Development and validation of a prognostic model based on RNA binding proteins in patients with esophageal cancer.

Background: RNA-binding proteins (RBPs) play a crucial role in regulating RNA turnover and are associated with cancer development. However, little is known about the role of RBPs in esophageal cancer (ESCA). The present study focuses on the association between RBP gene expression and survival in ESCA, addressing the clinical relevance of an RBPs-based prediction model for prognosis. Methods: RNA-sequencing data and clinical information of patients with ESCA were obtained from The Cancer Genome Atlas (TCGA) database. We identified differentially expressed genes in ESCA and intersected them with RBP-encoding genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed with the identified differentially expressed RBPs. Then, a protein-protein interaction (PPI) network was constructed through the STRING database to determine the hub RBPs. Univariate Cox regression analysis and multivariate Cox regression analysis were applied to construct a novel prognostic model based on RBPs. Based on the R package "Caret", we divided patients into the training set and validation set. The efficacy of the prognostic model was evaluated by the area under the receiver operating characteristic (ROC) curve. A nomogram was developed for the prediction of patient survival outcomes. Results: A total of 158 ESCA patients from the TCGA database were included in our analysis. We screened out five prognostic RBPs (CLK1, CIRBP, MRPL13, TNRC6A, and TYW3) through univariate and multivariate Cox regression analysis. CLK1, CIRBP, TNRC6A and TYW3 were downregulated in tumor samples, while MRPL13 was upregulated. A prognostic model constructed with these five RBPs in the training data set accurately stratified ESCA patients into high- and low-risk groups. When the same prognostic model was applied to the test data set and entire cohort, the 5-RBP signature remained an independent prognostic factor in multivariate analysis. The areas under the time-dependent ROC curve of the prognostic model for predicting one-year survival in the training data set, test data set, and entire cohort were 0.789, 0.753, and 0.764, respectively, confirming that this model is a good prognostic model. The nomogram based on the five RBPs and clinical variables could improve individualized outcome predictions and highlight the importance of RBPs in the outcomes of patients with ESCA. Conclusions: Our study provides a potential prognostic model for predicting the prognosis of ESCA patients. The prognostic nomogram could improve individualized outcome predictions for patients with ESCA, therefore providing novel insights into future diagnosis and treatment.

Correction: Sepe et al. The Long Non-Coding RNA RP5-1024C24.1 and Its Associated-Gene MPPED2 Are Down-Regulated in Human Thyroid Neoplasias and Act as Tumour Suppressors. Cancers 2018, 10, 146.

In the original publication [...].

The lncRNA RMST is drastically downregulated in anaplastic thyroid carcinomas where exerts a tumor suppressor activity impairing epithelial-mesenchymal transition and stemness.

Thyroid cancer is the most prevalent endocrine malignancy and comprises a wide range of lesions subdivided into differentiated (DTC) and undifferentiated thyroid cancer (UTC), mainly represented by the anaplastic thyroid carcinoma (ATC). This is one of the most lethal malignancies in humankind leading invariably to patient death in few months. Then, a better comprehension of the mechanisms underlying the development of ATC is required to set up new therapeutic approaches. Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides in length that do not code for proteins. They show a strong regulatory function at both transcriptional and post-transcriptional level and are emerging as key players in regulating developmental processes. Their aberrant expression has been linked to several biological processes, including cancer, making them potential diagnostic and prognostic markers. We have recently analyzed the lncRNA expression profile in ATC through a microarray technique and have identified rhabdomyosarcoma 2-associated transcript (RMST) as one of the most downregulated lncRNA in ATC. RMST has been reported to be deregulated in a series of human cancers, to play an anti-oncogenic role in triple-negative breast cancer, and to modulate neurogenesis by interacting with SOX2. Therefore, these findings prompted us to investigate the role of RMST in ATC development. In this study we show that RMST levels are strongly decreased in ATC, but only slightly in DTC, indicating that the loss of this lncRNA could be related to the loss of the differentiation and high aggressiveness. We also report a concomitant increase of SOX2 levels in the same subset of ATC, that inversely correlated with RMST levels, further supporting the RMST/SOX2 relationship. Finally, functional studies demonstrate that the restoration of RMST in ATC cells reduces cell growth, migration and the stemness properties of ATC stem cells. In conclusion, these findings support a critical role of RMST downregulation in ATC development.

Differential Expression of LncRNA in Bladder Cancer Development.

Bladder cancer (BC) is the tenth most common cancer, with urothelial carcinoma representing about 90% of all BC, including neoplasms and carcinomas of different grades of malignancy. Urinary cytology has a significant role in BC screening and surveillance, although it has a low detection rate and high dependence on the pathologist's experience. The currently available biomarkers are not implemented into routine clinical practice due to high costs or low sensitivity. In recent years, the role of lncRNAs in BC has emerged, even though it is still poorly explored. We have previously shown that the lncRNAs Metallophosphoesterase Domain-Containing 2 Antisense RNA 1 (MPPED2-AS1), Rhabdomyosarcoma-2 Associated Transcript (RMST), Kelch-like protein 14 antisense (Klhl14AS) and Prader Willi/Angelman region RNA 5 (PAR5) are involved in the progression of different types of cancers. Here, we investigated the expression of these molecules in BC, first by interrogating the GEPIA database and observing a different distribution of expression levels between normal and cancer specimens. We then measured them in a cohort of neoplastic bladder lesions, either benign or malignant, from patients with suspicion of BC undergoing transurethral resection of bladder tumor (TURBT). The total RNA from biopsies was analyzed using qRT-PCR for the expression of the four lncRNA genes, showing differential expression of the investigated lncRNAs between normal tissue, benign lesions and cancers. In conclusion, the data reported here highlight the involvement of novel lncRNAs in BC development, whose altered expression could potentially affect the regulatory circuits in which these molecules are involved. Our study paves the way for testing lncRNA genes as markers for BC diagnosis and/or follow-up.

Microsatellite instability evaluation of patients with solid tumour: routine practice insight from a large series of Italian referral centre.

DNA mismatch repair complex is involved in the maintenance of DNA stability. In the recent years, a plethora of technical approaches for microsatellite instability (MSI) analysis emerged. Here, we review the results of our MSI status evaluation by adopting a customised workflow on microfluidic system obtained in 4 years of diagnostic routine practice. Data from MSI status were retrieved from our institutional archive covering the period from January 2017 to December 2021. Microfluidic analysis was carried out on microfluidic platform. Results were inspected with a proprietary software. Overall, microsatellite stability (MSS) and MSI-high (MSI-H) profile was detected in n=423/458 (92.36%) and n=35/458 (7.64%) patients with metastatic CRC (mCRC), respectively. In addition, n=78/86 (90.70%) and n=8/86 (9.30%) patients without CRC showed an MSS and MSI-H profile. This review highlights the suitability of microfluidic approach in patients with cancer for MSI testing.

Drug-induced inhibition of HMGA and EZH2 activity as a possible therapy for anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal neoplasms in humans, and just limited progresses have been made to extend patient survival and decrease ATC-associated mortality. Thus, the identification of novel therapeutic strategies for treating ATC is needed. Recently, our group has identified two proteins with oncogenic activity, namely HMGA1 and EZH2, with pivotal roles in ATC cancer progression. Therefore, we tested the ability of trabectedin, a HMGA1-targeting drug, and GSK126, an inhibitor of EZH2 enzymatic activity, to impair cell viability of four ATC-derived cell lines. In the present study, we first confirmed the overexpression of HMGA1 and EZH2 in all ATC-derived cell lines and tissues compared to the normal primary thyroid cells and tissues. Then, treatment of the ATC cell lines with trabectedin and GSK126 resulted in a drastic induction of apoptotic cell death, which increased when the ATC cell lines were treated with a combination of both drugs. Conversely, normal primary human thyroid cells did not show any significant reduction in their viability when exposed to the same drugs. Noteworthy, both drugs induced the deregulation of EZH2- and HMGA1-controlled genes. Altogether, these findings propose the combination of trabectedin and GSK126 as possible novel strategy for ATC therapy.

Noncoding RNAs in Thyroid-Follicular-Cell-Derived Carcinomas.

Among the thyroid neoplasias originating from follicular cells, we can include well-differentiated carcinomas, papillary (PTC) and follicular (FTC) thyroid carcinomas, and the undifferentiated anaplastic (ATC) carcinomas. Several mutations in oncogenes and tumor suppressor genes have already been observed in these malignancies; however, we are still far from the comprehension of their full regulation-altered landscape. Even if only 2% of the human genome has the ability to code for proteins, most of the noncoding genome is transcribed, constituting the heterogeneous class of noncoding RNAs (ncRNAs), whose alterations are associated with the development of several human diseases, including cancer. Hence, many scientific efforts are currently focused on the elucidation of their biological role. In this review, we analyze the scientific literature regarding the involvement of microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and pseudogenes in FTC, PTC, and ATC. Recent findings emphasized the role of lncRNAs in all steps of cancer progression. In particular, lncRNAs may control progression steps by regulating the expression of genes and miRNAs involved in cell proliferation, apoptosis, epithelial-mesenchymal transition, and metastatization. In conclusion, the determination of the diagnosis, prognosis, and treatment of cancer based on the evaluation of the ncRNA network could allow the implementation of a more personalized approach to fighting thyroid tumors.

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.].

The role of HMGA1 protein in gastroenteropancreatic neuroendocrine tumors.

Neuroendocrine tumors (NETs) are neoplasms derived from neuroendocrine cells. One of their main features is to often remain asymptomatic and clinically undetectable. High Mobility Group A (HMGA) proteins belong to a family of non-histone chromatinic proteins able to modulate gene expression through the interaction with DNA and transcription factors. They are overexpressed in most of the human malignancies, playing a critical role in carcinogenesis. However, their expression levels and their role in neuroendocrine carcinogenesis has not been exhaustively evaluated until now. Therefore, in this study, we have addressed the validity of using the expression of HMGA1 as a diagnostic marker and have investigated its role in NET carcinogenesis. The expression of HMGA1 has been evaluated by qRT-PCR and immunohistochemistry, using NET tissue microarrays, in a cohort of gastroenteropancreatic (GEP)-NET samples. The expression levels of HMGA1 have been then correlated with the main clinical features of NET samples. Finally, the contribution of HMGA1 overexpression to NET development has been addressed as far as the modulation of proliferation and migration abilities of NET cells is concerned. Here, we report that HMGA1 is overexpressed in GEP-NET samples, at both mRNA and protein levels, and that the silencing of HMGA1 protein expression interferes with the ability of NET cells to proliferate and migrate through the downregulation of Cyclin E, Cyclin B1 and EZH2. These results propose the HMGA proteins as new diagnostic and prognostic markers.

BRCA1/2 NGS Somatic Testing in Clinical Practice: A Short Report.

High-grade serous ovarian carcinoma (HGSOC) is the most common subtype of all ovarian carcinomas. HGSOC harboring BRCA1/2 germline or somatic mutations are sensitive to the poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi). Therefore, detecting these mutations is crucial to identifying patients for PARPi-targeted treatment. In the clinical setting, next generation sequencing (NGS) has proven to be a reliable diagnostic approach BRCA1/2 molecular evaluation. Here, we review the results of our BRCA1/2 NGS analysis obtained in a year and a half of diagnostic routine practice. BRCA1/2 molecular NGS records of HGSOC patients were retrieved from our institutional archive covering the period from January 2020 to September 2021. NGS analysis was performed on the Ion S5™ System (Thermo Fisher Scientific, Waltham, MA, USA) with the Oncomine™ BRCA Research Assay panel (Thermo Fisher Scientific). Variants were classified as pathogenic or likely pathogenic according to the guidelines of the American College of Medical Genetics and Genomics by using the inspection of Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) and ClinVar (NCBI) databases. Sixty-five HGSOC patient samples were successfully analyzed. Overall, 11 (16.9%) out of 65 cases harbored a pathogenic alteration in BRCA1/2, in particular, six BRCA1 and five BRCA2 pathogenic variations. This study confirms the efficiency and high sensitivity of NGS analysis in detecting BRCA1/2 germline or somatic variations in patients with HGSOC.

Liquid Biopsy Is a Promising Tool for Genetic Testing in Idiopathic Pulmonary Fibrosis.

Liquid biopsy, which allows the isolation of circulating cell-free (ccf) DNA from blood, is an emerging noninvasive tool widely used in oncology for diagnostic and prognosis purposes. Previous data have shown that serum cfDNA discriminates idiopathic pulmonary fibrosis (IPF) from other interstitial lung diseases. Our study aimed to measure plasma levels of ccfDNA in 59 consecutive therapy-naive and clinically stable IPF patients. The single nucleotide polymorphism (SNP) of the MUC5B gene promoter (rs35705950), associated with increased susceptibility of developing IPF, has been sought in plasma cfDNA and genomic DNA for comparison. Thirty-five age- and sex-matched healthy volunteers were recruited as the control group. Our results show that concentrations of small-size ccfDNA fragments were significantly higher in IPF patients than in controls and inversely correlated with lung function deterioration. Moreover, the median level of 104 ng/mL allowed discriminating patients with mild disease from those more advanced. The rs35705950 polymorphism was found in 11.8% of IPF patients and 8% of controls, with no differences. Complete concordance between ccfDNA and genomic DNA was detected in all control samples, while four out of seven IPF cases (57%) carrying the rs35705950 polymorphism were discordant from genomic DNA (7% of total IPF). Liquid biopsy is a suitable tool with optimistic expectations of application in the field of IPF. In analogy with cancer biology, finding some discrepancies between ccfDNA and genomic DNA in IPF patients suggests that the former may convey specific genetic information present in the primary site of the disease.

MPPED2 is downregulated in glioblastoma, and its restoration inhibits proliferation and increases the sensitivity to temozolomide of glioblastoma cells.

Glioblastoma (GBM) is the most aggressive and lethal neoplasia of the central nervous system in adults. Based on the molecular signature genes, GBM has been classified in proneural, neural, mesenchymal and classical subtypes. The Metallophosphoesterase-domain-containing protein 2 (MPPED2) gene encodes a metallophosphodiesterase protein highly conserved throughout the evolution. MPPED2 downregulation, likely due to its promoter hypermethylation, has been found in several malignant neoplasias and correlated with a poor prognosis. In this study, we aimed to investigate the expression and the functional role of MPPED2 in GBM. TCGA and Gravendeel databases were employed to explore the MPPED2 expression levels in this type of tumor. We have found that MPPED2 expression is downregulated in GBM patients, showing a positive correlation with survival. Moreover, TCGA and Gravendeel data also revealed that MPPED2 expression negatively correlates with the most aggressive mesenchymal subtype. Additionally, the restoration of MPPED2 expression in U251 and GLI36 GBM cell lines decreases cell growth, migration and enhanced the sensitivity to the temozolomide, inducing apoptotic cell death, of GBM cells. These findings suggest that the restoration of MPPED2 function can be taken into consideration for an innovative GBM therapy.

Long non-coding RNAs regulating multiple proliferative pathways in cancer cell.

Long non-coding RNAs (lncRNAs) belong to an extremely heterogeneous class of non-coding RNAs with a length ranging from 200 to 100,000 bp. They modulate a series of cellular pathways in both physiological and pathological context. It is no coincidence that they are expressed in an aberrant way in pathologies such as cancer, so as to deserve to be subclassified as oncogenes or tumor suppressors. These molecules are also involved in the regulation of cancer cell proliferation. Several lncRNAs are able to modulate cell growth both positively and negatively, and in this review we have focused on a small group of them, characterized by the simultaneous action on different pathways regulating cell proliferation. They have been considered in the light of their behavior in three different subtypes of proliferative pathways that we can define as (I) tumor suppressor, (II) oncogenic and (III) transcriptionally-driven. More specifically, we have characterized some lncRNAs considered oncogenes (such as H19, linc-ROR, MALAT1, HULC, HOTAIR and ANRIL), tumor suppressors (such as MEG3 and lincRNA-p21), and both oncogenes/tumor suppressors (UCA1 and TUG1) in a little more detail. As can be understood from the review, the interactions between lncRNAs and their molecular targets, only in the context of controlling cell proliferation, give rise to an intricate molecular network, the understanding of which, in the future, will certainly be of help for the treatment of molecular diseases such as cancer.

The Long Non-Coding RNA Prader Willi/Angelman Region RNA5 (PAR5) Is Downregulated in Anaplastic Thyroid Carcinomas Where It Acts as a Tumor Suppressor by Reducing EZH2 Activity.

Anaplastic thyroid carcinoma (ATC) represents one the most aggressive neoplasias in humans, and, nowadays, limited advances have been made to extend the survival and reduce the mortality of ATC. Thus, the identification of molecular mechanism underlying its progression is needed. Here, we evaluated the long non-coding RNA (lncRNA) expression profile of nine ATC in comparison with five normal thyroid tissues by a lncRNA microarray. By this analysis, we identified 19 upregulated and 28 downregulated lncRNAs with a fold change >1.1 or <-1.1 and p-value < 0.05, in ATC samples. Some of them were subsequently validated by qRT-PCR. Then, we investigated the role of the lncRNA Prader Willi/Angelman region RNA5 (PAR5), drastically and specifically downregulated in ATC. The restoration of PAR5 reduces proliferation and migration rates of ATC-derived cell lines indicating that its downregulation contributes to thyroid cancer progression. Our results suggest that PAR5 exerts its anti-oncogenic role by impairing Enhancer of Zeste Homolog 2 (EZH2) oncogenic activity since we demonstrated that PAR5 interacts with it in thyroid cancer cell lines, reducing EZH2 protein levels and its binding on the E-cadherin promoter, relieving E-cadherin from the negative regulation by EZH2. Consistently, EZH2 is overexpressed in ATC, but not in differentiated thyroid carcinomas. The results reported here define a tumor suppressor role for PAR5 in undifferentiated thyroid neoplasias, further highlighting the pivotal role of lncRNAs in thyroid carcinogenesis.

The Metallophosphoesterase-Domain-Containing Protein 2 (MPPED2) Gene Acts as Tumor Suppressor in Breast Cancer.

Background: We have recently reported the downregulation of the Metallophosphoesterase-domain-containing protein 2 (MPPED2) gene and its cognate long non-coding RNA, MPPED2-AS1, in papillary thyroid carcinomas. Functional studies supported a tumor suppressor role of both these genes in thyroid carcinogenesis. We then decided to investigate their role in breast carcinogenesis. Methods: In order to verify MPPED2 expression, 45 human breast carcinoma samples have been investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Then, MPPED2 has been transfected in several human breast carcinoma cell lines, analyzing its role in cell proliferation, migration and invasion. To study the regulation of MPPED2 expression the methylation of its promoter was investigated by targeted bisulfite sequencing. Results: MPPED2 expression was decreased in breast cancer samples, and this was confirmed by the analysis of data available in The Cancer Genome Atlas (TCGA). Interestingly, the hypermethylation of MPPED2 promoter likely accounted for its downregulation in breast cancer. Additionally, MPPED2-AS1 was also found downregulated in breast cancer tissues and, intriguingly, its expression decreased the hypermethylation of the MPPED2 promoter by inhibiting DNA methyltransferase 1 (DNMT1). Furthermore, the restoration of MPPED2 expression reduced cell proliferation, migration and invasion capability of breast carcinoma cell lines. Conclusion: Taken together, these results propose MPPED2 downregulation as a critical event in breast carcinogenesis.

Oncogenic Properties of the Antisense lncRNA COMET in BRAF- and RET-Driven Papillary Thyroid Carcinomas.

RET rearrangements as well as BRAF and RAS mutations drive differential pathway activation in papillary thyroid carcinomas, leading to different tumor phenotypes and prognoses. Although The Cancer Genome Atlas Consortium has identified tumor subgroups based on protein-coding gene signatures, neither expression of long noncoding RNAs (lncRNA) nor their correlation with specific tumor-driving mutations and rearrangements have been systematically assessed. Here, we reanalyzed our RNA-sequencing data using a de novo discovery approach to identify lncRNAs and define tumor subtype-specific signatures of annotated lncRNAs. Among them, we identified COMET (Correlated-to-MET), a natural antisense transcript that was highly expressed in carcinomas harboring BRAF V600E mutation or RET gene rearrangements (i.e., BRAF-like tumors) and induced the downstream MAPK pathway. In papillary thyroid carcinomas, COMET was part of a coexpression network including different oncogenes belonging to the MAPK pathway, and its expression highly correlated with MET expression. Depletion of COMET resulted in reduced expression of genes within this network, including the MET oncogene. COMET repression inhibited viability and proliferation of tumor cells harboring BRAF V600E somatic mutation or RET oncogene rearrangement and dramatically reduced motility and invasiveness of tumor cells. Moreover, silencing COMET markedly increased sensitivity to vemurafenib, a common inhibitor of mutated B-raf. Collectively, our results suggest COMET as a new target to improve drug-based cancer therapies, especially in BRAF-mutated and MET-addicted papillary thyroid carcinomas. SIGNIFICANCE: These results highlight the oncogenic role of lncRNA COMET in thyroid and indicate it as a potential new target to overcome vemurafenib resistance in BRAF-mutated and MET-addicted carcinomas.

Circulating Tumour Cells in Predictive Molecular Pathology: Focus on Drug-Sensitive Assays and 3D Culture.

Molecular cytopathology is a rapidly evolving field of cytopathology that provides biological information about the response to personalised therapy and about the prognosis of neoplasms diagnosed on cytological samples. Biomarkers such as circulating tumour cells and circulating tumour DNA are increasingly being evaluated in blood and in other body fluids. Such liquid biopsies are non-invasive, repeatable, and feasible also in patients with severe comorbidities. However, liquid biopsy may be challenging due to a low concentration of biomarkers. In such cases, biomarkers can be detected with highly sensitive molecular techniques, which in turn should be validated and integrated in a complex algorithm that includes tissue-based molecular assessments. The aim of this review is to provide the cytopathologist with practical information that is relevant to daily practice, particularly regarding the emerging role of circulating tumour cells in the field of predictive molecular pathology.

Setting up and exploitation of a nano/technological platform for the evaluation of HMGA1b protein in peripheral blood of cancer patients.

Even if cancer specific biomarkers are present in peripheral blood of cancer patients, it is very difficult to detect them with conventional technology because of their low concentration. A potential cancer biomarker is the HMGA1b protein, whose overexpression is a feature of several human malignant neoplasias. By taking advantage of the surface plasmon resonance (SPR) phenomenon, we realized a specific nano/technology-based assay for cancer detection. More in details, anti-HMGA1b monoclonal antibodies, whose affinity was previously defined by ELISA, were immobilized onto metallic surfaces to develop a direct SPR-based assay. After having analyzed blood samples from colorectal cancer patients and healthy people for the presence of HMGA1b, we observed a 2-fold increase of the HMGA1b levels in the blood of cancer patients with respect to the healthy control people. We conclude that the set-up technology might allow to detect a tumoral mass through the evaluation of HMGA1b protein blood levels.