The importance of being (slightly) modified: The role of rRNA editing on gene expression control and its connections with cancer.

In human ribosomal RNAs, over 200 residues are modified by specific, RNA-driven enzymatic complexes or stand-alone, RNA-independent enzymes. In most cases, modification sites are placed in specific positions within important functional areas of the ribosome. Some evidence indicates that the altered control in ribosomal RNA modifications may affect ribosomal function during mRNA translation. Here we provide an overview of the connections linking ribosomal RNA modifications to ribosome function, and suggest how aberrant modifications may affect the control of the expression of key cancer genes, thus contributing to tumor development. In addition, the future perspectives in this field are discussed.

JHDM1B expression regulates ribosome biogenesis and cancer cell growth in a p53 dependent manner.

Tumors characterized by an intense ribosome biogenesis often display a more aggressive behavior. Ribosomal RNA (rRNA) synthesis is controlled at several levels, including the epigenetic regulation of the condensation of chromatin portions containing rRNA genes. JHDM1B (Jumonji C histone demethylase 1B) is a histone demethylase able to regulate the accessibility of rRNA genes. In this study, we aimed to define the contribution of JHDM1B expression to the features of breast cancer, a tumor type whose behavior is related to the rate of ribosome biogenesis. We show that, in breast cancer-derived cell lines, the increase in rRNA transcription that follows JHDM1B knock-down is mirrored by an augmented cell proliferation only in p53 compromised cells, while p53 competent cells undergo cellular senescence and death. The latter effect appears to be mediated by a p38-dependent phosphorylation of p53, inducing the expression of p15(Ink4b) and p21(Waf1). In breast cancers, lower JHDM1B expression correlates with an increased size of specifically stained nucleolar organized regions, a morphological parameter directly related to the rate of ribosome biogenesis and with a poorer prognosis. In addition, in tumors lacking the controller function of p53, a lower expression of JHDM1B is associated with an increased tumor size at diagnosis. Altogether, our data indicate that epigenetic activation of rDNA genes induced by JHDM1B depletion is associated with a p53-dependent growth arrest, but may promote cancer cell growth when p53 is lacking.

Dyskerin expression in human fetal, adult and neoplastic intrahepatic bile ducts: correlations with cholangiocarcinoma aggressiveness.

AIMS: To investigate the immunohistochemical expression of dyskerin, a biomarker involved in ribosome production and telomere maintenance, in human fetal, adult and neoplastic bile ducts, and possible correlations with cholangiocarcinoma aggressiveness. METHODS AND RESULTS: Sixty consecutive intrahepatic cholangiocarcinomas were collected and used for tissue microarray construction (total: 176 cores); clinical data and follow-up were also collected. Five fetal and 10 normal adult livers were included as controls. Automated immunohistochemistry for dyskerin, p53, and Ki67, and nucleolar silver staining, were performed. In normal livers, dyskerin expression was negative in smaller bile ducts (mean 44.8 µm) and positive in bile ducts of larger diameter (mean 116.1 µm; P < 0.001). Expression was positive in 56.7% of cholangiocarcinomas, and correlated with p53 mutation (P = 0.008) and a higher proliferative (Ki67) index (P = 0.003), which were included as markers of tumour aggressiveness. Finally, dyskerin-positive cholangiocarcinomas showed a negative trend in disease-free survival (P = 0.078) on univariate analysis. CONCLUSIONS: The non-neoplastic biliary tree seems to progressively lose dyskerin expression from the major branches to the peripheral portal bile ducts. Similarly, intrahepatic cholangiocarcinomas showed two patterns of dyskerin expression, and the dyskerin-positive phenotype seemed to characterize more aggressive cholangiocarcinomas.

Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation.

Dyskerin is a nucleolar protein encoded by the DKC1 gene that (i) stabilizes the RNA component of the telomerase complex, and (ii) drives the site-specific pseudouridilation of rRNA. It is known that the partial lack of dyskerin function causes a defect in the translation of a subgroup of mRNAs containing internal ribosome entry site (IRES) elements such as those encoding for the tumor suppressors p27 and p53. In this study, we aimed to analyze what is the effect of the lack of dyskerin on the IRES-mediated translation of mRNAs encoding for vascular endothelial growth factor (VEGF). We transiently reduced dyskerin expression and measured the levels of the IRES-mediated translation of the mRNA encoding for VEGF in vitro in transformed and primary cells. We demonstrated a significant increase in the VEGF IRES-mediated translation after dyskerin knock-down. This translational modulation induces an increase in VEGF production in the absence of a significant upregulation in VEGF mRNA levels. The analysis of a list of viral and cellular IRESs indicated that dyskerin depletion can differentially affect IRES-mediated translation. These results indicate for the first time that dyskerin inhibition can upregulate the IRES translation initiation of specific mRNAs.

The nucleolar size is associated to the methylation status of ribosomal DNA in breast carcinomas.

BACKGROUND: There is a body of evidence that shows a link between tumorigenesis and ribosome biogenesis. The precursor of mature 18S, 28S and 5.8S ribosomal RNAs is transcribed from the ribosomal DNA gene (rDNA), which exists as 300-400 copies in the human diploid genome. Approximately one half of these copies are epigenetically silenced, but the exact role of epigenetic regulation on ribosome biogenesis is not completely understood. In this study we analyzed the methylation profiles of the rDNA promoter and of the 5' regions of 18S and 28S in breast cancer. METHODS: We analyzed rDNA methylation in 68 breast cancer tissues of which the normal counterpart was partially available (45/68 samples) using the MassARRAY EpiTYPER assay, a sensitive and quantitative method with single base resolution. RESULTS: We found that rDNA locus tended to be hypermethylated in tumor compared to matched normal breast tissues and that the DNA methylation level of several CpG units within the rDNA locus was associated to nuclear grade and to nucleolar size of tumor tissues. In addition we identified a subgroup of samples in which large nucleoli were associated with very limited or absent rDNA hypermethylation in tumor respect to matched normal tissue. CONCLUSIONS: In conclusion, we suggest that rDNA is an important target of epigenetic regulation in breast tumors and that rDNA methylation level is associated to nucleolar size.

Changes in ribosome biogenesis may induce cancer by down-regulating the cell tumor suppressor potential.

Many human pathological conditions, not linked to genetic alterations of oncogenes or tumor suppressors, are nevertheless associated with an increased risk of developing cancer, and some of them are characterized by quantitative and/or qualitative changes in ribosome biogenesis. Indeed, there is evidence that both an up-regulation of ribosome biogenesis, such as that occurring during the abnormal stimulation of cell growth, and intrinsic dysfunctions of ribosomes, such as those characterizing a series of inherited disorders, show an increased incidence of tumor onset. Here we discuss some recent insights into the mechanisms by which these alterations in ribosome biogenesis may facilitate tumorigenesis.

Selective inhibition of rRNA transcription downregulates E2F-1: a new p53-independent mechanism linking cell growth to cell proliferation.

The tumour suppressor p53 negatively controls cell cycle progression in response to perturbed ribosome biogenesis in mammalian cells, thus coordinating growth with proliferation. Unlike mammalian cells, p53 is not involved in the growth control of proliferation in yeasts and flies. We investigated whether a p53-independent mechanism of response to inadequate ribosome biogenesis rate is also present in mammalian cells. We studied the effect of specific inhibition of rRNA synthesis on cell cycle progression in human cancer cell lines using the small-interfering RNA procedure to silence the POLR1A gene, which encodes the catalytic subunit of RNA polymerase I. We found that interference of POLR1A inhibited the synthesis of rRNA and hindered cell cycle progression in cells with inactivated p53, as a consequence of downregulation of the transcription factor E2F-1. Downregulation of E2F-1 was due to release of the ribosomal protein L11, which inactivated the E2F-1-stabilising function of the E3 ubiquitin protein ligase MDM2. These results demonstrated the existence of a p53-independent mechanism that links cell growth to cell proliferation in mammalian cells, and suggested that selective targeting of the RNA polymerase I transcription machinery might be advisable to hinder proliferation of p53-deficient cancer cells.

SnoRNA U50 levels are regulated by cell proliferation and rRNA transcription.

rRNA post transcriptional modifications play a role in cancer development by affecting ribosomal function. In particular, the snoRNA U50, mediating the methylation of C2848 in 28S rRNA, has been suggested as a potential tumor suppressor-like gene playing a role in breast and prostate cancers and B-cell lymphoma. Indeed, we observed the downregulation of U50 in colon cancer cell lines as well as tumors. We then investigated the relationship between U50 and proliferation in lymphocytes stimulated by phytohemagglutinin (PHA) and observed a strong decrease in U50 levels associated with a reduced C2848 methylation. This reduction was due to an alteration of U50 stability and to an increase of its consumption. Indeed, the blockade of ribosome biogenesis induced only an early decrease in U50 followed by a stabilization of U50 levels when ribosome biogenesis was almost completely blocked. Similar results were found with other snoRNAs. Lastly, we observed that U50 modulation affects ribosome efficiency in IRES-mediated translation, demonstrating that changes in the methylation levels of a single specific site on 28S rRNA may alter ribosome function. In conclusion, our results link U50 to the cellular proliferation rate and ribosome biogenesis and these findings may explain why its levels are often greatly reduced in cancers.

Liquid Biopsy in the Management of Breast Cancer Patients: Where Are We Now and Where Are We Going.

Liquid biopsy (LB) is an emerging diagnostic tool that analyzes biomarkers in the blood (and possibly in other body fluids) to provide information about tumor genetics and response to therapy. This review article provides an overview of LB applications in human cancer with a focus on breast cancer patients. LB methods include circulating tumor cells and cell-free tumor products, such as circulating tumor DNA. LB has shown potential in detecting cancer at an early stage, monitoring tumor progression and recurrence, and predicting patient response to therapy. Several studies have demonstrated its clinical utility in breast cancer patients. However, there are limitations to LB, including the lack of standardized assays and the need for further validation. Future potential applications of LB include identifying the minimal residual disease, early detection of recurrence, and monitoring treatment response in various cancer types. LB represents a promising non-invasive diagnostic tool with potential applications in breast cancer diagnosis, treatment, and management. Further research is necessary to fully understand its clinical utility and overcome its current limitations.

An innovative hyperbaric hypothermic machine perfusion protects the liver from experimental preservation injury.

PURPOSE: Hypothermic machine perfusion systems seem more effective than the current static storage to prevent cold ischemic liver injury. Thus, we test an innovative hyperbaric hypothermic machine perfusion (HHMP), which combines hyperbaric oxygenation of the preservation solution and continuous perfusion of the graft. METHODS: Rat livers were preserved with Celsior solution according to 4 different modalities: normobaric static preservation; hyperbaric static preservation at 2 atmosphere absolute (ATA); normobaric dynamic preservation, with continuous perfusion; hyperbaric dynamic preservation, with continuous perfusion at 2 ATA. After 24 h cold preservation, we assessed different parameters. RESULTS: Compared to baseline, livers preserved with the current static storage showed severe ultrastructural damage, glycogen depletion and an increased oxidative stress. Normobaric perfused livers showed improved hepatocyte ultrastructure and ameliorated glycogen stores, but they still suffered a significant oxidative damage. The addition of hyperbaric oxygen produces an extra benefit by improving oxidative injury and by inducing endothelial NO synthase (eNOS) gene expression. CONCLUSIONS: Preservation by means of the present innovative HHMP reduced the liver injury occurring after the current static cold storage by lowering glycogen depletion and oxidative damage. Interestingly, only the use of hyperbaric oxygen was associated to a blunted oxidative stress and an increased eNOS gene expression.

MicroRNAs at the Crossroad between Immunoediting and Oncogenic Drivers in Hepatocellular Carcinoma.

Treatments aimed to reverse the tumor-induced immune tolerance represent a promising approach for advanced hepatocellular carcinoma (HCC). Notwithstanding, primary nonresponse, early, and late disease reactivation still represent major clinical challenges. Here, we focused on microRNAs (miRNAs) acting both as modulators of cancer cell hallmarks and immune system response. We outlined the bidirectional function that some oncogenic miRNAs play in the differentiation and program activation of the immune system development and, at the same time, in the progression of HCC. Indeed, the multifaceted spectrum of miRNA targets allows the modulation of both immune-associated factors and oncogenic or tumor suppressor drivers at the same time. Understanding the molecular changes contributing to disease onset, progression, and resistance to treatments might help to identify possible novel biomarkers for selecting patient subgroups, and to design combined tailored treatments to potentiate antitumor approaches. Preliminary findings seem to argue in favor of a bidirectional function of some miRNAs, which enact an effective modulation of molecular pathways driving oncogenic and immune-skipping phenotypes associated with cancer aggressiveness. The identification of these miRNAs and the characterization of their 'dual' role might help to unravel novel biomarkers identifying those patients more likely to respond to immune checkpoint inhibitors and to identify possible therapeutic targets with both antitumor and immunomodulatory functions. In the present review, we will focus on the restricted panel of miRNAs playing a bidirectional role in HCC, influencing oncogenic and immune-related pathways at once. Even though this field is still poorly investigated in HCC, it might represent a source of candidate molecules acting as both biomarkers and therapeutic targets in the setting of immune-based treatments.

Human dyskerin binds to cytoplasmic H/ACA-box-containing transcripts affecting nuclear hormone receptor dependence.

BACKGROUND: Dyskerin is a nuclear protein involved in H/ACA box snoRNA-guided uridine modification of RNA. In humans, its defective function is associated with cancer development and induces specific post-transcriptional alterations of gene expression. In this study, we seek to unbiasedly identify mRNAs regulated by dyskerin in human breast cancer-derived cells. RESULTS: We find that dyskerin depletion affects the expression and the association with polysomes of selected mRNA isoforms characterized by the retention of H/ACA box snoRNA-containing introns. These snoRNA retaining transcripts (snoRTs) are bound by dyskerin in the cytoplasm in the form of shorter 3' snoRT fragments. We then characterize the whole cytoplasmic dyskerin RNA interactome and find both H/ACA box snoRTs and protein-coding transcripts which may be targeted by the snoRTs' guide properties. Since a fraction of these protein-coding transcripts is involved in the nuclear hormone receptor binding, we test to see if this specific activity is affected by dyskerin. Obtained results indicate that dyskerin dysregulation may alter the dependence on nuclear hormone receptor ligands in breast cancer cells. These results are paralleled by consistent observations on the outcome of primary breast cancer patients stratified according to their tumor hormonal status. Accordingly, experiments in nude mice show that the reduction of dyskerin levels in estrogen-dependent cells favors xenograft development in the absence of estrogen supplementation. CONCLUSIONS: Our work suggests a cytoplasmic function for dyskerin which could affect mRNA post-transcriptional networks relevant for nuclear hormone receptor functions.

Location of rRNA transcription to the nucleolar components: disappearance of the fibrillar centers in nucleoli of regenerating rat hepatocytes.

The precise location of rDNA transcription to the components of mammalian cell nucleolus is still debated. This was due to the fact that all the molecules necessary for rRNA synthesis are located in two of the three components, the fibrillar centers (FCs) and the dense fibrillar component (DFC), which together with the granular component (GC) are considered to be constantly present in mammalian cell nucleoli. In the present study we demonstrated that in nucleoli of many regenerating rat hepatocytes at 15 h after partial hepatectomy the FCs were no longer present, only the DFC and the GC being detected. At this time of regeneration the rRNA transcriptional activity was three fold that of resting hepatocytes, while the synthesis of DNA was not yet significantly increased, indicating that these nucleolar changes were due to the rRNA synthesis up-regulation. The DFC appeared to be organized in numerous, small, roundish tufts of fibrils. The silver staining procedure for AgNOR proteins, which are associated with the ribosomal genes, selectively and homogeneously stained these fibrillar tufts. Immuno-gold visualization of the Upstream Binding Factor (UBF), which is associated with the promoter region and the transcribed portion of the rRNA 45S gene, demonstrated that UBF was selectively located in the fibrillar tufts. We concluded that in proliferating rat hepatocytes the increased synthesis of rRNA induced an activation of the rRNA transcription machinery located in the fibrillar centers which, by becoming associated with the ribonucleoprotein transcripts, assumed the morphological pattern of the DFC.

Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms.

The ribosomal RNA 5.8S is one of the four rRNAs that constitute ribosomes. In human cells, like in all eukaryotes, it derives from the extensive processing of a long precursor containing the sequence of 18S, 5.8S and 28S rRNAs. It has been confirmed also in human cells the presence of three isoforms of 5.8S rRNA: one more abundant called 5.8S short, one called 5.8S long bearing 5 extra-nucleotides at its 5' end and one 10 nucleotide shorter called 5.8S cropped. So far, little is known about 5.8S long specific role in cell biology and its function in human pathology. The lack of studies on the three 5.8S isoforms could be due to the techniques usually applied to study ribosome biogenesis, such as Northern blot with radioactively labelled probes, that require strict protective measures, and abundant and high-quality samples. To overcome this issue, we optimized a method that combines primer extension with a fluorescently labeled reverse primer designed on the 3' of 5.8S rRNA sequence and fragment analysis. The resulting electropherogram shows the peaks corresponding to the three isoforms of 5.8S rRNA. The estimation of the area underneath the peaks allows to directly quantify the isoforms and to express their relative abundance. The relative abundance of 5.8S long and 5.8S short remains constant using scalar dilution of RNA and in samples subjected to partial degradation. 5.8S cropped abundance varies significantly in lower concentrate RNA samples. This method allows to analyze rapidly and safely the abundance of 5.8S rRNA isoforms in samples that have been so far considered not suitable such as poorly concentrated samples, RNA derived from frozen tissue or unique samples.

Assessing the Role of Trust in Information Sources, Adoption of Preventive Practices, Volunteering and Degree of Training on Biological Risk Prevention, on Perceived Risk of Infection and Usage of Personal Protective Equipment Among Italian Medical Students During the SARS-CoV-2 Pandemic.

Background: During the initial phase of the COVID-19 pandemic, the University of Bologna Medical School surveyed medical students to learn more about their preparation to confront challenges posed by the pandemic and whether it affects perceptions of viral infection risk. This information could help design risk-reduction interventions with training to mitigate possible viral exposure. Method: A cross-sectional online survey examining students' characteristics, volunteer status, adoption of evidence-based preventive measures, trust in information sources used, infectious disease training, and knowledge of PPE usage in relation to perceived risk of infection from SARS-CoV-2 in daily living, academic, and healthcare activities. A multivariate path model estimated the simultaneous influences of all exogenous factors on perceived risk. A Poisson regression model assessed the same multivariate effects on knowledge of PPE usage. Results: The analysis sample included 537 respondents. Perceived risk of infection was highest in hospital activities. On average, students were able to use only four out of seven types of PPE albeit they adopted most of the evidence-based preventive measures. Adoption of preventive measures was positively associated with perceived risk of COVID infection. Conversely, training on PPE usage and volunteer work were associated with lower perceived risk in healthcare setting and higher PPE knowledge. Conclusion: Implementing early safety-based educational programs remedy students' lack of knowledge in infectious disease prevention and mitigate their risk of infection. Voluntary work should be encouraged with potential benefit for both their continued medical training and strengthening the healthcare system's response to public health emergencies.

MicroRNA expression profiling with a droplet digital PCR assay enables molecular diagnosis and prognosis of cancers of unknown primary.

Metastasis is responsible for the majority of cancer-related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin-fixed paraffin-embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions.

The p53-mediated sensitivity of cancer cells to chemotherapeutic agents is conditioned by the status of the retinoblastoma protein.

Despite the well-established function of p53 in determining cell cycle arrest and/or apoptosis in response to cytostatic/cytotoxic stresses, the role of the p53 status in the response to chemotherapeutic agents in human cancers has been not clearly defined. We wondered whether this was due to the fact that the p53-mediated response to chemotherapy drugs might be conditioned by the status of the retinoblastoma protein (pRb), a downstream factor of the pathway activated by p53 stabilization, which is frequently disrupted in cancer. The dependence of p53-mediated chemosensitivity on pRb status was first investigated in a prospective study on the prognostic relevance of p53 in breast cancer patients treated with adjuvant chemotherapy (5-fluorouracil, methotrexate and cyclophosphamide). Univariate analysis of disease-free survival (DFS) indicated that the p53 status, immunohistochemically evaluated, had no predictive value if considered independently of the pRb status. However, in patients with cancer with pRb neither lost nor hyperphosphorylated, p53 was significantly associated with the prognosis and, in a multivariate analysis of DFS including the established clinical and histopathological prognostic parameters, was found to be the only factor predicting the progression of the disease. We then studied the role of pRb status in the p53-mediated response to 5-fluorouracil and methotrexate or doxorubicin treatment in three human cancer cell lines. We found that in these cells the chemosensitivity was strictly dependent on the p53 status. However, either RB1 silencing or pRb hyperphosphorylation, caused by p16(INK4a) silencing, strongly reduced the p53-mediated response to chemotherapeutic agents. These results demonstrated that: (a) the p53-mediated response to chemotherapeutic agents induces a cytostatic/cytotoxic effect only in cancers with unaltered pRb pathway; and (b) the p53 status can actually predict the clinical outcome in this group of cancer patients.

Separated Siamese Twins: Intronic Small Nucleolar RNAs and Matched Host Genes May be Altered in Conjunction or Separately in Multiple Cancer Types.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs involved in RNA modification and processing. Approximately half of the so far identified snoRNA genes map within the intronic regions of host genes, and their expression, as well as the expression of their host genes, is dependent on transcript splicing and maturation. Growing evidence indicates that mutations and/or deregulations that affect snoRNAs, as well as host genes, play a significant role in oncogenesis. Among the possible factors underlying snoRNA/host gene expression deregulation is copy number alteration (CNA). We analyzed the data available in The Cancer Genome Atlas database, relative to CNA and expression of 295 snoRNA/host gene couples in 10 cancer types, to understand whether the genetic or expression alteration of snoRNAs and their matched host genes would have overlapping trends. Our results show that, counterintuitively, copy number and expression alterations of snoRNAs and matched host genes are not necessarily coupled. In addition, some snoRNA/host genes are mutated and overexpressed recurrently in multiple cancer types. Our findings suggest that the differential contribution to cancer development of both snoRNAs and host genes should always be considered, and that snoRNAs and their host genes may contribute to cancer development in conjunction or independently.

Combined expression levels of KDM2A and KDM2B correlate with nucleolar size and prognosis in primary breast carcinomas.

Ribosome biogenesis is a fine-tuned cellular process and its deregulation is linked to cancer progression: tumors characterized by an intense ribosome biogenesis often display a more aggressive behavior. Ribosomal RNA (rRNA) synthesis is controlled at several levels, the higher one being the epigenetic regulation of the condensation of chromatin portions containing rRNA genes. KDM2A and KDM2B (Lysine (K)-specific demethylase 2A / B) are histone demethylases modulating the accessibility of ribosomal genes, thereby regulating their transcription. Both enzymes are able to demethylate lysins at relevant sites (e.g. K4, K36) on histone H3. We previously demonstrated that KDM2B is one of the factors regulating ribosome biogenesis in human breast cancer. In this study we aimed to define the combined contribution of KDM2A and KDM2B to breast cancer outcome. KDM2A and KDM2B mRNA levels, nucleolar area as a marker of ribosome biogenesis, and patients' prognosis were retrospectively assessed in a series of primary breast carcinomas. We observed that tumors characterized by reduced levels of both KDM2A and KDM2B displayed a particularly aggressive clinical behavior and increased nucleolar size. Our results suggest that KDM2A and KDM2B may cooperate in regulating ribosome biogenesis thus influencing the biological behavior and clinical outcome of human breast cancers.

Direct Antiviral Treatments for Hepatitis C Virus Have Off-Target Effects of Oncologic Relevance in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: HCV eradication by direct-acting antiviral agents (DAAs) reduces de novo hepatocellular carcinoma (HCC) incidence in cirrhosis; however, contrasting evidence about beneficial or detrimental effects still exists in patients who have already developed HCC. METHODS: we investigated whether sofosbuvir and daclatasvir modulate cell proliferation, invasion capability and gene expression (RNA-seq) in HCC-derived cell lines, hypothesizing possible off-target effects of these drugs. Results observed in HCC cell lines were validated in non-HCC cancer-derived cell lines and a preliminary series of human HCC tissues by qPCR and IHC. RESULTS: DAAs can affect HCC cell proliferation and migration capability by either increasing or reducing them, showing transcriptomic changes consistent with some unexpected drug-associated effects. Off-target gene modulation, mainly affecting ribosomal genes, mitochondrial functions and histones, points to epigenetics and proliferation as relevant events, consistent with matched phenotypic changes. A preliminary validation of in vitro findings was performed in a restricted cohort of HCC patients previously treated with DAAs, with immunohistochemical correlations suggesting DAA-treated HCCs to be more aggressive in terms of migration and epidermal-to-mesenchymal transition. CONCLUSIONS: Our findings suggested the possible occurrence of off-target effects ultimately modulating cell proliferation and/or migration and potentially justified previous findings showing some instances of particularly aggressive HCC recurrence as well as reduced incidence of recurrence of HCC following treatment with DAAs.