Role of microRNAs in Chronic Lymphocytic Leukemia.

Chronic Lymphocytic Leukemia (CLL) is the most common form of leukemia in adults, with a highly variable clinical course. Improvement in the knowledge of the molecular pathways behind this disease has led to the development of increasingly specific therapies, such as BCR signaling inhibitors and BCL-2 inhibitors. In this context, the emerging role of microRNAs (miRNAs) in CLL pathophysiology and their possible application in therapy is worth noting. MiRNAs are one of the most important regulatory molecules of gene expression. In CLL, they can act both as oncogenes and tumor suppressor genes, and the deregulation of specific miRNAs has been associated with prognosis, progression, and drug resistance. In this review, we describe the role of the miRNAs that primarily impact the disease, and how these miRNAs could be used as therapeutic tools. Certainly, the use of miRNAs in clinical practice is still limited in CLL. Many issues still need to be solved, particularly regarding their biological and safety profile, even if several studies have suggested their efficacy on the disease, alone or in combination with other drugs.

Epigenetics and MicroRNAs in Cancer.

The ability to reprogram the transcriptional circuitry by remodeling the three-dimensional structure of the genome is exploited by cancer cells to promote tumorigenesis. This reprogramming occurs because of hereditable chromatin chemical modifications and the consequent formation of RNA-protein-DNA complexes that represent the principal actors of the epigenetic phenomena. In this regard, the deregulation of a transcribed non-coding RNA may be both cause and consequence of a cancer-related epigenetic alteration. This review summarizes recent findings that implicate microRNAs in the aberrant epigenetic regulation of cancer cells.

MicroRNAs in Autoimmunity and Hematological Malignancies.

Autoimmunity and hematological malignancies are often concomitant in patients. A causal bidirectional relationship exists between them. Loss of immunological tolerance with inappropriate activation of the immune system, likely due to environmental and genetic factors, can represent a breeding ground for the appearance of cancer cells and, on the other hand, blood cancers are characterized by imbalanced immune cell subsets that could support the development of the autoimmune clone. Considerable effort has been made for understanding the proteins that have a relevant role in both processes; however, literature advances demonstrate that microRNAs (miRNAs) surface as the epigenetic regulators of those proteins and control networks linked to both autoimmunity and hematological malignancies. Here we review the most up-to-date findings regarding the miRNA-based molecular mechanisms that underpin autoimmunity and hematological malignancies.

Differential Regulation of Wingless-Wnt/c-Jun N-Terminal Kinase Crosstalk via Oxidative Eustress in Primary and Metastatic Colorectal Cancer Cells.

In the tumor microenvironment (TME), ROS production affects survival, progression, and therapy resistance in colorectal cancer (CRC). H2O2-mediated oxidative stress can modulate Wnt/ß-catenin signaling and metabolic reprogramming of the TME. Currently, it is unclear how mild/moderate oxidative stress (eustress) modulates Wnt/ß-catenin/APC and JNK signaling relationships in primary and metastatic CRC cells. In this study, we determined the effects of the H2O2 concentration inducing eustress on isogenic SW480 and SW620 cells, also in combination with JNK inhibition. We assessed cell viability, mitochondrial respiration, glycolysis, and Wnt/ß-catenin/APC/JNK gene and protein expression. Primary CRC cells were more sensitive to H2O2 eustress combined with JNK inhibition, showing a reduction in viability compared to metastatic cells. JNK inhibition under eustress reduced both glycolytic and respiratory capacity in SW620 cells, indicating a greater capacity to adapt to TME. In primary CRC cells, H2O2 alone significantly increased APC, LEF1, LRP6, cMYC and IL8 gene expression, whereas in metastatic CRC cells, this effect occurred after JNK inhibition. In metastatic but not in primary tumor cells, eustress and inhibition of JNK reduced APC, ß-catenin, and pJNK protein. The results showed differential cross-regulation of Wnt/JNK in primary and metastatic tumor cells under environmental eustress conditions. Further studies would be useful to validate these findings and explore their therapeutic potential.

Exploring the Immunomodulatory Potential of Pancreatic Cancer-Derived Extracellular Vesicles through Proteomic and Functional Analyses.

Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A better understanding of tumor-derived extracellular vesicle (EV) effects on immune responses might contribute to improved immunotherapy. EVs derived from Capan-2 and BxPC-3 PC cells isolated by ultracentrifugation were characterized by atomic force microscopy, Western blot (WB), nanoparticle tracking analysis, and label-free proteomics. Fresh PBMCs from healthy donors were treated with PC- or control-derived heterologous EVs, followed by flow cytometry analysis of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated or untreated PBMCs was performed, and the IFN-γ concentration was measured by ELISA. Notably, most of the proteins identified in Capan-2 and BxPC-3 EVs by the proteomic analysis were connected in a single functional network (p = 1 × 10-16) and were involved in the "Immune System" (FDR: 1.10 × 10-24 and 3.69 × 10-19, respectively). Interestingly, the treatment of healthy donor-derived PBMCs with Capan-2 EVs but not with BxPC-3 EVs or heterologous control EVs induced early activation of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated PBMCs was consistent with their activation by Capan-2 EVs, indicating IFN-γ among the major upstream regulators, as confirmed by ELISA. The proteomic and functional analyses indicate that PC-EVs have pleiotropic effects, and some may activate early immune responses, which might be relevant for the development of highly needed immunotherapeutic strategies in this immune-cold tumor.

miR-125b targets erythropoietin and its receptor and their expression correlates with metastatic potential and ERBB2/HER2 expression.

BACKGROUND: The microRNA 125b is a double-faced gene expression regulator described both as a tumor suppressor gene (in solid tumors) and an oncogene (in hematologic malignancies). In human breast cancer, it is one of the most down-regulated miRNAs and is able to modulate ERBB2/3 expression. Here, we investigated its targets in breast cancer cell lines after miRNA-mimic transfection. We examined the interactions of the validated targets with ERBB2 oncogene and the correlation of miR-125b expression with clinical variables. METHODS: MiR-125b possible targets were identified after transfecting a miRNA-mimic in MCF7 cell line and analyzing gene expression modifications with Agilent microarrays and Sylamer bioinformatic tool. Erythropoietin (EPO) and its receptor (EPOR) were validated as targets of miR-125b by luciferase assay and their expression was assessed by RT-qPCR in 42 breast cancers and 13 normal samples. The molecular talk between EPOR and ERBB2 transcripts, through miR-125b, was explored transfecting MDA-MD-453 and MDA-MB-157 with ERBB2 RNA and using RT-qPCR. RESULTS: We identified a panel of genes down-regulated after miR-125b transfection and putative targets of miR-125b. Among them, we validated erythropoietin (EPO) and its receptor (EPOR) - frequently overexpressed in breast cancer--as true targets of miR-125b. Moreover, we explored possible correlations with clinical variables and we found a down-regulation of miR-125b in metastatic breast cancers and a significant positive correlation between EPOR and ERBB2/HER2 levels, that are both targets of miR-125b and function as competing endogenous RNAs (ceRNAs). CONCLUSIONS: Taken together our results show a mechanism for EPO/EPOR and ERBB2 co-regulation in breast cancer and confirm the importance of miR-125b in controlling clinically-relevant cancer features.

CAR-T-Derived Extracellular Vesicles: A Promising Development of CAR-T Anti-Tumor Therapy.

Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

Targeted metabolomics detects a putatively diagnostic signature in plasma and dried blood spots from head and neck paraganglioma patients.

Head and neck paragangliomas (HNPGLs), rare chemoresistant tumors curable only with surgery, are strongly influenced by genetic predisposition, hence patients and relatives require lifetime follow-up with MRI and/or PET-CT because of de novo disease risk. This entails exposure to electromagnetic/ionizing radiation, costs, and organizational challenges, because patients and relatives are scattered far from reference centers. Simplified first-line screening strategies are needed. We employed flow injection analysis tandem mass spectrometry, as used in newborn metabolic screening, to compare the plasma metabolic profile of HNPGL patients (59 samples, 56 cases) and healthy controls (24 samples, 24 cases). Principal Component Analysis (PCA) and Partial Least Discriminant Analysis (PLS-DA) highlighted a distinctive HNPGL signature, likely reflecting the anaplerotic conversion of the TCA cycle to glutaminolysis and catabolism of branched amino acids, DNA damage and deoxyadenosine (dAdo) accumulation, impairment of fatty acid oxidation, switch towards the Warburg effect and proinflammatory lysophosphatidylcholines (LPCs) signaling. Statistical analysis of the metabolites that most impacted on PLS-DA was extended to 10 acoustic neuroma and 2 cholesteatoma patients, confirming significant differences relative to the HNPGL plasma metabolomic profile. The best confusion matrix from the ROC curve built on 2 metabolites, dAdo and C26:0-LPC, provided specificity of 94.29% and sensitivity of 89.29%, with positive and negative predictive values of 96.2% and 84.6%, respectively. Analysis of dAdo and C26:0-LPC levels in dried venous and capillary blood confirmed that dAdo, likely deriving from 2'-deoxy-ATP accumulated in HNPGL cells following endogenous genotoxic damage, efficiently discriminated HNPGL patients from healthy controls and acoustic neuroma/cholesteatoma patients on easily manageable dried blood spots.

Analysis of cavitation artifacts in Magnetic Resonance Imaging Thermometry during laser ablation monitoring.

Magnetic Resonance Thermometry Imaging (MRTI) holds great potential in laser ablation (LA) monitoring. It provides the real-time multidimensional visualization of the treatment effect inside the body, thus enabling accurate intraoperative prediction of the thermal damage induced. Despite its great potential., thermal maps obtained with MRTI may be affected by numerous artifacts. Among the sources of error producing artifacts in the images., the cavitation phenomena which could occur in the tissue during LA induces dipole-structured artifacts. In this work., an analysis of the cavitation artifacts occurring during LA in a gelatin phantom in terms of symmetry in space and symmetry of temperature values was performed. Results of 2 Wand 4 W laser power were compared finding higher symmetry for the 2 W case in terms of both dimensions of artifact-lobes and difference in temperature values extracted in specular pixels in the image. This preliminary investigation of artifact features may provide a step forward in the identification of the best strategy to correct and avoid artifact occurrence during thermal therapy monitoring. Clinical Relevance- This work presents an analysis of cavitation artifacts in MRTI from LA which must be corrected to avoid error in the prediction of thermal damage during LA monitoring.

Tgf-ß1 transcriptionally promotes 90K expression: possible implications for cancer progression.

The 90K protein, also known as Mac-2 BP or LGALS3BP, can activate the immune response in part by increasing major histocompatibility (MHC) class I levels. In studies on a non-immune cell model, the rat FRTL-5 cell line, we observed that transforming growth factor (TGF)-ß1, like γ-interferon (IFN), increased 90K levels, despite its immunosuppressive functions and the ability to decrease MHC class I. To explain this paradoxical result, we investigated the mechanisms involved in the TGF-ß1 regulation of 90K expression with the aim to demonstrate that TGF-ß1 utilizes different molecular pathways to regulate the two genes. We found that TGF-ß1 was able to increase the binding of Upstream Stimulatory Factors, USF1 and USF2, to an E-box element, CANNTG, at -1926 to -1921 bp, upstream of the interferon response element (IRE) in the 90K promoter. Thyrotropin (TSH) suppressed constitutive and γ-IFN-induced 90K expression by decreasing USF binding to the E-box. TGF-ß1 was able to overcome TSH suppression at the transcriptional level by increasing USF binding to the E-box. We suggest that the ability of TGF-ß1 to increase 90K did not result in an increase in MHC class I because of a separate suppressive action of TGF-ß1 directly on the MHC class I gene. We propose that the increased levels of 90K may play a role, rather than in immune response, in the context of the TGF-ß1-induced changing of the cellular microenvironment that predisposes to cell motility and cancer progression. Consistently, analyzing the publicly available cancer patient data sets cBioPortal, we found that 90K expression directly correlated with TGF-ß1 and USFs and that high levels of 90K were significantly associated with increased mortality in patients affected by different types of cancer.

Enhanced Expression of miR-181b in B Cells of CLL Improves the Anti-Tumor Cytotoxic T Cell Response.

The clinical progression of B cell chronic lymphocytic leukemia (CLL) is associated with immune cell dysfunction and a strong decrease of miR-181b-5p (miR-181b), promoting the death of CLL cells. Here we investigated whether the reduction of miR-181b impairs the immune response in CLL. We demonstrate that activated CD4+ T cells increase miR-181b expression in CLL through CD40-CD40L signaling, which enhances the maturation and activity of cytotoxic T cells and, consequently, the apoptotic response of CLL cells. The cytotoxic response is facilitated by a depletion of the anti-inflammatory cytokine interleukin 10, targeted by miR-181b. In vivo experiments in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice confirmed that miR-181b promotes the apoptotic death of CLL cells only when functional T cells are restored. Overall, our findings suggest that the reinstatement of miR-181b in CLL cells could be an exploitable adjuvant therapeutic option for the treatment of CLL.

The Benzimidazole-Based Anthelmintic Parbendazole: A Repurposed Drug Candidate That Synergizes with Gemcitabine in Pancreatic Cancer.

Pancreatic cancer (PC) is one of the most lethal, chemoresistant malignancies and it is of paramount importance to find more effective therapeutic agents. Repurposing of non-anticancer drugs may expand the repertoire of effective molecules. Studies on repurposing of benzimidazole-based anthelmintics in PC and on their interaction with agents approved for PC therapy are lacking. We analyzed the effects of four Food and Drug Administration (FDA)-approved benzimidazoles on AsPC-1 and Capan-2 pancreatic cancer cell line viability. Notably, parbendazole was the most potent benzimidazole affecting PC cell viability, with half maximal inhibitory concentration (IC50) values in the nanomolar range. The drug markedly inhibited proliferation, clonogenicity and migration of PC cell lines through mechanisms involving alteration of microtubule organization and formation of irregular mitotic spindles. Moreover, parbendazole interfered with cell cycle progression promoting G2/M arrest, followed by the emergence of enlarged, polyploid cells. These abnormalities, suggesting a mitotic catastrophe, culminated in PC cell apoptosis, are also associated with DNA damage in PC cell lines. Remarkably, combinations of parbendazole with gemcitabine, a drug employed as first-line treatment in PC, synergistically decreased PC cell viability. In conclusion, this is the first study providing evidence that parbendazole as a single agent, or in combination with gemcitabine, is a repurposing candidate in the currently dismal PC therapy.

HNRNPL Restrains miR-155 Targeting of BUB1 to Stabilize Aberrant Karyotypes of Transformed Cells in Chronic Lymphocytic Leukemia.

Aneuploidy and overexpression of hsa-miR-155-5p (miR-155) characterize most solid and hematological malignancies. We recently demonstrated that miR-155 sustains aneuploidy at early stages of in vitro cellular transformation. During in vitro transformation of normal human fibroblast, upregulation of miR-155 downregulates spindle checkpoint proteins as the mitotic checkpoint serine/threonine kinase budding uninhibited by benzimidazoles 1 (BUB1), the centromere protein F (CENPF) and the zw10 kinetochore protein (ZW10), compromising the chromosome alignment at the metaphase plate and leading to aneuploidy in daughter cells. Here we show that the heterogeneous nuclear ribonucleoprotein L (HNRNPL) binds to the polymorphic marker D2S1888 at the 3'UTR of BUB1 gene, impairs the miR-155 targeting, and restores BUB1 expression in chronic lymphocytic leukemia. This mechanism occurs at advanced passages of cell transformation and allows the expansion of more favorable clones. Our findings have revealed, at least in part, the molecular mechanisms behind the chromosomal stabilization of cell lines and the concept that, to survive, tumor cells cannot continuously change their genetic heritage but need to stabilize the most suitable karyotype.

DNA methylation of shelf, shore and open sea CpG positions distinguish high microsatellite instability from low or stable microsatellite status colon cancer stem cells.

Aim: To investigate the genome-wide methylation of genetically characterized colorectal cancer stem cell (CR-CSC) lines. Materials & methods: Eight CR-CSC lines were isolated from primary colorectal cancer (CRC) tissues, cultured and characterized for aneuploidy, mutational status of CRC-related genes and microsatellite instability (MSI). Genome-wide DNA methylation was assessed by MethylationEPIC microarray. Results: We describe a distinctive methylation pattern that is maintained following in vivo passages in immune-compromised mice. We identified an epigenetic CR-CSC signature associated with MSI. We noticed that the preponderance of the differentially methylated positions do not reside at CpG islands, but spread to shelf and open sea regions. Conclusion: Given that CRCs with MSI-high status have a lower metastatic potential, the identification of a MSI-related methylation signature could provide new insights and possible targets into metastatic CRC.

Genetic dynamics in untreated CLL patients with either stable or progressive disease: a longitudinal study.

Clonal evolution of chronic lymphocytic leukemia (CLL) often follows chemotherapy and is associated with adverse outcome, but also occurs in untreated patients, in which case its predictive role is debated. We investigated whether the selection and expansion of CLL clone(s) precede an aggressive disease shift. We found that clonal evolution occurs in all CLL patients, irrespective of the clinical outcome, but is faster during disease progression. In particular, changes in the frequency of nucleotide variants (NVs) in specific CLL-related genes may represent an indicator of poor clinical outcome.

Hsa-miR-155-5p drives aneuploidy at early stages of cellular transformation.

Hsa-miR-155-5p (miR-155) is overexpressed in most solid and hematological malignancies. It promotes loss of genomic integrity in cancer cells by targeting genes involved in microsatellite instability and DNA repair; however, the link between miR-155 and aneuploidy has been scarcely investigated. Here we describe a novel mechanism by which miR-155 causes chromosomal instability. Using osteosarcoma cells (U2OS) and normal human dermal fibroblast (HDF), two well-established models for the study of chromosome congression, we demonstrate that miR-155 targets the spindle checkpoint proteins BUB1, CENP-F, and ZW10, thus compromising chromosome alignment at the metaphase plate. In U2OS cells, exogenous miR-155 expression reduced the recruitment of BUB1, CENP-F, and ZW10 to the kinetochores which resulted in defective chromosome congression. In contrast, during in vitro transformation of HDF by enforced expression of SV40 Large T antigen and human telomerase (HDFLT/hTERT), inhibition of miR-155 reduced chromosome congression errors and aneuploidy at early passages. Using live-cell imaging we observed that miR-155 delays progression through mitosis, indicating an activated mitotic spindle checkpoint, which likely fails to reduce aneuploidy. Overall, this study provides insight into a mechanism that generates aneuploidy at early stages of cellular transformation, pointing to a role for miR-155 in chromosomal instability at tumor onset.

Over-expression of the miR-483-3p overcomes the miR-145/TP53 pro-apoptotic loop in hepatocellular carcinoma.

The miR-145-5p, which induces TP53-dependent apoptosis, is down-regulated in several tumors, including hepatocellular carcinomas (HCCs), but some HCCs show physiological expression of this miR. Here we demonstrate that in HCC cells carrying wild-type TP53 the steady activation of the miR-145 signaling selects clones resistant to apoptosis via up-regulation of the oncogenic miR-483-3p. Expression of the miR-145-5p and of the miR-483-3p correlated negatively in non-neoplastic liver (n=41; ρ=-0.342, P=0.028), but positively in HCCs (n=21; ρ=0.791, P<0.0001), which we hypothesized to be due to impaired glucose metabolism in HCCs versus normal liver. In fact, when liver cancer cells were grown in low glucose, miR-145-5p lowered miR-483-3p expression, allowing apoptosis, whereas when cells were grown in high glucose the levels of miR-483-3p increased, reducing the apoptotic rate. This indicates that depending on glucose availability the miR-145-5p has double effects on the miR-483-3p, either inhibitory or stimulatory. Moreover, resistance to apoptosis in clones overexpressing both miR-145-5p and miR-483-3p was abrogated by silencing the miR-483-3p. Our data highlight a novel mechanism of resistance to apoptosis in liver cancer cells harbouring wild type TP53 and suggest a potential role of miR-145-5p and miR-483-3p as druggable targets in a subset of HCCs.

MicroRNA fingerprints in juvenile myelomonocytic leukemia (JMML) identified miR-150-5p as a tumor suppressor and potential target for treatment.

Juvenile myelomonocytic leukemia (JMML) is an aggressive leukemia of early childhood characterized by aberrant proliferation of myelomonocytic cells and hypersensitivity to GM-CSF stimulation. Mutually exclusive mutations in the RAS/ERK pathway genes such as PTPN11, NRAS, KRAS, CBL, or NF1 are found in ~90% of the cases. These mutations give rise to disease at least in part by activating STAT5 through phosphorylation and by promoting cell growth. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression, which are often deregulated in leukemia. However, little is known about their role in JMML. Here, we report distinctive miR expression signatures associated with the molecular subgroups of JMML. Among the downregulated miRs in JMML, miR-150-5p was found to target STAT5b, a gene which is often over-activated in JMML, and contributes to the characteristic aberrant signaling of this disorder. Moreover, loss of miR-150-5p and upregulation of STAT5b expression were also identified in a murine model of JMML. Ectopic overexpression of miR-150-5p in mononuclear cells from three JMML patients significantly decreased cell proliferation. Altogether, our data indicate that miR expression is deregulated in JMML and may play a role in the pathogenesis of this disorder by modulating key effectors of cytokine receptor pathways.