Genome-wide aberrant methylation in primary metastatic UM and their matched metastases

Uveal melanoma (UM) is an aggressive intra-ocular cancer with a strong tendency to metastasize. Metastatic UM is associated with mutations in BAP1 and SF3B1, however only little is known about the epigenetic modifications that arise in metastatic UM. In this study we aim to unravel epigenetic changes contributing to UM metastasis using a new genome-wide methylation analysis technique that covers over 50% of all CpG’s. We identified aberrant methylation contributing to BAP1 and SF3B1-mediated UM metastasis. The methylation data was integrated with expression data and surveyed in matched UM metastases from the liver, skin and bone. UM metastases showed no commonly shared novel epigenetic modifications, implying that epigenetic changes contributing to metastatic spreading and colonization in distant tissues occur early in the development of UM and epigenetic changes that occur after metastasis are mainly patient-specific. Our findings reveal a plethora of epigenetic modifications in metastatic UM and its metastases, which could subsequently result in aberrant repression or activation of many tumor-related genes. This observation points towards additional layers of complexity at the level of gene expression regulation, which may explain the low mutational burden of UM.

P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.

Communication between cells: exosomes as a delivery system in prostate cancer

Despite the considerable efforts in screening and diagnostic protocols, prostate cancer still represents the second leading cause of cancer-related death in men. Many patients with localized disease and low risk of recurrence have a favourable outcome. In a substantial proportion of patients, however, the disease progresses and becomes aggressive. The mechanisms that promote prostate cancer progression remain still debated. Many findings point to the role of cross-communication between prostate tumor cells and their surrounding microenvironment during the disease progression. Such a connection fosters survival, proliferation, angiogenesis, metastatic spreading and drug-resistance of prostate cancer. Recent years have seen a profound interest in understanding the way by which prostate cancer cells communicate with the surrounding cells in the microenvironment. In this regard, direct cell-to-cell contacts and soluble factors have been identified. Increasing evidence indicates that PC cells communicate with the surrounding cells through the release of extracellular vesicles, mainly the exosomes. By directly acting in stromal or prostate cancer epithelial cells, exosomes represent a critical intercellular communication system. By querying the public database (https://pubmed.ncbi.nlm.nih.gov) for the past 10 years, we have found more than four hundred papers. Among them, we have extrapolated the most relevant about the role of exosomes in prostate cancer malignancy and progression. Emerging data concerning the use of these vesicles in diagnostic management and therapeutic guidance of PC patients are also presented. Graphical Abstract

Blood Vessels and Peripheral Nerves as Key Players in Cancer Progression and Therapy Resistance

Cancer cells continuously interact with the tumor microenvironment (TME), a heterogeneous milieu that surrounds the tumor mass and impinges on its phenotype. Among the components of the TME, blood vessels and peripheral nerves have been extensively studied in recent years for their prominent role in tumor development from tumor initiation. Cancer cells were shown to actively promote their own vascularization and innervation through the processes of angiogenesis and axonogenesis. Indeed, sprouting vessels and axons deliver several factors needed by cancer cells to survive and proliferate, including nutrients, oxygen, and growth signals, to the expanding tumor mass. Nerves and vessels are also fundamental for the process of metastatic spreading, as they provide both the pro-metastatic signals to the tumor and the scaffold through which cancer cells can reach distant organs. Not surprisingly, continuously growing attention is devoted to the development of therapies specifically targeting these structures, with promising initial results. In this review, we summarize the latest evidence that supports the importance of blood vessels and peripheral nerves in cancer pathogenesis, therapy resistance, and innovative treatments.

Interactions of platelets with circulating tumor cells contribute to cancer metastasis

Recent studies have suggested that platelets have a crucial role in enhancing the survival of circulating tumor cells in the bloodstream and aggravating cancer metastasis. The main function of platelets is to bind to the sites of the damaged vessels to stop bleeding. However, in cancer patients, activated platelets adhere to circulating tumor cells and exacerbate metastatic spreading. Several hypotheses have been proposed about the platelet–cancer cell interactions, but the underlying mechanisms of these interactions are not completely understood yet. In this work, we quantitatively investigated the interactions between circulating tumor cells, red blood cells, platelets, plasma flow and microvessel walls via computational modelling at the cellular scale. Our highly detailed computational model allowed us to understand and quantitatively explain the role of platelets in deformation, adhesion and survival of tumor cells in their active arrest to the endothelium.

Adapter Chimeric Antigen Receptor (AdCAR)-Engineered NK-92 Cells for the Multiplex Targeting of Bone Metastases

Background: Since metastatic spreading of solid tumor cells often leads to a fatal outcome for most cancer patients, new approaches for patient-individualized, targeted immunotherapy are urgently needed. Methods: Here, we established cell lines from four bone metastases of different tumor entities. We assessed AdCAR NK-92-mediated cytotoxicity in vitro in standard cytotoxicity assays as well as 3D spheroid models Results: AdCAR-engineered NK-92 cells successfully demonstrated distinct and specific cytotoxic potential targeting different tumor antigens expressed on cell lines established from bone metastases of mammary, renal cell and colorectal carcinoma as well as melanomas. In that process AdCAR NK-92 cells produced a multitude of NK effector molecules as well as pro inflammatory cytokines. Furthermore, AdCAR NK-92 showed increased cytotoxicity in 3D spheroid models which can recapitulate in vivo architecture, thereby bridging the gap between in vitro and in vivo models. Conclusions: AdCAR NK-92 cells may provide an interesting and promising “off-the-shelf” cellular product for the targeted therapy of cancers metastasizing to the bone, while utilization of clinically approved, therapeutic antibodies, as exchangeable adapter molecules can facilitate quick clinical translation.

Inactivation of E-cadherin by Trop-2 drives colon cancer metastasis.

105 Background: Tumor metastasis is the main cause of death of colon cancer patients and the biggest hurdle for cancer cure. We set to identify decisive drivers and of pivotal therapy targets for colon cancer metastasis. Methods: IHC analysis quantified the expression of target molecules in primary tumors and metastases. Cell-cell adhesion capacity was assessed in vitro and in HCT116 colon cancer cell spheroids. Pre-clinical models of orthotopic growth of KM12SM colon cancer cells and metastatic diffusion to the liver were utilized to assess metastatic spreading force of wtTrop-2 and of the constitutively-active, tail-less form of Trop-2 (Δcyto). Xenotransplant and metastasis transcriptomes were analyzed for differential induction of EMT determinants. Kaplan–Meier plots were used to illustrate survival and metastatic relapse in independent case series of colon cancer patients. Results: wtTrop-2 was shown to induce wound-healing. ΔcytoTrop-2 further increased cell migration ability. Both wtTrop-2 and ΔcytoTrop-2 induced resistance to apoptosis in vitro and in vivo. wtTrop-2 strikingly increased the metastatic capacity of KM12SM cells, raising metastasis rates from 45% for control cells to 90% for wtTrop-2 transfectants. The constitutively-active ΔcytoTrop-2 further boosted metastatic spreading, with metastatic livers reaching up to four times their normal size. Cancer metastases revealed high levels of E-cadherin, in the absence of transcriptional down-regulation. EMT transcription factors were largely missing from Trop-2-activated cells. Rather, binding to Trop-2 was shown to cause the release of E-cadherin from the cytoskeleton, loss of cell-cell adhesion and activation of β-catenin. This global, Trop-2/E-cadherin/β-catenin-driven pro-metastatic program was recapitulated in colon cancer patients and was shown to impact on colon cancer metastatic relapse and overall patient survival. Conclusions: We identify Trop-2-driven functional inactivation of E-cadherin as a widespread driver of metastatic diffusion in colon cancer, opening novel avenues for personalized diagnostic procedures and anti-cancer therapies. [Table: see text]

Genetic Alterations of Metastatic Colorectal Cancer

Genome sequencing studies have characterized the genetic alterations of different tumor types, highlighting the diversity of the molecular processes driving tumor development. Comprehensive sequencing studies have defined molecular subtypes of colorectal cancers (CRCs) through the identification of genetic events associated with microsatellite stability (MSS), microsatellite-instability-high (MSI-H), and hypermutation. Most of these studies characterized primary tumors. Only recent studies have addressed the characterization of the genetic and clinical heterogeneity of metastatic CRC. Metastatic CRC genomes were found to be not fundamentally different from primary CRCs in terms of the mutational landscape or of genes that drive tumorigenesis, and a genomic heterogeneity associated with tumor location of primary tumors helps to define different clinical behaviors of metastatic CRCs. Although CRC metastatic spreading was traditionally seen as a late-occurring event, growing evidence suggests that this process can begin early during tumor development and the clonal architecture of these tumors is consistently influenced by cancer treatment. Although the survival rate of patients with metastatic CRC patients improved in the last years, the response to current treatments and prognosis of many of these patients remain still poor, indicating the need to discover new improvements for therapeutic vulnerabilities and to formulate a rational prospective of personalized therapies.

Tetraploidization of Immortalized Myoblasts Induced by Cell Fusion Drives Myogenic Sarcoma Development with DMD Deletion

Whole-genome doubling is the second most frequent genomic event, after TP53 alterations, in advanced solid tumors and is associated with poor prognosis. Tetraploidization step will lead to aneuploidy and chromosomic rearrangements. The mechanism leading to tetraploid cells is important since endoreplication, abortive cytokinesis and cell fusion could have distinct consequences. Unlike processes based on duplication, cell fusion involves the merging of two different genomes, epigenomes and cellular states. Since it is involved in muscle differentiation, we hypothesized that it could play a role in the oncogenesis of myogenic cancers. Spontaneous hybrids, but not their non-fused immortalized myoblast counterparts they are generated from, induced tumors in mice. Unstable upon fusion, the hybrid genome evolved from initial mitosis to tumors with a highly rearranged genome. This genome remodeling finally produced targeted DMD deletions associated with replicative stress, isoform relocalization and metastatic spreading, exactly as observed in human myogenic sarcomas. In conclusion, these results draw a model of myogenic oncogenesis in which cell fusion and oncogene activation combine to produce pleomorphic aggressive sarcomas.