Role of Epithelial-to-Mesenchymal Transition for the Generation of Circulating Tumors Cells and Cancer Cell Dissemination.

Tumor-related death is primarily caused by metastasis; consequently, understanding, preventing, and treating metastasis is essential to improving clinical outcomes. Metastasis is mainly governed by the dissemination of tumor cells in the systemic circulation: so-called circulating tumor cells (CTCs). CTCs typically arise from epithelial tumor cells that undergo epithelial-to-mesenchymal transition (EMT), resulting in the loss of cell-cell adhesions and polarity, and the reorganization of the cytoskeleton. Various oncogenic factors can induce EMT, among them the transforming growth factor (TGF)-ß, as well as Wnt and Notch signaling pathways. This entails the activation of numerous transcription factors, including ZEB, TWIST, and Snail proteins, acting as transcriptional repressors of epithelial markers, such as E-cadherin and inducers of mesenchymal markers such as vimentin. These genetic and phenotypic changes ultimately facilitate cancer cell migration. However, to successfully form distant metastases, CTCs must primarily withstand the hostile environment of circulation. This includes adaption to shear stress, avoiding being trapped by coagulation and surviving attacks of the immune system. Several applications of CTCs, from cancer diagnosis and screening to monitoring and even guided therapy, seek their way into clinical practice. This review describes the process leading to tumor metastasis, from the generation of CTCs in primary tumors to their dissemination into distant organs, as well as the importance of subtyping CTCs to improve personalized and targeted cancer therapy.

Nanoparticle-based immunotherapy of pancreatic cancer.

Pancreatic cancer (PC) has a complex and unique tumor microenvironment (TME). Due to the physical barrier formed by the desmoplastic stroma, the delivery of drugs to the tumor tissue is limited. The TME also contributes to resistance to various immunotherapies such as cancer vaccines, chimeric antigen receptor T cell therapy and immune checkpoint inhibitors. Overcoming and/or modulating the TME is therefore one of the greatest challenges in developing new therapeutic strategies for PC. Nanoparticles have been successfully used as drug carriers and delivery systems in cancer therapy. Recent experimental and engineering developments in nanotechnology have resulted in increased drug delivery and improved immunotherapy for PC. In this review we discuss and analyze the current nanoparticle-based immunotherapy approaches that are at the verge of clinical application. Particularly, we focus on nanoparticle-based delivery systems that improve the effectiveness of PC immunotherapy. We also highlight current clinical research that will help to develop new therapeutic strategies for PC and especially targeted immunotherapies based on immune checkpoint inhibitors.

Macrophage Differentiation and Polarization Regulate the Release of the Immune Checkpoint Protein V-Domain Ig Suppressor of T Cell Activation.

Recently, the V-domain immunoglobulin suppressor of T-cell activation (VISTA) was identified as a negative immune checkpoint regulator (NCR) that is mainly expressed in hematopoietic cells. Preclinical studies have shown that VISTA blockade results in impeded tumor growth and improved survival. Nevertheless, little is known about the physiological role of VISTA expression in macrophages. This study focused on the differential expression of VISTA in human monocytes and macrophages in order to elucidate a putative role of VISTA regulation upon macrophage polarization and activation. We observed that human peripheral monocytes constitutively release soluble VISTA, which was regulated via matrix metalloproteinases. However, monocyte stimulation with cytokines that induce macrophage differentiation, such as granulocyte-macrophage colony-stimulating (GM-CSF) and macrophage colony-stimulating factor (M-CSF), substantially reduced soluble VISTA release. VISTA release was further affected by various pro- and anti-inflammatory stimuli that led to macrophage polarization, where activated M1 macrophages generally released more VISTA than M2 macrophages. Additionally, we observed that stimulation of activated macrophages with the toll-like receptor 4 ligand lipopolysaccharide (LPS) led to a further decrease of soluble VISTA release. Moreover, we found that soluble VISTA impairs T cell cytotoxic activity but did not induce their programmed death. Our results suggest that VISTA is constantly produced and released in the peripheral blood where it may contribute to peripheral tolerance.

Expression of the Immune Checkpoint Protein VISTA Is Differentially Regulated by the TGF-ß1 - Smad3 Signaling Pathway in Rapidly Proliferating Human Cells and T Lymphocytes.

Immune checkpoint proteins play crucial roles in human embryonic development but are also used by cancer cells to escape immune surveillance. These proteins and biochemical pathways associated with them form a complex machinery capable of blocking the ability of cytotoxic immune lymphoid cells to attack cancer cells and, ultimately, to fully suppress anti-tumor immunity. One of the more recently discovered immune checkpoint proteins is V-domain Ig-containing suppressor of T cell activation (VISTA), which plays a crucial role in anti-cancer immune evasion pathways. The biochemical mechanisms underlying regulation of VISTA expression remain unknown. Here, we report for the first time that VISTA expression is controlled by the transforming growth factor beta type 1 (TGF-ß)-Smad3 signaling pathway. However, in T lymphocytes, we found that VISTA expression was differentially regulated by TGF-ß depending on their immune profile. Taken together, our results demonstrate the differential biochemical control of VISTA expression in human T cells and various types of rapidly proliferating cells, including cancer cells, fetal cells and keratinocytes.

Functional role of galectin-9 in directing human innate immune reactions to Gram-negative bacteria and T cell apoptosis.

Galectin-9 is a member of the galectin family of proteins, which were first identified to specifically bind to carbohydrates containing ß-galactosides. Galectin-9 is conserved through evolution and recent evidence demonstrated its involvement in innate immune reactions to bacterial infections as well as the suppression of cytotoxic immune responses of T and natural killer cells. However, the molecular mechanisms underlying such differential immunological functions of galectin-9 remain largely unknown. In this work we confirmed that soluble galectin-9 derived from macrophages binds to Gram-negative bacteria by interacting with lipopolysaccharide (LPS), which forms their cell wall. This opsonisation effect most likely interferes with the mobility of bacteria leading to their phagocytosis by innate immune cells. Galectin-9-dependent opsonisation also promotes the innate immune reactions of macrophages to these bacteria and significantly enhances the production of pro-inflammatory cytokines - interleukin (IL) 6, IL-1ß and tumour necrosis factor alpha (TNF-α). In contrast, galectin-9 did not bind peptidoglycan (PGN), which forms the cell wall of Gram-positive bacteria. Moreover, galectin-9 associated with cellular surfaces (studied in primary human embryonic cells) was not involved in the interaction with bacteria or bacterial colonisation. However, galectin-9 expressed on the surface of primary human embryonic cells, as well as soluble forms of galectin-9, were able to target T lymphocytes and caused apoptosis in T cells expressing granzyme B. Furthermore, "opsonisation" of T cells by galectin-9 led to the translocation of phosphatidylserine onto the cell surface and subsequent phagocytosis by macrophages through Tim-3, the receptor, which recognises both galectin-9 and phosphatidylserine as ligands.

Ligand-Receptor Interactions of Galectin-9 and VISTA Suppress Human T Lymphocyte Cytotoxic Activity.

Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.

Increased Aggregation Tendency of Alpha-Synuclein in a Fully Disordered Protein Complex.

The recent discovery of biologically active fully disordered, so called random fuzzy protein-protein interactions leads to the question of how the high flexibility of these protein complexes correlates to aggregation and pathologic misfolding. We identify the structural mechanism by which a random fuzzy protein complex composed of the intrinsically disordered proteins alpha-Synuclein and SERF1a is able to potentiate cytotoxic aggregation. A structural model derived from an integrated NMR/SAXS analysis of the reconstituted aSyn:SERF1a complex enabled us to observe the partial deprotection of one precise aSyn amyloid nucleation element in the fully unstructured ensemble. This minimal exposure was sufficient to increase the amyloidogenic tendency of SERF1a-bound aSyn. Our findings provide a structural explanation of the previously observed pro-amyloid activity of SERF1a. They further demonstrate that random fuzziness can trigger a structurally organized disease-associated reaction such as amyloid polymerization.

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68.

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome.

Structural basis for homodimerization of the Src-associated during mitosis, 68-kDa protein (Sam68) Qua1 domain.

Sam68 (Src-associated during mitosis, 68 kDa) is a prototypical member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. STAR proteins bind mRNA targets and modulate cellular processes such as cell cycle regulation and tissue development in response to extracellular signals. Sam68 has been shown to modulate alternative splicing of the pre-mRNAs of CD44 and Bcl-xL, which are linked to tumor progression and apoptosis. Sam68 and other STAR proteins recognize bipartite RNA sequences and are thought to function as homodimers. However, the structural and functional roles of the self-association are not known. Here, we present the solution structure of the Sam68 Qua1 homodimerization domain. Each monomer consists of two antiparallel alpha-helices connected by a short loop. The two subunits are arranged perpendicular to each other in an unusual four-helix topology. Mutational analysis of Sam68 in vitro and in a cell-based assay revealed that the Qua1 domain and residues within the dimerization interface are essential for alternative splicing of a CD44 minigene. Together, our results indicate that the Qua1 homodimerization domain is required for regulation of alternative splicing by Sam68.