a rational combination therapy, it is necessary to depict the molecular basis behind the cytotoxic effect of MGCD0103. In this study, we found that MGCD0103 was substantially more toxic in neoplastic B cells relative to normal cells, and we described the death pathways activated by MGCD0103 in B-cell chronic lymphocytic leukemia (CLL) cells from 32 patients. MGCD0103 decreased the expression of Mcl-1 and induced translocation of Bax to the mitochondria, mitochondrial depolarization, and release of cytochrome c in the cytosol. Caspase processing in the presence of the caspase inhibitor Q-VD-OPh and time course experiments showed that caspase-9 was the apical caspase. Thus, MGCD0103 induced the intrinsic pathway of apoptosis in CLL cells. Moreover, MGCD0103 treatment resulted in the activation of a caspase cascade downstream of caspase-9, caspase-dependent amplification of mitochondrial depolarization, activation of calpain, and Bax cleavage. We propose a model whereby the intrinsic pathway of apoptosis triggered by MGCD0103 in CLL is associated with a mitochondrial death amplification loop. Mol Cancer Ther; 9(5); 1349–60. ©2010 AACR.

Autophagy: An adaptive metabolic response to stress shaping the antitumor immunity

Several environmental-associated stress conditions, including hypoxia, starvation, oxidative stress, fast growth and cell death suppression, modulate both cellular metabolism and autophagy to enable cancer cells to rapidly adapt to environmental stressors, maintain proliferation and evade therapies. It is now widely accepted that autophagy is essential to support cancer cell growth and metabolism and that metabolic reprogramming in cancer can also favor autophagy induction. Therefore, this complex interplay between autophagy and tumor cell metabolism will provide unique opportunities to identify new therapeutic targets. As the regulation of the autophagic activity is related to metabolism, it is important to elucidate the exact molecular mechanism which drives it and the functional consequence of its activation in the context of cancer therapy. In this review, we will summarize the role of autophagy in shaping the cellular response to an abnormal tumor microenvironment and discuss some recent results on the molecular mechanism by which autophagy plays such a role in the context of the anti-tumor immune response. We will also describe how autophagy activation can behave as a double-edged sword, by activating the immune response in some circumstances, and impairing the anti-tumor immunity in others. These findings imply that defining the precise context-specific role for autophagy in cancer is critical to guide autophagy-based therapeutics which are becoming key strategies to overcome tumor resistance to therapies.

Tumor-derived exosomes modulate PD-L1 expression in monocytes.

Transfer of exosomal RNA from leukemic cells to monocytes induces immunosuppression. Messaging with RNAs Understanding interactions between tumor cells and immune cells is essential for tailoring immunocentric therapies to tumors. Here, Haderk et al. have identified a key role for tumor-derived exosomes in modulating immune responses to chronic lymphocytic leukemia (CLL). They report that CLL-derived exosomal RNAs promote monocytes in CLL patients to adopt an immunosuppressive phenotype, including promoting expression of PD-L1. They identify noncoding RNA hY4 as a key functional component of CLL-derived exosomes and show that hY4 promotes exosome-dependent skewing of monocytes in a TLR7-dependent manner. Using mouse models, they found that inhibition of TLR7 delayed progression of CLL, opening up the possibility that the TLR7 pathway could be therapeutically targeted in CLL. In chronic lymphocytic leukemia (CLL), monocytes and macrophages are skewed toward protumorigenic phenotypes, including the release of tumor-supportive cytokines and the expression of immunosuppressive molecules such as programmed cell death 1 ligand 1 (PD-L1). To understand the mechanism driving protumorigenic skewing in CLL, we evaluated the role of tumor cell–derived exosomes in the cross-talk with monocytes. We carried out RNA sequencing and proteome analyses of CLL-derived exosomes and identified noncoding Y RNA hY4 as a highly abundant RNA species that is enriched in exosomes from plasma of CLL patients compared with healthy donor samples. Transfer of CLL-derived exosomes or hY4 alone to monocytes resulted in key CLL-associated phenotypes, including the release of cytokines, such as C-C motif chemokine ligand 2 (CCL2), CCL4, and interleukin-6, and the expression of PD-L1. These responses were abolished in Toll-like receptor 7 (TLR7)–deficient monocytes, suggesting exosomal hY4 as a driver of TLR7 signaling. Pharmacologic inhibition of endosomal TLR signaling resulted in a substantially reduced activation of monocytes in vitro and attenuated CLL development in vivo. Our results indicate that exosome-mediated transfer of noncoding RNAs to monocytes contributes to cancer-related inflammation and concurrent immune escape via PD-L1 expression.

The multifaceted role of autophagy in tumor evasion from immune surveillance

While autophagy is constitutively executed at basal level in all cells, it is activated in cancer cells in response to various microenvironmental stresses including hypoxia. It is now well established that autophagy can act both as tumor suppressor or tumor promoter. In this regard, several reports indicate that the tumor suppressor function of autophagy is associated with its ability to scavenge damaged oxidative organelles, thereby preventing the accumulation of toxic oxygen radicals and limiting the genome instability. Paradoxically, in developed tumors, autophagy can promote the survival of cancer cells and therefore operates as a cell resistance mechanism. The consensus appears to be that autophagy has a dual role in suppressing tumor initiation and in promoting the survival of established tumors. This has inspired significant interest in applying anti-autophagy therapies as an entirely new approach to cancer treatment. While much remains to be learned about the regulation and context-dependent biological role of autophagy, it is now well established that modulation of this process could be an attractive approach for the development of novel anticancer therapeutic strategies. In this review, we will summarize recent reports describing how tumor cells, by activating autophagy, manage to resist the immune cell attack. Data described in this review strongly argue that targeting autophagy may represent a conceptual realm for new immunotherapeutic strategies aiming to block the immune escape and therefore providing rational approach to future tumor immunotherapy design.

Role of the Low-Density Lipoprotein Receptor in Entry of Bovine Viral Diarrhea Virus

ABSTRACT Among several proposed cellular receptors for bovine viral diarrhea virus (BVDV), the low-density lipoprotein (LDL) receptor is of special interest because it is also considered a receptor for the related hepatitis C virus. It has been reported that an anti-LDL receptor monoclonal antibody blocked the infection of bovine cells by BVDV and that the resistance of bovine CRIB cells (cells resistant to infection with BVDV) (E. F. Flores and R. O. Donis, Virology 208:565-575, 1995) to BVDV infection was due to a lack of the LDL receptor (V. Agnello et al., Proc. Natl. Acad. Sci. USA 96:12766-12771, 1999). In connection with our studies on BVDV entry, we reevaluated the putative role of the LDL receptor as a cellular receptor for BVDV. It was first clearly demonstrated that neither of two monoclonal antibodies against the LDL receptor inhibited BVDV infection of two bovine cell lines. Furthermore, the LDL receptor was detected on the surface of CRIB cells. The functionality of the LDL receptor on CRIB cells was demonstrated by the internalization of fluorescently labeled LDL. In conclusion, at present no experimental evidence supports an involvement of the LDL receptor in BVDV invasion.