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A retrospective observational study utilising cancer incidence data from a population-based registry investigated determinants affecting primary liver cancer survival in a southern Italian region with high hepatitis viral infection rates and obesity prevalence. Among 2687 patients diagnosed between 2006 and 2019 (65.3% male), a flexible hazard-based regression model revealed factors influencing 5-year survival rates. High deprivation levels [HR = 1.41 (95%CI = 1.15-1.76); p < 0.001], poor access to care [HR = 1.99 (95%IC = 1.70-2.35); p < 0.0001], age between 65 and 75 [HR = 1.48 (95%IC = 1.09-2.01); p < 0.05] or >75 [HR = 2.21 (95%CI = 1.62-3.01); p < 0.0001] and residing in non-urban areas [HR = 1.35 (95%CI = 1.08-1.69); p < 0.01] were associated with poorer survival estimates. While deprivation appeared to be a risk factor for primary liver cancer patients residing within the urban area, the geographic distance from specialised treatment centres emerged as a potential determinant of lower survival estimates for residents in the non-urban areas. After balancing the groups of easy and poor access to care using a propensity score approach, poor access to care and a lower socioeconomic status resulted in potentially having a negative impact on primary liver cancer survival, particularly among urban residents. We emphasise the need to interoperate cancer registries with other data sources and to deploy innovative digital solutions to improve cancer prevention.

Neutrophil gelatinase-associated lipocalin (NGAL), a siderophore-mediated iron binding protein, is highly expressed in human anaplastic thyroid carcinomas (ATCs) where it plays pleiotropic protumorigenic roles including that of a prosurvival protein. Here we show that NGAL inhibits FAS/CD95 death receptor to control ATC cell survival. FAS/CD95 expression in human specimens from patients with ATC and in ATC-derived cell lines negatively correlate with NGAL expression. Silencing of NGAL in ATC cells leads to FAS/CD95 upregulation, whereas NGAL overexpression determines the opposite effect. As a result, an agonist anti-FAS/CD95 antibody induces cell death in NGAL-silenced cells while it is ineffective on NGAL-overexpressing cells. Interestingly, the inhibitory activity of NGAL on FAS/CD95 is due to its iron carrier property given that perturbing iron homeostasis of NGAL-proficient and -deficient ATC cells directly influences FAS/CD95 expression. Accordingly, conditioned media containing a mutant form of NGAL unable to bind siderophores cannot rescue cells from FAS/CD95-dependent death, whereas NGAL wild type-containing conditioned media abolish the effects of the agonist antibody. We also find that downregulation of FAS/CD95 expression is mediated by iron-dependent NGAL suppression of p53 transcriptional activity. Our results indicate that NGAL contributes to ATC cell survival by iron-mediated inhibition of p53-dependent FAS/CD95 expression and suggest that restoring FAS/CD95 by NGAL suppression could be a helpful strategy to kill ATC cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility., (© 2023. The Author(s).)

Inflammation is a biological response involving immune cells, blood vessels and mediators induced by endogenous and exogenous stimuli, such as pathogens, damaged cells or chemicals. Unresolved (chronic) inflammation is characterized by the secretion of cytokines that maintain inflammation and redox stress. Mitochondrial or nuclear redox imbalance induces DNA damage, which triggers the DNA damage response (DDR) that is orchestrated by ATM and ATR kinases, which modify gene expression and metabolism and, eventually, establish the senescent phenotype. DDR-mediated senescence is induced by the signalling proteins p53, p16 and p21, which arrest the cell cycle in G1 or G2 and promote cytokine secretion, producing the senescence-associated secretory phenotype. Senescence and inflammation phenotypes are intimately associated, but highly heterogeneous because they vary according to the cell type that is involved. The vicious cycle of inflammation, DNA damage and DDR-mediated senescence, along with the constitutive activation of the immune system, is the core of an evolutionarily conserved circuitry, which arrests the cell cycle to reduce the accumulation of mutations generated by DNA replication during redox stress caused by infection or inflammation. Evidence suggests that specific organ dysfunctions in apparently unrelated diseases of autoimmune, rheumatic, degenerative and vascular origins are caused by inflammation resulting from DNA damage-induced senescence., (© 2023. Springer Nature Limited.)

Conventional and targeted cancer therapies may induce a cellular senescence program termed therapy-induced senescence. However, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, driving tumor recurrence after treatments. Cells that escape from therapy-induced senescence are characterized by a plastic, cancer stem cell-like phenotype, and recent studies are beginning to define their unique metabolic features, such as glutamine dependence. Here, we show that the antineoplastic drug trabectedin suppresses escape from therapy-induced senescence in all cell lines studied, and reduces breast cancer stem-like cells, at concentrations that do not affect the viability of senescent tumor cells. We demonstrate that trabectedin downregulates both the glutamine transporter SLC1A5 and glutamine synthetase, thereby interfering with glutamine metabolism. On the whole, our results indicate that trabectedin targets a glutamine-dependent cancer stem-like cell population involved in evasion from therapy-induced senescence and suggest a therapeutic potential for trabectedin combined with pro-senescence chemotherapy in tumor treatment., (Copyright © 2022. Published by Elsevier Inc.)

Mutations of the rearranged during transfection (RET) kinase are frequently reported in cancer, which make it as an attractive therapeutic target. Herein, we discovered a series of N-trisubstituted pyrimidine derivatives as potent inhibitors for both wild-type ( wt ) RET and RET V804M , which is a resistant mutant for several FDA-approved inhibitors. The X-ray structure of a representative inhibitor with RET revealed that the compound binds in a unique pose that bifurcates beneath the P-loop and confirmed the compound as a type I inhibitor. Through the structure-activity relationship (SAR) study, compound 20 was identified as a lead compound, showing potent inhibition of both RET and RET V804M . Additionally, compound 20 displayed potent antiproliferative activity of CCDC6-RET-driven LC-2/ad cells. Analysis of RET phosphorylation indicated that biological activity was mediated by RET inhibition. Collectively, N-trisubstituted pyrimidine derivatives could serve as scaffolds for the discovery and development of potent inhibitors of type I RET and its gatekeeper mutant for the treatment of RET-driven cancers.

Therapy-induced senescence (TIS) is a major cellular response to anticancer therapies. While induction of a persistent growth arrest would be a desirable outcome in cancer therapy, it has been shown that, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, likely contributing to tumor relapse. Notably, cells that escape from TIS acquire a plastic, stem cell-like phenotype. The metabolic dependencies of cells that evade senescence have not been thoroughly studied. In this study, we show that glutamine depletion inhibits escape from TIS in all cell lines studied, and reduces the stem cell subpopulation. In line with a metabolic reliance on glutamine, escaped clones overexpress the glutamine transporter SLC1A5. We also demonstrate a central role of glutamine synthetase that mediates resistance to glutamine deprivation, conferring independence from exogenous glutamine. Finally, rescue experiments demonstrate that glutamine provides nitrogen for nucleotides biosynthesis in cells that escape from TIS, but also suggest a critical involvement of glutamine in other metabolic and non-metabolic pathways. On the whole, these results reveal a metabolic vulnerability of cancer stem cells that recover proliferation after exposure to anticancer therapies, which could be exploited to prevent tumor recurrence.

We have recently described Pz-1, a benzimidazole-based type-2 RET and VEGFR2 inhibitor. Based on a kinome scan, here we show that Pz-1 is also a potent (IC 50  < 1 nM) TRKA/B/C inhibitor. Pz-1 potently inhibited proliferation of human cancer cells carrying either RET- or TRKA oncoproteins (IC 50  ~ 1 nM), with a negligible effect against RET- and TRKA-negative cells. By testing mutations, known to mediate resistance to other compounds, RET G810R/S, but not L730I/V, E732K, V738A and Y806N, showed some degree of resistance to Pz-1. In the case of TRKA, G595R and F589L, but not G667C, showed some degree of resistance. In xenograft models, orally administered Pz-1 almost completely inhibited RET- and TRKA-mutant tumours at 1-3 mg/kg/day but showed a reduced effect on RET/TRKA-negative cancer models. The activity, albeit reduced, on RET/TRKA-negative tumours may be justified by VEGFR2 inhibition. Tumours induced by NIH3T3 cells transfected by RET G810R and TRKA G595R featured resistance to Pz-1, demonstrating that RET or TRKA inhibition is critical for its anti-tumourigenic effect. In conclusion, Pz-1 represents a new powerful kinase inhibitor with distinct activity towards cancers induced by oncogenic RET and TRKA variants, including some mutants displaying resistance to other drugs., (© 2021. The Author(s).)

Aptamers, synthetic single-strand oligonucleotides that are similar in function to antibodies, are promising as therapeutics because of their minimal side effects. However, the stability and bioavailability of the aptamers pose a challenge. We developed aptamers converted from RNA aptamer to modified DNA aptamers that target phospho-AXL with improved stability and bioavailability. On the basis of the comparative analysis of a library of 17 converted modified DNA aptamers, we selected aptamer candidates, GLB-G25 and GLB-A04, that exhibited the highest bioavailability, stability, and robust antitumor effect in in vitro experiments. Backbone modifications such as thiophosphate or dithiophosphate and a covalent modification of the 5'-end of the aptamer with polyethylene glycol optimized the pharmacokinetic properties, improved the stability of the aptamers in vivo by reducing nuclease hydrolysis and renal clearance, and achieved high and sustained inhibition of AXL at a very low dose. Treatment with these modified aptamers in ovarian cancer orthotopic mouse models significantly reduced tumor growth and the number of metastases. This effective silencing of the phospho-AXL target thus demonstrated that aptamer specificity and bioavailability can be improved by the chemical modification of existing aptamers for phospho-AXL. These results lay the foundation for the translation of these aptamer candidates and companion biomarkers to the clinic.

Testicular germ cell tumors (TGCTs) are the leading form of solid cancer and death affecting males between the ages of 20 and 40. Today, their surgical resection and chemotherapy are the treatments of first choice, even if sometimes this is not enough to save the lives of patients with TGCT. As seen for several tumors, the deregulation of microRNAs (miRNAs) is also a key feature in TGCTs. miRNAs are small molecules of RNA with biological activity that are released into biological fluids by testicular cancer cells. Their presence, therefore, can be detected and monitored by considering miRNAs as diagnostic and prognostic markers for TGCTs. The purpose of this review is to collect all the studies executed on miRNAs that have a potential role as biomarkers for testicular tumors.

Therapy-induced senescence (TIS or therapy-induced premature senescence) is a key cellular program triggered in the course of cancer radiotherapy and chemotherapy with genotoxic drugs, both in cancer cells and in normal cells, whose activation critically affects the outcome of cancer therapy. Drug-induced senescent cells undergo a permanent cell cycle arrest, acquire distinctive morphological and biochemical alterations, and an enhanced secretory ability, referred to as senescence-associated secretory phenotype (SASP). The transcription factor NF-κB acts as a master regulator of the SASP, driving the expression of senescence-associated secretome components.Here we describe protocols for the establishment of a tetracycline-regulated cell system for the investigation of the role of NF-κB in TIS. We also describe protocols routinely used in our laboratory, to investigate TIS in this Tet-On inducible expression system. Finally, we describe techniques for the validation of TIS induction.

Background: Phosphorylation of the intracellular domain of the EPHA2 receptor tyrosine kinase (RTK) on serine 897 (S897) has been demonstrated to mediate EPHA2 oncogenic activity. Here, we show that in thyroid cancer cells harboring driver oncogenes that signal through the extracellular regulated kinase (ERK1/2) signaling pathway [rearranged RET RTK (RET/PTC), KRAS(G12R), or BRAF V600E oncogenes], EPHA2 is robustly phosphorylated on S897. EPHA2 S897 is embedded in a consensus sequence for phosphorylation by the AGC family kinases, including p90RSK (ribosomal protein S6 kinase), a direct ERK1/2 target. Methods: We show that recombinant p90RSK phosphorylates in vitro EPHA2 S897 and that treatment with chemical inhibitors targeting p90RSK or other components of the ERK1/2 pathway blunts S897 phosphorylation. Results: RNA interference-mediated knockdown combined with rescue experiments demonstrated that EPHA2 S897 phosphorylation mediates thyroid cancer cell proliferation and motility. Conclusions: These findings point to EPHA2 S897 as a crucial mediator of the oncogenic activity of the ERK1/2 signaling cascade in thyroid cancer.

The endoplasmic reticulum stress and the unfolded protein response are triggered following an imbalance between protein load and protein folding. Until recently, two possible outcomes of the unfolded protein response have been considered: life or death. We sought to substantiate a third alternative, dedifferentiation, mesenchymal shift, and activation of the antioxidant response by using typical endocrine cells, i.e. thyroid cells. The thyroid is a unique system both of endoplasmic reticulum stress (a single protein, thyroglobulin represents the majority of proteins synthesized in the endoplasmic reticulum by the thyrocyte) and of polarized epithelium (the single layer of thyrocytes delimiting the follicle). Following endoplasmic reticulum stress, in thyroid cells the folding of thyroglobulin was disrupted. The mRNAs of unfolded protein response were induced or spliced (X-box binding protein-1). Differentiation was inhibited: mRNA levels of thyroid specific genes, and of thyroid transcription factors were dramatically downregulated, at least in part, transcriptionally. The dedifferentiating response was accompanied by an upregulation of mRNAs of antioxidant genes. Moreover, cadherin-1, and the thyroid (and kidney)-specific cadherin-16 mRNAs were downregulated, vimentin, and SNAI1 mRNAs were upregulated. In addition, loss of cortical actin and stress fibers formation were observed. Together, these data indicate that ER stress in thyroid cells induces dedifferentiation, loss of epithelial organization, shift towards a mesenchymal phenotype, and activation of the antioxidant response, highlighting, at the same time, a new and wide strategy to achieve survival following ER stress, and, as a sort of the other side of the coin, a possible new molecular mechanism of decline/loss of function leading to a deficit of thyroid hormones formation., (Copyright © 2020 Ulianich, Mirra, Garbi, Calì, Conza, Treglia, Miraglia, Punzi, Miele, Raciti, Beguinot, Consiglio and Di Jeso.)

We have recently reported that transgenic mice overexpressing the HMGA1-pseudogene7 develop hematological neoplasia marked by monoclonal B-cell populations, and diagnosed as Diffuse Large B-cell Lymphoma. These findings prove the HMGA1-pseudogene7 oncogenic role in vivo .

The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor β1 (TGF-β1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-β1 during EMT initiation and establishment. TGF-β1 triggered, 30-90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-β1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-β1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-β1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-β1 and the priming role of DNA oxidation that marks TGF-β1-induced and -repressed genes involved in the EMT., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Latest studies have shown that deregulated pseudogene transcripts contribute to cancer working as competing endogenous RNAs. Our research group has recently demonstrated that the overexpression of two HMGA1 pseudogenes, HMGA1P6 and HMGA1P7 , has a critical role in cancer progression. These pseudogenes work sustaining the expression of HMGA1 and other cancer-related genes. We generated a mouse model overexpressing HMGA1P6 to better study the HMGA1 -pseudogene function in a more physiological context. Here, we show the proliferation rate and the susceptibility to senescence of mouse embryonic fibroblasts obtained from HMGA1P6 -overexpressing mice to better characterize the HMGA1-pseudogene function. Indeed, our study reports that mouse embryonic fibroblasts (MEFs) derived from HMGA1P6 mice express higher HMGA1 mRNA and protein levels. Moreover, these cells grow faster and senesce later than wild-type sustaining the oncogenic role of ceRNA crosstalk mediated by HMGA1Ps .

Testicular germ cell tumors (TGCTs) are the most frequent solid malignant tumors in men 20- 40 years of age and the most frequent cause of death from solid tumors in this age group. Recent studies have underscored the fact that miRNA deregulation is a feature of carcinogenesis, including TGCT development and progression. MiRNAs are a group of small noncoding RNAs that bind to the 3'-untranslated region (UTR) of the targeted mRNAs, thus causing mRNA degradation or the inhibition of its translation, regulating gene expression in a temporal and tissue-specific manner. However, few miRNAs have been found to play key roles in TGCTs; recently, other miRNAs have been identified, representing novel potential therapeutic targets., (2020, International Research and Cooperation Association for Bio & Socio - Sciences Advancement.)

An important drawback in the management of glioblastoma (GBM) patients is the frequent relapse upon surgery and therapy. A likely explanation is that conventional therapies poorly affect a small population of stem-like cancer cells (glioblastoma stem cells, GSCs) that remain capable of repopulating the tumour mass. Indeed, the development of therapeutic strategies able to hit GSCs while reducing the tumour burden has become an important challenge to increase a patient's survival. The signal transducer and activator of transcription-3 (STAT3) has been reported to play a pivotal role in maintaining the tumour initiating capacity of the GSC population. Therefore, in order to impair the renewal and propagation of the PDGFRβ-expressing GSC population, here we took advantage of the aptamer-siRNA chimera (AsiC), named Gint4.T-STAT3, that we previously have shown to efficiently antagonize STAT3 in subcutaneous PDGFRβ-positive GBM xenografts. We demonstrate that the aptamer conjugate is able to effectively and specifically prevent patient-derived GSC function and expansion. Moreover, because of the therapeutic potential of using miR-10b inhibitors and of the broad expression of the Axl receptor in GBM, we used the GL21.T anti-Axl aptamer as the targeting moiety for anti-miR-10b, showing that, in combination with the STAT3 AsiC, the aptamer-miR-10b antagonist treatment further enhances the inhibition of GSC sphere formation. Our results highlight the potential to use a combined approach with targeted RNA therapeutics to inhibit GBM tumour dissemination and relapse.

RET receptor tyrosine kinase is a driver oncogene in human cancer. We recently identified the clinical drug candidate Pz-1, which targets RET and VEGFR2. A key in vivo metabolite of Pz-1 is its less active demethylated pyrazole analogue. Using bioisosteric substitution methods, here, we report the identification of NPA101.3, lacking the structural liability for demethylation. NPA101.3 showed a selective inhibitory profile and an inhibitory concentration 50 (IC 50 ) of <0.003 μM for both RET and VEGFR2. NPA101.3 inhibited phosphorylation of all tested RET oncoproteins as well as VEGFR2 and proliferation of cells transformed by RET. Oral administration of NPA101.3 (10 mg/kg/day) completely prevented formation of tumors induced by RET/C634Y-transformed cells, while it weakened, but did not abrogate, formation of tumors induced by a control oncogene (HRAS/G12V). The balanced synchronous inhibition of both RET and VEGFR2, as well the resistance to demethylation, renders NPA101.3 a potential clinical candidate for RET-driven cancers.

We have recently identified and characterized two pseudogenes (HMGA1P6 and HMGA1P7) of the HMGA1 gene, which has a critical role in malignant cell transformation and cancer progression. HMGA1P6 and HMGAP17 act as microRNA decoy for HMGA1 and other cancer-related genes upregulating their protein levels. We have previously shown that they are upregulated in several human carcinomas, and their expression positively correlates with a poor prognosis and an advanced cancer stage. To evaluate in vivo oncogenic activity of HMGA1 pseudogenes, we have generated a HMGA1P7 transgenic mouse line overexpressing this pseudogene. By a mean age of 12 months, about 50% of the transgenic mice developed splenomegaly and accumulation of lymphoid cells in several body compartments. For these mice FACS and immunohistochemical analyses suggested the diagnosis of B-cell lymphoma that was further supported by clonality analyses and RNA expression profile of the pathological tissues of the HMGA1P7 transgenic tissues. Therefore, these results clearly demonstrate the oncogenic activity of HMGA1 pseudogenes in vivo.

Background: Recent studies have underlined HMGA protein's key role in the onset of testicular germ cell tumors, where HMGA1 is differently expressed with respect to the state of differentiation, suggesting its fine regulation as master regulator in testicular tumorigenesis. Several studies have highlighted that the HMGA1 transcript is strictly regulated by a set of inhibitory microRNAs. Thus, the aim of this study is to test whether HMGA1 overexpression in human seminomas may be induced by the deregulation of miR-26a and Let-7a-two HMGA1 -targeting microRNAs., Methods: HMGA1 mRNA and Let-7a and miR-26a levels were measured in a seminoma dataset available in the Cancer Genome Atlas database and confirmed in a subset of seminomas by qRT-PCR and western blot. A TCam-2 seminoma cell line was then transfected with Let-7a and miR-26a and tested for proliferation and motility abilities., Results: an inverse correlation was found between the expression of miR-26a and Let-7a and HMGA1 expression levels in seminomas samples, suggesting a critical role of these microRNAs in HMGA1 levels regulation. Accordingly, functional studies showed that miR-26a and Let-7a inhibited the proliferation, migration and invasion capabilities of the human seminoma derived cell line TCam-2., Conclusions: these data strongly support that the upregulation of HMGA1 levels occurring in seminoma is-at least in part-due to the downregulation of HMGA1 -targeting microRNAs.

Tumor suppressor genes in the CDKN2A/B locus ( p15INK4b , p16INK4a , and p14ARF ) function as biological barriers to transformation and are the most frequently silenced or deleted genes in human cancers. This gene silencing frequently occurs due to DNA methylation of the promoter regions, although the underlying mechanism is currently unknown. We present evidence that methylation of p16INK4a promoter is associated with DNA damage caused by interference between transcription and replication processes. Inhibition of replication or transcription significantly reduces the DNA damage and CpGs methylation of the p16INK4a promoter. We conclude that de novo methylation of the promoter regions is dependent on local DNA damage. DNA methylation reduces the expression of p16INK4a and ultimately removes this barrier to oncogene-induced senescence.

Introduction : Testicular germ cell tumors (TGCTs) are the most common neoplasia in the young male population, and the incidence has been constantly increasing in many parts of the world. These tumors are classified into seminomas and non-seminomas, and those divided, in turn, into yolk sac tumors, embryonal cell carcinomas, choriocarcinomas, and teratomas. Although therapeutic approaches have improved, approximately 25% of the patients relapse or, in a small number of cases, show platinum-resistant disease. Areas covered : We review several molecular targets that have recently emerged as powerful tools for both diagnosis and therapy of TGCTs. Moreover, we reviewed the most frequent deregulated pathways involved in TGCT tumorigenesis, reporting drugs that may emerge as novel therapeutic agents. Expert opinion : TGCT treatment is mainly based on platinum-derivative therapy with high cure rates. However, in the refractory patients, there are few alternative treatments. Thus, different pharmacological approaches have to be thoroughly investigated to shed new light on TGCT pathogenesis and treatment.

The tendency of individual CpG sites to be methylated is distinctive, non-random and well-regulated throughout the genome. We investigated the structural and spatial factors influencing CpGs methylation by performing an ultra-deep targeted methylation analysis on human, mouse and zebrafish genes. We found that methylation is not a random process and that closer neighboring CpG sites are more likely to share the same methylation status. Moreover, if the distance between CpGs increases, the degree of co-methylation decreases. We set up a simulation model to analyze the contribution of both the intrinsic susceptibility and the distance effect on the probability of a CpG to be methylated. Our finding suggests that the establishment of a specific methylation pattern follows a universal rule that must take into account of the synergistic and dynamic interplay of these two main factors: the intrinsic methylation susceptibility of specific CpG and the nucleotide distance between two CpG sites., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Programmed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as D-serine and D-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of D-amino acids are found. We recently demonstrated that Ddo, the D-aspartate degradation gene, is actively demethylated to ultimately reduce D-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate D-ser levels during brain development has been poorly investigated to date., Methods: We performed comprehensive ultradeep DNA methylation and hydroxymethylation profiling along with mRNA expression and HPLC-based D-amino acids level analyses of genes controlling the mammalian brain levels of D-serine and D-aspartate. DNA methylation changes occurring in specific cerebellar cell types were also investigated. We conducted high coverage targeted bisulfite sequencing by next-generation sequencing and single-molecule bioinformatic analysis., Results: We report consistent spatiotemporal modifications occurring at the Dao gene during neonatal development in a specific brain region (the cerebellum) and within specific cell types (astrocytes) for the first time. Dynamic demethylation at two specific CpG sites located just downstream of the transcription start site was sufficient to strongly activate the Dao gene, ultimately promoting the complete physiological degradation of cerebellar D-serine a few days after mouse birth. High amount of 5'-hydroxymethylcytosine, exclusively detected at relevant CpG sites, strongly evoked the occurrence of an active demethylation process., Conclusion: The present investigation demonstrates that robust and selective demethylation of two CpG sites is associated with postnatal activation of the Dao gene and consequent removal of D-serine within the mouse cerebellum. A single-molecule methylation approach applied at the Dao locus promises to identify different cell-type compositions and functions in different brain areas and developmental stages.

Due to the progress made in the area of precision and personalized medicine in the field of cancer therapy, strategies to selectively and specifically identify target molecules causative of the diseases are urgently needed. Efforts are being made by a number of different laboratories, companies, and researchers to develop therapeutic molecules that selectively recognize the tissues and the cells of interest, exhibit few or no off-target and side effects, are non-immunogenic, and have a strong action. Aptamers, artificially selected single-stranded DNA or RNA oligonucleotides, are promising molecules satisfying many of the requirements needed for diagnosis and precision medicine. Aptamers can also couple to their native mechanism of action the delivery of additional molecules (oligonucleotides, siRNAs, miRNAs) to target cells. In this review, we summarize recent progress in the aptamer-mediated strategy for the specific delivery of therapeutic oligonucleotides.

Non-small-cell lung cancer (NSCLC) accounts for 85%-90% of all cases of lung cancer that is the most deadly type of cancer. Despite advances in chemotherapy and radiotherapy, severe side effects and frequent drug resistance limit the success of the treatments, and the identification of new therapeutic options still represents a crucial challenge. Here, we provide the evidence for the therapeutic potential of an aptamer-microRNA (miR) complex (AmiC) composed by an aptamer (GL21.T), able to bind and antagonize the oncogenic receptor Axl, and the miR-137, downregulated in lung cancer and involved in cell survival and proliferation. We found that, when applied to Axl-expressing NSCLC cancer cells, the complex is effectively internalized, increasing miR cellular levels and downregulating miR targets. Most importantly, the complex combines the inhibitory function of the GL21.T aptamer and miR-137, leading to a negative impact on NSCLC migration and growth. The described AmiC thus represents a promising tool for the development of new therapeutic approaches for NSCLC., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Two novel molecular mechanisms with a driver role in pituitary tumorigenesis have been recently identified. They are (a) mutations in the Ubiquitin-Specific Protease 8 (USP8) gene in corticotroph tumors and (b) overexpression of the HMGA1 and HMGA2 genes in most of the pituitary tumors. Moreover, deregulated expression of the non-coding RNAs has been very frequently observed in this neoplasia. The aim of this review is to better elucidate the role, the mechanisms, and the possible clinical impact of these novel alterations in the development of pituitary neoplasia.

Glioblastoma (GBM) is a lethal, fast-growing brain cancer, affecting 2-3 per 100,000 adults per year. It arises from multipotent neural stem cells which have reduced their ability to divide asymmetrically and hence divide symmetrically, generating increasing number of cancer stem cells, fostering tumor growth. We have previously demonstrated that the architectural transcription factor HMGA1 is highly expressed in brain tumor stem cells (BTSCs) and that its silencing increases stem cell quiescence, reduces self-renewal and sphere-forming efficiency in serial passages, suggesting a shift from symmetric to asymmetric division. Since NUMB expression is fundamental for the fulfillment of asymmetric division in stem cells, and is lost or reduced in many tumors, including GBM, we have investigated the ability of HMGA1 to regulate NUMB expression. Here, we show that HMGA1 negatively regulates NUMB expression at transcriptional level, by binding its promoter and counteracting c/EBP-β and at posttranscriptional level, by regulating the expression of MSI1 and of miR-146a. Finally, we report that HMGA1 knockdown-induced NUMB upregulation leads to the downregulation of the NOTCH1 pathway. Therefore, the data reported here indicate that HMGA1 negatively regulates NUMB expression in BTSCs, further supporting HMGA1 targeting as innovative and effective anti-cancer therapy.

The Chromobox protein homolog 7 (CBX7) belongs to the Polycomb Group (PcG) family, and, as part of the Polycomb repressive complex (PRC1), contributes to maintain transcriptional gene repression. Loss of CBX7 expression has been reported in several human malignant neoplasias, where it often correlates with an advanced cancer state and poor survival, proposing CBX7 as a candidate tumor-suppressor gene in cancer progression. Indeed, CBX7 is able to positively or negatively regulate the expression of genes involved in cell proliferation and cancer progression, such as E-cadherin, cyclin E, osteopontin, EGR1. To understand the molecular mechanisms that underlie the involvement of CBX7 in cancer progression, we designed a functional proteomic experiment based on CHIP-MS to identify novel CBX7 protein partners. Among the identified CBX7-interacting proteins we focused our attention on the Protein Arginine Methyltransferase 1 (PRMT1) whose critical role in epithelial-mesenchymal transition (EMT), cancer cell migration and invasion has been already reported. We confirmed the interaction between CBX7 and PRMT1 and demonstrated that this interaction is crucial for PRMT1 enzymatic activity both in vitro and in vivo and for the regulation of E-cadherin expression, an important hallmark of EMT. These results suggest a general mechanism by which CBX7 interacting with histone modification enzymes like HDAC2 and PRMT1 enhances E-cadherin expression. Therefore, disruption of this equilibrium may induce impairment of E-cadherin expression and increased cell migration eventually leading to EMT and, then, cancer progression., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Molecular differentiation between benign (follicular thyroid adenoma, FTA) and malignant (follicular thyroid carcinoma, FTC) thyroid neoplasms is challenging. Here, we explored the genome-wide DNA methylation profile of FTA (n.10) and FTC (n.11) compared to normal thyroid (NT) (n.7) tissues. FTC featured 3,564 differentially-methylated CpGs (DMCpG), most (84%) of them hypermethylated, with respect to normal controls. At the principal component analysis (PCA), the methylation profile of FTA occupied an intermediate position between FTC and normal tissue. A large fraction (n. 2,385) of FTC-associated DMCpG were related (intragenic or within 1500 bp from the transcription start site) to annotated genes (n. 1,786). FTC-hypermethylated genes were enriched for targets of the Polycomb transcriptional repressor complex and the specific histone H3 marks (H3K4me2/me3-H3K27me3) found in chromatin domains known as "bivalent". Transcriptome profiling by RNAseq showed that 7.9% of the DMCpGs-associated genes were differentially expressed in FTC compared to NT, suggesting that altered DNA methylation may contribute to their altered expression. Overall, this study suggests that perturbed DNA methylation, in particular hypermethylation, is a component of the molecular mechanisms leading to the formation of FTC and that DNA methylation profiling may help differentiating FTCs from their benign counterpart.

Pulmonary sarcomatoid carcinomas (PSC) are a rare group of lung cancer with a median overall survival of 9-12 months. PSC are divided into five histotypes, challenging to diagnose and treat. The identification of PSC biomarkers is warranted, but PSC molecular profile remains to be defined. Herein, a targeted whole transcriptome analysis was performed on 14 PSC samples, evaluated also for the presence of the main oncogene mutations and rearrangements. PSC expression data were compared with transcriptome data of lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC) from The Cancer Genome Atlas. Deregulated genes were used for pathway enrichment analysis; the most representative genes were tested by immunohistochemistry (IHC) in an independent cohort (30 PSC, 31 LUAD, 31 LUSC). All PSC cases were investigated for PD-L1 expression. Thirty-eight genes deregulated in PSC were identified, among these IGJ and SLMAP were confirmed by IHC. Moreover, Forkhead box signaling and Fanconi anemia pathways were specifically enriched in PSC. Finally, some PSC harboured alterations in genes targetable by tyrosine kinase inhibitors, as EGFR and MET. We provide a deep molecular characterization of PSC; the identification of specific molecular profiles, besides increasing our knowledge on PSC biology, might suggest new strategies to improve patients management.

MicroRNAs (miRNAs) are key regulators of different human processes that represent a new promising class of cancer therapeutics or therapeutic targets. Indeed, in several tumor types, including non-small-cell lung carcinoma (NSCLC), the deregulated expression of specific miRNAs has been implicated in cell malignancy. As expression levels of the oncosuppressor miR-34c-3p are decreased in NSCLC compared to normal lung, we show that reintroduction of miR-34c-3p reduces NSCLC cell survival in vitro. Further, in order to deliver the miR-34c-based therapeutic selectively to tumor cells, we took advantage of a reported nucleic acid aptamer (GL21.T) that binds and inhibits the AXL transmembrane receptor and is rapidly internalized in the target cells. By applying methods successfully used in our laboratory, we conjugated miR-34c to the GL21.T aptamer as targeting moiety for the selective delivery to AXL-expressing NSCLC cells. We demonstrate that miR-34c-3p and the GL21.T/miR-34c chimera affect NSCLC cell proliferation and are able to overcome acquired RTK-inhibitor resistance by targeting AXL receptor. Thus, the GL21.T/miR-34c chimera exerts dual inhibition of AXL at functional and transcriptional levels and represents a novel therapeutic tool for the treatment of NSCLC., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Nucleic acid-based aptamers have emerged as efficient delivery carriers of therapeutics. Thanks to their unique features, they can be, to date, considered one of the best targeting moieties, allowing the specific recognition of diseased cells and avoiding unwanted off-target effects on healthy tissues. In this review, we revise the most recent contributes on bispecific and multifunctional aptamer therapeutic chimeras. We will discuss key examples of aptamer-mediated delivery of nucleic acid and peptide-based therapeutics underlying their great potentiality and versatility. Achieved objectives and challenges will be highlighted as well.

Nucleic acid therapeutics, including siRNAs, miRNAs/antimiRs, gRNAs and ASO, represent innovative and highly promising molecules for the safe treatment of a wide range of pathologies. The efficiency of systemic treatments is impeded by 1) the need to overcome physical and functional barriers in the organism, and 2) to accumulate in the intracellular active site at therapeutic concentrations. Although oligonucleotides either as modified naked molecules or complexed with delivery carriers have revealed to be effectively delivered to the affected target cells, this is restricted to topic treatments or to a few highly vascularized tissues. Therefore, the development of effective strategies for therapeutic nucleic acid selective delivery to target tissues is of primary importance in order to reduce the occurrence of undesired effects on non-target healthy tissues and to permit their translation to clinic. Due to their high affinity for specific ligands, high tissue penetration and chemical flexibility, short single-stranded nucleic acid aptamers are emerging as very attractive carriers for various therapeutic oligonucleotides. Yet, different aptamer-based bioconjugates, able to provide accumulation into target tissues, as well as efficient processing of therapeutic oligonucleotides, have been developed. In this respect, nucleic acid aptamer-mediated delivery strategies represent a powerful approach able to increase the therapeutic efficacy also highly reducing the overall toxicity. In this review, we will summarize recent progress in the field and discuss achieved objectives and optimization of aptamers as delivery carriers of short oligonucleotides., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We have previously shown that Neutrophil Gelatinase-Associated Lipocalin (NGAL) is strongly expressed in thyroid carcinomas, especially of anaplastic type, where it protects neoplastic cells from serum deprivation-induced apoptosis and enhances tumor invasivity by regulating MMP-9 activity. Here we demonstrate that NGAL-containing conditioned medium from human anaplastic thyroid carcinoma (ATC) cells is able to induce monocyte migration via up-regulation of a number of different chemokines. The enhanced chemokines transcription is due to the NGAL-mediated intracellular iron uptake. Very importantly, mice tumor allografts raised from subcutaneous injection of syngeneic colon carcinoma cell lines, expressing high levels of NGAL, show a dense leukocyte infiltrate which strongly decreases in tumor allografts from NGAL-depleted cell injected mice. Our results indicate that the NGAL promotes leukocytes recruitment in tumor microenvironment through iron-mediated chemokines production., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Aims: Malignant tumours from the upper aerodigestive tract are grouped collectively in the class of head and neck squamous cell carcinoma (HNSCC). The head and neck tumours were responsible for more than 500 000 cancer cases in 2012, accounting for the sixth highest incidence rate and mortality worldwide among all tumour types. Laryngeal squamous cell carcinoma (LSCC) possesses the second highest incidence rate among all HNSCC. Despite significant advances in surgery and radiotherapy during the last few decades, no treatment has been shown to achieve a satisfactory therapeutic outcome and the mortality rate of LSCC is still high, with a 5-year survival rate of 64%. Therefore, further investigations are required to identify the pathogenesis of LSCC., Methods and Results: In order to search for new LSCC biomarkers, we have analysed the expression of the HMGA family members, HMGA1 and HMGA2, by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. HMGA proteins are usually absent in the healthy adult tissues. In contrast, their constitutive expression is a feature of several neoplasias, being associated with a highly malignant phenotype and reduced survival. Here, we report HMGA2 overexpression in larynx carcinomas. Conversely, HMGA1 does not show any differences in its expression between normal and carcinoma tissues. Interestingly, HMGA2 overexpression appears associated with that of two HMGA1-pseudogenes, HMGA1P6 and HMGA1P7, acting as a sponge for HMGA1- and HMGA2-targeting microRNAs and involved in several human cancers., Conclusions: Therefore, HMGA2 overexpression appears to be a strong feature of larynx carcinoma, supporting its detection as a valid tool for the diagnosis of these malignancies., (© 2017 John Wiley & Sons Ltd.)

Background: Ionizing radiation (IR) is a well-known risk factor for papillary thyroid cancer, and it has been reported to deregulate microRNA expression, which is important to thyroid carcinogenesis. Therefore, this study investigated the impact of IR on microRNA expression profile of the normal thyroid cell line (FRTL-5 CL2), as well as its effect on radiosensitivity of thyroid cancer cell lines, especially the human anaplastic thyroid carcinoma cell line (8505c)., Methods: The global microRNA expression profile of irradiated FRTL-5 CL2 cells (5 Gy X-ray) was characterized, and data were confirmed by quantitative real-time polymerase chain reaction evaluating the expression of rno-miR-10b-5p, rno-miR-33-5p, rno-miR-128-1-5p, rno-miR-199a-3p, rno-miR-296-5p, rno-miR-328a-3p, and rno-miR-541-5p in irradiated cells. The miR-199a-3p and miR-10b-5p targets were validated by quantitative real-time polymerase chain reaction, Western blot, and luciferase target assays. The effects of miR-199a-3p and miR-10b-5p on DNA repair were determined by evaluating the activation of the protein kinases ataxia-telangiectasia mutated, ataxia telangiectasia, and Rad3-related and the serine 39 phosphorylation of variant histone H2AX as an indirect measure of double-strand DNA breaks in irradiated FRTL-5 CL2 cells. The impact of miR-10b-5p on radiosensitivity was analyzed by cell counting and MTT assays in FRTL-5 CL2, Kras-transformed FRTL-5 CL2 (FRTL KiKi), and 8505c cell lines., Results: The results reveal that miR-10b-5p and miR-199a-3p display the most pronounced alterations in expression in irradiated FRTL-5 CL2 cells. Dicer1 and Lin28b were validated as targets of miR-10b-5p and miR-199a-3p, respectively. Functional studies demonstrate that miR-10b-5p increases the growth rate of FRTL-5 CL2 cells, while miR-199a-3p inhibits their proliferation. Moreover, both of these microRNAs negatively affect homologous recombination repair, reducing activated ataxia-telangiectasia mutated and Rad3-related protein levels, consequently leading to an accumulation of the serine 39 phosphorylation of variant histone H2AX. Interestingly, the overexpression of miR-10b-5p decreases the viability of the irradiated FRTL5-CL2 and 8505c cell lines. Consistent with this observation, its inhibition in FRTL KiKi cells, which display high basal expression levels of miR-10b-5p, leads to the opposite effect., Conclusions: These results demonstrate that IR deregulates microRNA expression, affecting the double-strand DNA breaks repair efficiency of irradiated thyroid cells, and suggest that miR-10b-5p overexpression may be an innovative approach for anaplastic thyroid cancer therapy by increasing cancer cell radiosensitivity.

The extraordinary boost in the understanding of the genetic and epigenetic mechanisms underlying the development and progression of different types of cancer, is offering an unprecedented hope for the development of precise therapeutics able to interfere or replace the expression of target genes. In the last decade, the design of stable, safe and effective RNA-based therapeutics has been significantly improved increasing the number of molecules now in preclinical or in clinical trials for cancer gene therapy. However, with few exclusions as liver and hematological malignancies which are easy accessible to drugs, the development of effective systemic approaches for the delivery of RNA therapeutics to target cells is still unmet. To be effective, targeting carriers must be able to overcome both functional and physical barriers to safely carry and accumulate the therapeutic through the organism selectively to the tumor site, penetrate the target cancer mass, promote the uptake and localization in the appropriate intracellular compartment ultimately leading to the effective modulation of gene expression. Nucleic acid aptamers are folded single stranded oligonucleotides that bind at high affinity and high specificity their targets (proteins, lipids, small molecules etc), coupling the advantages of binding specificity proper of antibodies to the chemical nature of nucleic acids, sometimes also termed "nucleic acid antibodies". In several cases, aptamers targeting cell surface receptors are recycled into the cell together with the bound receptor enabling to drive conjugated therapeutics to cancer cells in a receptor-dependent manner. Therefore, besides other in vivo delivery strategies, the use of aptamers as precise and effective targeting moieties for anticancer RNA-based therapeutics has rapidly emerged and has been successfully addressed by several laboratories. In this Review, we will focus on the most recent and challenging progresses in the field that highlights the precision and flexibility of aptamer-based chimeras paving the way to the development of safe and effective carriers for cancer gene therapeutics., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Medullary thyroid carcinomas (MTC) arise from thyroid parafollicular, calcitonin-producing C-cells and can occur either as sporadic or as hereditary diseases in the context of familial syndromes, including multiple endocrine neoplasia 2A (MEN2A), multiple endocrine neoplasia 2B (MEN2B) and familial MTC (FMTC). In a large fraction of sporadic cases, and virtually in all inherited cases of MTC, activating point mutations of the RET proto-oncogene are found. RET encodes for a receptor tyrosine kinase protein endowed with transforming potential on thyroid parafollicular cells. As in other cancer types, microenvironmental factors play a critical role in MTC. Tumor-associated extracellular matrix, stromal cells and immune cells interact and influence the behavior of cancer cells both in a tumor-promoting and in a tumor-suppressing manner. Several studies have shown that, besides the neoplastic transformation of thyroid C-cells, a profound modification of tumor microenvironment has been associated to the RET FMTC/MEN2-associated oncoproteins. They influence the surrounding stroma, activating cancer-associated fibroblasts (CAFs), promoting cancer-associated inflammation and suppressing anti-cancer immune response. These mechanisms might be exploited to develop innovative anti-cancer therapies and novel prognostic tools in the context of familial, RET-associated MTC., (© 2018 Society for Endocrinology.)

Despite substantial improvements in the treatment strategies, ovarian cancer is still the most lethal gynecological malignancy. Identification of drug treatable therapeutic targets and their safe and effective targeting is critical to improve patient survival in ovarian cancer. AXL receptor tyrosine kinase (RTK) has been proposed to be an important therapeutic target for metastatic and advanced-stage human ovarian cancer. We found that AXL-RTK expression is associated with significantly shorter patient survival based on the The Cancer Genome Atlas patient database. To target AXL-RTK, we developed a chemically modified serum nuclease-stable AXL aptamer (AXL-APTAMER), and we evaluated its in vitro and in vivo antitumor activity using in vitro assays as well as two intraperitoneal animal models. AXL-aptamer treatment inhibited the phosphorylation and the activity of AXL, impaired the migration and invasion ability of ovarian cancer cells, and led to the inhibition of tumor growth and number of intraperitoneal metastatic nodules, which was associated with the inhibition of AXL activity and angiogenesis in tumors. When combined with paclitaxel, in vivo systemic (intravenous [i.v.]) administration of AXL-aptamer treatment markedly enhanced the antitumor efficacy of paclitaxel in mice. Taken together, our data indicate that AXL-aptamers successfully target in vivo AXL-RTK and inhibit its AXL activity and tumor growth and progression, representing a promising strategy for the treatment of ovarian cancer., (Copyright © 2017. Published by Elsevier Inc.)

Several studies have established that pseudogene mRNAs can work as competing endogenous RNAs and, when deregulated, play a key role in the onset of human neoplasias. Recently, we have isolated two HMGA1 pseudogenes, HMGA1P6 and HMGA1P7 . These pseudogenes have a critical role in cancer progression, acting as micro RNA (miRNA) sponges for HMGA1 and other cancer-related genes. HMGA1 pseudogenes were found overexpressed in several human carcinomas, and their expression levels positively correlate with an advanced cancer stage and a poor prognosis. In order to investigate the molecular alterations following HMGA1 pseudogene 7 overexpression, we carried out miRNA sequencing analysis on HMGA1P7 overexpressing mouse embryonic fibroblasts. Intriguingly, the most upregulated miRNAs were miR-483 and miR-675 that have been described as key regulators in cancer progression. Here, we report that HMGA1P7 upregulates miR-483 and miR-675 through a competing endogenous RNA mechanism with Egr1 , a transcriptional factor that positively regulates miR-483 and miR-675 expression., Competing Interests: The authors declare no conflict of interest.