The Conundrum of Cancer-Associated Thrombosis: Lesson Learned from Two Intriguing Cases and Literature Review.

The correlation between cancer and venous thromboembolism (VTE) is solid, whereas the knowledge about cancer-related arterial thromboembolism (ATE) still needs a deeper investigation to clarify its pathogenesis. We describe two cases that represent useful hints for a comprehensive review of the thrombotic issue. A 75-year-old man with advanced rectal cancer treated with fluoropyrimidines suffered two catheter-related VTE events managed according to current guidelines. There was no indication for "extended" anticoagulant therapy for him, but during antithrombotic wash-out and fluoropyrimidines plus panitumumab regimen, he suffered a massive right coronary artery (RCA) thrombosis. Another patient with no cardiovascular (CV) risk factors and affected by advanced bladder cancer was treated with a platinum-containing regimen and suffered an acute inferior myocardial infarction 2 days after chemotherapy administration. He was successfully treated with primary Percutaneous Transluminal Coronary Angioplasty of RCA, discontinuing platinum-based therapy. Our observations raise the issue of cancer-associated thrombosis (CAT) complexity and the potential correlation between arterial and venous thrombotic events. Moreover, physicians should be aware of the thrombotic risk associated with anticancer therapies, suggesting that an appropriate prophylaxis should be considered.

Comparative analyses of DNA extraction methods for whole blood quantification of HCMV DNAemia in patients with hematological diseases: false negative cases in manual method.

Human cytomegalovirus (HCMV) DNA quantitation in whole blood (WB) by real-time or quantitative polymerase chain reaction (qPCR) is a highly sensitive and reproducible diagnostic procedure for monitoring HCMV DNAemia (DNAemia is the detection of DNA in samples of plasma, whole blood, isolated peripheral blood leukocytes or in buffy-coat specimens) in patients. We provided a comparative analysis of HCMV DNA extraction performance by two different techniques, one performed by an automated extractor and the other by a manual method. We observed that the automated extraction method allowed HCMV DNA detection in the presence of weak viremia while no differences are observed when the viral load is greater. Therefore, automated DNA extraction is a suitable and recommended protocol not only for early detection of HCMV infection but also for more accurate monitoring of HCMV DNAemia during post-therapy follow-up.

Role of IL-6/STAT3 Axis in Resistance to Cisplatin in Gastric Cancers.

Gastric cancer, the second most common cause of death worldwide, is characterized by poor prognosis and low responsiveness to chemotherapy. Indeed, multidrug resistance, based mainly on cellular and molecular factors, remains one of the most limiting factors of the current approach to gastric cancer (GC) therapy. We employed a comprehensive gene expression analysis through data mining of publicly available databases to assess the role of the signal transducer and activator of transcription 3 (STAT3) in gastric cancer drug efficiency. It has been proposed that gastric cancer cells are less sensitive to these drugs because they develop resistance to these agents through activating alternative signalling pathways responsible for overcoming pharmacological inhibition. Our study evaluated the hypothesis that activating STAT3 signalling in response to cisplatin reduces the reaction to the drug. Consistent with this hypothesis, inhibition of interleukin 6 (IL-6)/STAT3 in combination therapy with cisplatin prevented both STAT3 activation and more lethality than induction by a single agent. The data suggest that the IL-6/STAT3 axis block associated with cisplatin treatment may represent a strategy to overcome resistance.

TRPM8 indicates poor prognosis in colorectal cancer patients and its pharmacological targeting reduces tumour growth in mice by inhibiting Wnt/ß-catenin signalling.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin type-8 (TRPM8) is a cold-sensitive cation channel protein belonging to the TRP superfamily of ion channels. Here, we reveal the molecular mechanism of TRPM8 and its clinical relevance in colorectal cancer (CRC). EXPERIMENTAL APPROACH: TRPM8 expression and its correlation with the survival rate of CRC patients was analysed. To identify the key pathways and genes related to TRPM8 high expression, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted in CRC patients. TRPM8 functional role was assessed by using Trpm8-/- mice in models of sporadic and colitis-associated colon cancer. TRPM8 pharmacological targeting by WS12 was evaluated in murine models of CRC. KEY RESULTS: TRPM8 is overexpressed in colon primary tumours and in CD326+ tumour cell fraction. TRPM8 high expression was related to lower survival rate of CRC patients, Wnt-Frizzled signalling hyperactivation and adenomatous polyposis coli down-regulation. In sporadic and colitis-associated models of colon cancer, either absence or pharmacological desensitization of TRPM8 reduced tumour development via inhibition of the oncogenic Wnt/ß-catenin signalling. TRPM8 pharmacological blockade reduced tumour growth in CRC xenograft mice by reducing the transcription of Wnt signalling regulators and the activation of ß-catenin and its target oncogenes such as C-Myc and Cyclin D1. CONCLUSION AND IMPLICATIONS: Human data provide valuable insights to propose TRPM8 as a prognostic marker with a negative predictive value for CRC patient survival. Animal experiments demonstrate TRPM8 involvement in colon cancer pathophysiology and its potential as a drug target for CRC.

Radiation-induced sarcomas: A single referral cancer center experience and literature review.

Background and objective: The oncogenic effect of ionizing radiation is widely known. Sarcomas developing after radiation therapy (RT), termed "iatrogenic disease of success", represent a growing problem, since the advancements in cancer management and screening programs have increased the number of long-term cancer survivors. Although many patients have been treated with radiation therapy, only few data are available on radiation-induced sarcomas (RIS). Methods: We examined the medical and radiological records of 186 patients with histologically proven soft tissue and bone sarcomas, which referred to IRCCS CROB Centro di Riferimento Oncologico della Basilicata from January 2009 to May 2022. Among them, seven patients received a histological diagnosis of secondary RIS, according to Cahan's criteria. Clinicopathological features and treatment follow-up data of RIS patients were retrospectively analyzed. Results: Among these secondary RIS, five arose in irradiated breast cancer (5/2,570, 0.19%) and two in irradiated head and neck cancer (2/1,986, 0.10%) patients, with a mean onset latency time of 7.3 years. The histology of RIS was one desmoid tumor, two angiosarcomas, one chondrosarcoma, two leiomyosarcomas, and one undifferentiated pleomorphic sarcoma. Out of the seven RIS, one received radiotherapy, one received electrochemotherapy (ECT), one received a second-line chemotherapy, three were subjected to three lines of chemotherapy, and one underwent radiofrequency ablation, chemotherapy, and ECT. Median survival time is 36 months. No significant survival differences were found stratifying patients for age at RT, latency time, and age at RIS diagnosis. Conclusions: RIS represents a possible complication for long-survivor cancer patients. Therefore, adherence to a strict follow-up after the radiation treatment is recommended to allow early diagnosis and optimal management of RIS patients. After the planned follow-up period, considering the long-term risk to develop a RIS, a specific multispecialty survivorship care plan could be of benefit for patients.

Gene Regulatory Network Characterization of Gastric Cancer's Histological Subtypes: Distinctive Biological and Clinically Relevant Master Regulators.

Gastric cancer (GC) molecular heterogeneity represents a major determinant for clinical outcomes, and although new molecular classifications have been introduced, they are not easy to translate from bench to bedside. We explored the data from GC public databases by performing differential gene expression analysis (DEGs) and gene network reconstruction to identify master regulators (MRs), as well as a gene set analysis (GSA) to reveal their biological features. Moreover, we evaluated the association of MRs with clinicopathological parameters. According to the GSA, the Diffuse group was characterized by an epithelial-mesenchymal transition (EMT) and inflammatory response, while the Intestinal group was associated with a cell cycle and drug resistance pathways. In particular, the regulons of Diffuse MRs, such as Vgll3 and Ciita, overlapped with the EMT and interferon-gamma response, while the regulons Top2a and Foxm1 were shared with the cell cycle pathways in the Intestinal group. We also found a strict association between MR activity and several clinicopathological features, such as survival. Our approach led to the identification of genes and pathways differentially regulated in the Intestinal and Diffuse GC histotypes, highlighting biologically interesting MRs and subnetworks associated with clinical features and prognosis, suggesting putative actionable candidates.

Identification of Cdk8 and Cdkn2d as New Prame-Target Genes in 2C-like Embryonic Stem Cells.

Embryonic stem cells (ESCs) present a characteristic pluripotency heterogeneity correspondent to specific metastates. We recently demonstrated that retinoic acid (RA) induces an increase in a specific 2C-like metastate marked by target genes specific to the two-cell embryo stage in preimplantation. Prame (Preferentially expressed antigen in melanoma) is one of the principal actors of the pluripotency stage with a specific role in RA responsiveness. Additionally, PRAME is overexpressed in a variety of cancers, but its molecular functions are poorly understood. To further investigate Prame's downstream targets, we used a chromatin immunoprecipitation sequencing (ChIP-seq) assay in RA-enriched 2C-like metastates and identified two specific target genes, Cdk8 and Cdkn2d, bound by Prame. These two targets, involved in cancer dedifferentiation and pluripotency, have been further validated in RA-resistant ESCs. Here, we observed for the first time that Prame controls the Cdk8 and Cdkn2d genes in ESCs after RA treatment, shedding light on the regulatory network behind the establishment of naïve pluripotency.

In vitro characterization of iridoid and phenylethanoid glycosides from Cistanche phelypaea for nutraceutical and pharmacological applications.

"Desert hyacinths" are a remarkable group of parasitic plants belonging to genus Cistanche, including more than 20 accepted species typically occurring in deserts or coastal dunes parasitizing roots of shrubs. Several Cistanche species have long been a source of traditional herbal medicine or food, being C. deserticola and C. tubulosa the most used in China. This manuscript reports the isolation and identification of some phenylethanoid and iridoid glycosides, obtained from the hydroalcoholic extract of C. phelypaea collected in Spain. The present study aims to characterize the antioxidant activity of C. phelypaea metabolites in the light of their application in nutraceutical and cosmeceutical industries and the effect of acetoside, the most abundant metabolite in C. phelypaea extract, on human keratinocyte and pluripotent stem cell proliferation and differentiation. Our study demonstrated that acetoside, besides its strong antioxidant potential, can preserve the proliferative potential of human basal keratinocytes and the stemness of mesenchymal progenitors necessary for tissue morphogenesis and renewal. Therefore, acetoside can be of practical relevance for the clinical application of human stem cell cultures in tissue engineering and regenerative medicine.

Correction: Analysis of miRNA profiles identified miR-196a as a crucial mediator of aberrant PI3K/AKT signaling in lung cancer cells.

[This corrects the article DOI: 10.18632/oncotarget.13432.].

FUS driven circCNOT6L biogenesis in mouse and human spermatozoa supports zygote development.

Circular RNA (circRNA) biogenesis requires a backsplicing reaction, promoted by inverted repeats in cis-flanking sequences and trans factors, such as RNA-binding proteins (RBPs). Among these, FUS plays a key role. During spermatogenesis and sperm maturation along the epididymis such a molecular mechanism has been poorly explored. With this in mind, we chose circCNOT6L as a study case and wild-type (WT) as well as cannabinoid receptor type-1 knock-out (Cb1-/-) male mice as animal models to analyze backsplicing mechanisms. Our results suggest that spermatozoa (SPZ) have an endogenous skill to circularize mRNAs, choosing FUS as modulator of backsplicing and under CB1 stimulation. A physical interaction between FUS and CNOT6L as well as a cooperation among FUS, RNA Polymerase II (RNApol2) and Quaking (QKI) take place in SPZ. Finally, to gain insight into FUS involvement in circCNOT6L biogenesis, FUS expression was reduced through RNA interference approach. Paternal transmission of FUS and CNOT6L to oocytes during fertilization was then assessed by using murine unfertilized oocytes (NF), one-cell zygotes (F) and murine oocytes undergoing parthenogenetic activation (PA) to exclude a maternal contribution. The role of circCNOT6L as an active regulator of zygote transition toward the 2-cell-like state was suggested using the Embryonic Stem Cell (ESC) system. Intriguingly, human SPZ exactly mirror murine SPZ.

Dihydrophenanthrenes from a Sicilian Accession of Himantoglossum robertianum (Loisel.) P. Delforge Showed Antioxidant, Antimicrobial, and Antiproliferative Activities.

The peculiar aspect that emerges from the study of Orchidaceae is the presence of various molecules, which are particularly interesting for pharmaceutical chemistry due to their wide range of biological resources. The aim of our study was to investigate the properties of two dihydrophenanthrenes, isolated, for the first time, from Himantoglossum robertianum (Loisel.) P. Delforge (Orchidaceae) bulbs and roots. Chemical and spectroscopic study of the bulbs and roots of Himantoglossumrobertianum (Loisel.) P. Delforge resulted in the isolation of two known dihydrophenanthrenes-loroglossol and hircinol-never isolated from this plant species. The structures were evaluated based on 1H-NMR, 13C-NMR, and two-dimensional spectra, and by comparison with the literature. These two molecules have been tested for their possible antioxidant, antimicrobial, antiproliferative, and proapoptotic activities. In particular, it has been shown that these molecules cause an increase in the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) in polymorphonuclear leukocytes (PMN); show antimicrobial activity against Escherichia coli and Staphylococcus aureus, and have anti-proliferative effects on gastric cancer cell lines, inducing apoptosis effects. Therefore, these two molecules could be considered promising candidates for pharmaceutical and nutraceutical preparations.

CHIR99021, trough GSK-3ß Targeting, Reduces Epithelioid Sarcoma Cell Proliferation by Activating Mitotic Catastrophe and Autophagy.

Epithelioid sarcoma (ES) is a rare disease representing <1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3ß, which results in up-regulation in tumor versus normal tissue samples and associated to poor prognosis in sarcoma patients. Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.

Cationic Channel TRPV2 Overexpression Promotes Resistance to Cisplatin-Induced Apoptosis in Gastric Cancer Cells.

Gastric cancer (GC) is characterized by poor efficacy and modest clinical impact of current therapies, in which apoptosis evasion is relevant. Intracellular calcium homeostasis dysregulation is associated with apoptosis escaping, and aberrant expression of calcium regulator genes could promote GC drug resistance. Since we previously found a prognostic value for TRPV2 calcium channel expression in GC, we aimed to characterize the role of TRPV2 in cisplatin resistance. Using the TCGA-STAD dataset, we performed a differential gene expression analysis between GC samples in upper and lower tertiles of TRPV2 expression, and then through a gene set analysis, we highlighted the enriched ontology and canonical pathways. We used qRT-PCR to assess TRPV2 expression in three GC cell lines and flow cytometry to evaluate cisplatin-induced cell death rates. Calcium green-1-AM assay was used to estimate differences in intracellular Ca2+ concentrations after inhibition of TRPV2. We engineered AGS cell line to overexpress TRPV2 and used confocal microscopy to quantify its overexpression and localization and flow cytometry to evaluate their sensitivity to cisplatin. Consistent with our hypothesis, among enriched gene sets, we found a significant number of those involved in the regulation of apoptosis. Subsequently, we found an inverse correlation between TRPV2 expression and sensitivity to cisplatin in GC cell lines. Moreover, we demonstrated that inhibition of TRPV2 activity by tranilast blocks the efflux of Ca2+ ions and, in combination with cisplatin, induced a significant increase of apoptotic cells (p = 0.004). We also demonstrated that TRPV2 exogenous expression confers a drug-resistant phenotype, and that tranilast is able to revert this phenotype, restoring cisplatin sensitivity. Our findings consistently suggested that TRPV2 could be a potential target for overcoming cisplatin resistance by promoting apoptosis. Notably, our data are a prerequisite for the potential reposition of tranilast to the treatment of GC patients and anticipate the in vivo evaluation.

Pancreatic Progenitor Commitment Is Marked by an Increase in Ink4a/Arf Expression.

The identification of the molecular mechanisms controlling early cell fate decisions in mammals is of paramount importance as the ability to determine specific lineage differentiation represents a significant opportunity for new therapies. Pancreatic Progenitor Cells (PPCs) constitute a regenerative reserve essential for the maintenance and regeneration of the pancreas. Besides, PPCs represent an excellent model for understanding pathological pancreatic cellular remodeling. Given the lack of valid markers of early endoderm, the identification of new ones is of fundamental importance. Both products of the Ink4a/Arf locus, in addition to being critical cell-cycle regulators, appear to be involved in several disease pathologies. Moreover, the locus' expression is epigenetically regulated in ES reprogramming processes, thus constituting the ideal candidates to modulate PPCs homeostasis. In this study, starting from mouse embryonic stem cells (mESCs), we analyzed the early stages of pancreatic commitment. By inducing mESCs commitment to the pancreatic lineage, we observed that both products of the Cdkn2a locus, Ink4a and Arf, mark a naïve pancreatic cellular state that resembled PPC-like specification. Treatment with epi-drugs suggests a role for chromatin remodeling in the CDKN2a (Cycline Dependent Kinase Inhibitor 2A) locus regulation in line with previous observations in other cellular systems. Our data considerably improve the comprehension of pancreatic cellular ontogeny, which could be critical for implementing pluripotent stem cells programming and reprogramming toward pancreatic lineage commitment.

Gastric Cancer Stem Cells: A Glimpse on Metabolic Reprogramming.

Gastric cancer (GC) is one of the most widespread causes of cancer-related death worldwide. Recently, emerging implied that gastric cancer stem cells (GCSCs) play an important role in the initiation and progression of GC. This subpopulation comprises cells with several features, such as self-renewal capability, high proliferating rate, and ability to modify their metabolic program, which allow them to resist current anticancer therapies. Metabolic pathway intermediates play a pivotal role in regulating cell differentiation both in tumorigenesis and during normal development. Thus, the dysregulation of both anabolic and catabolic pathways constitutes a significant opportunity to target GCSCs in order to eradicate the tumor progression. In this review, we discuss the current knowledge about metabolic phenotype that supports GCSC proliferation and we overview the compounds that selectively target metabolic intermediates of CSCs that can be used as a strategy in cancer therapy.

ZSCAN4+ mouse embryonic stem cells have an oxidative and flexible metabolic profile.

Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4+ mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4+ cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4+ stem cell state with potential applications in regenerative medicine.

Synthetic mycomelanin thin films as emergent bio-inspired interfaces controlling the fate of embryonic stem cells.

The fungal pathways of melanin synthesis have so far been little considered as a source of bio-inspiration in the field of functional materials, despite the interesting properties exhibited by Ascomycetes melanins from 1,8-dihydroxynaphthalene (1,8-DHN), including the ability to shield organisms from ionizing radiation. Herein, the processing techniques and characterizations of mycomelanin thin films obtained from the solid state polymerization of 1,8-DHN is reported for the first time. Overall, the results highlighted the role of synthetic mycomelanin thin films as a prototype of next generation bioinspired interfaces featuring high structural regularity and ultrasmooth morphology, high robustness against peroxidative bleaching and adhesion under water conditions, good biocompatibility and unprecedented effects in inducing the spontaneous differentiation of embryonic stem cells prevalently towards the endodermal lineages in the absence of added factors. These data open up new avenues towards the applications of this biomaterial in the fields of tissue engineering and regenerative medicine.

Prenatal expression of D-aspartate oxidase causes early cerebral D-aspartate depletion and influences brain morphology and cognitive functions at adulthood.

The free D-amino acid, D-aspartate, is abundant in the embryonic brain but significantly decreases after birth. Besides its intracellular occurrence, D-aspartate is also present at extracellular level and acts as an endogenous agonist for NMDA and mGlu5 receptors. These findings suggest that D-aspartate is a candidate signaling molecule involved in neural development, influencing brain morphology and behaviors at adulthood. To address this issue, we generated a knockin mouse model in which the enzyme regulating D-aspartate catabolism, D-aspartate oxidase (DDO), is expressed starting from the zygotic stage, to enable the removal of D-aspartate in prenatal and postnatal life. In line with our strategy, we found a severe depletion of cerebral D-aspartate levels (up to 95%), since the early stages of mouse prenatal life. Despite the loss of D-aspartate content, Ddo knockin mice are viable, fertile, and show normal gross brain morphology at adulthood. Interestingly, early D-aspartate depletion is associated with a selective increase in the number of parvalbumin-positive interneurons in the prefrontal cortex and also with improved memory performance in Ddo knockin mice. In conclusion, the present data indicate for the first time a biological significance of precocious D-aspartate in regulating mouse brain formation and function at adulthood.

Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study.

JAK2 V617F mutational status is an essential diagnostic index in myeloproliferative neoplasms (MPNs). Although widely used for detection of JAK2 V617F mutation in peripheral blood (PB), sensitive real-time quantitative PCR (qPCR) presents some methodological limitations. Recently, emerging alternative technologies, like digital droplet PCR (ddPCR), have been reported to overcome some of qPCR's technical drawbacks. The purpose of this study was to compare the diagnostic utility of ddPCR to qPCR for JAK2 V617F detection and quantification in samples from MPNs patients. Sensitivity and specificity of qPCR and ddPCR in the detection of the mutation were assessed by using a calibrator panel of mutated DNA on 195 JAK2 positive MPN samples. Based on our results, ddPCR proved to be a suitable, precise, and sensitive method for detection and quantification of the JAK2 V617F mutation.

Topographic Cues Impact on Embryonic Stem Cell Zscan4-Metastate.

The extracellular microenvironment proved to exert a potent regulatory effect over different aspects of Embryonic Stem Cells (ESCs) behavior. In particular, the employment of engineered culture surfaces aimed at modulating ESC self-organization resulted effective in directing ESCs toward specific fate decision. ESCs fluctuate among different levels of functional potency and in this context the Zscan4 gene marks the so-called "metastate," a cellular state in which ESCs retain both self-renewal and pluripotency capabilities. Here we investigated the impact of topographic cues on ESCs pluripotency, differentiation and organization capabilities. To this aim, we engineered culturing platforms of nanograted surfaces with different features size and we investigated their impact on ESCs multicellular organization and Zscan4 gene expression. We showed that the morphology of ESC-derived aggregates and Zscan4 expression are strictly intertwined. Our data suggest that ESC Zscan4 metastate can be promoted if the adhesive surface conditions guide cellular self-aggregation into 3D dome-like structure, in which both cell-material interactions and cell-cell contact are supportive for Zscan4 expression.