Showing papers by "Carlo M. Croce published in 2017"

Chronic lymphocytic leukaemia.

Abstract: Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5+ B cells that is characterized by the accumulation of small, mature-appearing lymphocytes in the blood, marrow and lymphoid tissues. Signalling via surface immunoglobulin, which constitutes the major part of the B cell receptor, and several genetic alterations play a part in CLL pathogenesis, in addition to interactions between CLL cells and other cell types, such as stromal cells, T cells and nurse-like cells in the lymph nodes. The clinical progression of CLL is heterogeneous and ranges from patients who require treatment soon after diagnosis to others who do not require therapy for many years, if at all. Several factors, including the immunoglobulin heavy-chain variable region gene (IGHV) mutational status, genomic changes, patient age and the presence of comorbidities, should be considered when defining the optimal management strategies, which include chemotherapy, chemoimmunotherapy and/or drugs targeting B cell receptor signalling or inhibitors of apoptosis, such as BCL-2. Research on the biology of CLL has profoundly enhanced our ability to identify patients who are at higher risk for disease progression and our capacity to treat patients with drugs that selectively target distinctive phenotypic or physiological features of CLL. How these and other advances have shaped our current understanding and treatment of patients with CLL is the subject of this Primer.

Small non-coding RNA and cancer

Abstract: The ENCODE project has reported that at least 80% of the human genome is biologically active, yet only a small part of human DNA encodes for protein. The massive amount of RNA transcribed but not translated into protein can be classified as housekeeping RNA (such as rRNA, tRNA) and regulatory RNA (such as miRNA, piRNA, lncRNA). Small non-coding RNAs, in particular, have been the focus of many studies in the last 20 years and their fundamental role in many human diseases is currently well established. Inter alia, their role in cancer development and progression, as well as in drug resistance, is being increasingly investigated. In this review, focusing our attention on recent research results, we provide an overview of the four large classes of small non-coding RNAs, namely, miRNAs, piRNAs, snoRNA and the new class of tRNA-derived fragments, highlighting their fundamental role in cancer and their potential as diagnostic and prognostic biomarkers.

tsRNA signatures in cancer.

Abstract: Small, noncoding RNAs are short untranslated RNA molecules, some of which have been associated with cancer development. Recently we showed that a class of small RNAs generated during the maturation process of tRNAs (tRNA-derived small RNAs, hereafter “tsRNAs”) is dysregulated in cancer. Specifically, we uncovered tsRNA signatures in chronic lymphocytic leukemia and lung cancer and demonstrated that the ts-4521/3676 cluster (now called “ts-101” and “ts-53,” respectively), ts-46, and ts-47 are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that ts-101 and ts-53 can associate with PiwiL2, a protein involved in the silencing of transposons. In this study, we extended our investigation on tsRNA signatures to samples collected from patients with colon, breast, or ovarian cancer and cell lines harboring specific oncogenic mutations and representing different stages of cancer progression. We detected tsRNA signatures in all patient samples and determined that tsRNA expression is altered upon oncogene activation and during cancer staging. In addition, we generated a knocked-out cell model for ts-101 and ts-46 in HEK-293 cells and found significant differences in gene-expression patterns, with activation of genes involved in cell survival and down-regulation of genes involved in apoptosis and chromatin structure. Finally, we overexpressed ts-46 and ts-47 in two lung cancer cell lines and performed a clonogenic assay to examine their role in cell proliferation. We observed a strong inhibition of colony formation in cells overexpressing these tsRNAs compared with untreated cells, confirming that tsRNAs affect cell growth and survival.

Exosome-Derived miR-25-3p and miR-92a-3p Stimulate Liposarcoma Progression.

Abstract: Despite the development of combined modality treatments against liposarcoma in recent years, a significant proportion of patients respond only modestly to such approaches, possibly contributing to local or distant recurrence. Early detection of recurrent or metastatic disease could improve patient prognosis by triggering earlier clinical intervention. However, useful biomarkers for such purposes are lacking. Using both patient plasma samples and cell lines, we demonstrate here that miR-25-3p and miR-92a-3p are secreted by liposarcoma cells through extracellular vesicles and may be useful as potential biomarkers of disease. Both miR-25-3p and miR-92a-3p stimulated secretion of proinflammatory cytokine IL6 from tumor-associated macrophages in a TLR7/8-dependent manner, which in turn promoted liposarcoma cell proliferation, invasion, and metastasis via this interaction with the surrounding microenvironment. Our findings provide novel and previously unreported insight into liposarcoma progression, identifying communication between liposarcoma cells and their microenvironment as a process critically involved in liposarcoma progression. This study establishes the possibility that the pattern of circulating miRNAs may identify recurrence prior to radiological detectability while providing insight into disease outcome and as a possible approach to monitor treatment efficacy. Cancer Res; 77(14); 3846-56. ©2017 AACR.

MicroRNAs in melanoma development and resistance to target therapy

Abstract: // Luigi Fattore 1 , Susan Costantini 2 , Debora Malpicci 3 , Ciro Francesco Ruggiero 3 , Paolo Antonio Ascierto 1 , Carlo M. Croce 4 , Rita Mancini 5 and Gennaro Ciliberto 1,6 1 Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione G. Pascale”, Naples, Italy 2 CROM, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy 3 Dipartimento di Medicina Sperimentale e Clinica, Universita degli Studi di Catanzaro “Magna Graecia”, Catanzaro, Italy 4 Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA 5 Dipartimento di Medicina Clinica e Molecolare, Sapienza Universita di Roma, Rome, Italy 6 IRCCS Istituto Nazionale Tumori “Regina Elena”, Rome, Italy Correspondence to: Gennaro Ciliberto, email: // Keywords : melanoma, miRNA, target therapy, drug resistance, intracellular pathways Received : August 04, 2016 Accepted : January 10, 2017 Published : January 19, 2017 Abstract microRNAs constitute a complex class of pleiotropic post-transcriptional regulators of gene expression involved in the control of several physiologic and pathologic processes. Their mechanism of action is primarily based on the imperfect matching of a seed region located at the 5’ end of a 21-23 nt sequence with a partially complementary sequence located in the 3’ untranslated region of target mRNAs. This leads to inhibition of mRNA translation and eventually to its degradation. Individual miRNAs are capable of binding to several mRNAs and several miRNAs are capable of influencing the function of the same mRNAs. In recent years networks of miRNAs are emerging as capable of controlling key signaling pathways responsible for the growth and propagation of cancer cells. Furthermore several examples have been provided which highlight the involvement of miRNAs in the development of resistance to targeted drug therapies. In this review we provide an updated overview of the role of miRNAs in the development of melanoma and the identification of the main downstream pathways controlled by these miRNAs. Furthermore we discuss a group of miRNAs capable to influence through their respective up- or down-modulation the development of resistance to BRAF and MEK inhibitors.

MicroRNAs and Cancer: A Long Story for Short RNAs

Abstract: More than six decades ago Watson and Crick published the chemical structure of DNA. This discovery revolutionized our approach to medical science and opened new perspectives for the diagnosis and treatment of many diseases including cancer. Since then, progress in molecular biology, together with the rapid advance of technologies, allowed to clone hundreds of protein-coding genes that were found mutated in all types of cancer. Normal and aberrant gene functions, interactions, and mechanisms of mutations were studied to identify the intricate network of pathways leading to cancer. With the acknowledgment of the genetic nature of cancer, new diagnostic, prognostic, and therapeutic strategies have been attempted and developed, but very few have found their way in the clinical field. In an effort to identify new translational targets, another great discovery has changed our way to look at genes and their functions. MicroRNAs have been the first noncoding genes involved in cancer. This review is a brief chronological history of microRNAs and cancer. Through the work of few of the greatest scientists of our times, this chapter describes the discovery of microRNAs from C. elegans to their debut in cancer and in the medical field, the concurrent development of technologies, and their future translational applications. The purpose was to share the exciting path that lead to one of the most important discoveries in cancer genetics in the past 20 years.

Role of the tRNA-Derived Small RNAs in Cancer: New Potential Biomarkers and Target for Therapy

Abstract: Noncoding RNAs are untranslated RNA molecules that can be divided into two main types: infrastructural, including transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), and regulatory, including long ncRNAs (lncRNAs) and small ncRNAs (sRNA). Among small ncRNA, the role of microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) in cancer is well documented. Recently, other small ncRNAs have been described. In particular, tRNA-derived small RNAs (tsRNA) have been found to be frequently dysregulated in cancer. Since tsRNAs can be considered unique sequences and are able to bind both Argonaute proteins (like miRNAs) and Piwi proteins (like piRNAs), their dysregulation could play a critical role in cancer by interfering with gene expression regulation at different levels. Like microRNAs, ts-53 (previously known as miR-3676) interacts with the 3'UTR of TCL1, therefore supporting a role for tsRNAs on the posttranscriptional regulation of gene expression. Like piRNAs, tsRNAs are produced as single-stranded molecules and can interact with DNA and histone methylation machinery, suggesting a role in the pretranscriptional regulation of gene expression. Herein, we describe the most recent findings about the role of tsRNAs in cancer.

HIF-1α promotes autophagic proteolysis of Dicer and enhances tumor metastasis

Abstract: HIF-1α, one of the most extensively studied oncogenes, is activated by a variety of microenvironmental factors. The resulting biological effects are thought to depend on its transcriptional activity. The RNAse enzyme Dicer is frequently downregulated in human cancers, which has been functionally linked to enhanced metastatic properties; however, current knowledge of the upstream mechanisms regulating Dicer is limited. In the present study, we identified Dicer as a HIF-1α-interacting protein in multiple types of cancer cell lines and different human tumors. HIF-1α downregulated Dicer expression by facilitating its ubiquitination by the E3 ligase Parkin, thereby enhancing autophagy-mediated degradation of Dicer, which further suppressed the maturation of known tumor suppressors, such as the microRNA let-7 and microRNA-200b. Consequently, expression of HIF-1α facilitated epithelial-mesenchymal transition (EMT) and metastasis in tumor-bearing mice. Thus, this study uncovered a connection between oncogenic HIF-1α and the tumor-suppressive Dicer. This function of HIF-1α is transcription independent and occurs through previously unrecognized protein interaction-mediated ubiquitination and autophagic proteolysis.

miR-130a Deregulates PTEN and Stimulates Tumor Growth

Abstract: H-RasV12 oncogene has been shown to promote autophagic cell death. Here, we provide evidence of a contextual role for H-RasV12 in cell death that is varied by its effects on miR-130a. In E1A-immortalized murine embryo fibroblasts, acute expression of H-RasV12 promoted apoptosis, but not autophagic cell death. miRNA screens in this system showed that miR-130a was strongly downregulated by H-RasV12 in this model system. Enforced expression of miR-130a increased cell proliferation in part via repression of PTEN. Consistent with this effect, miR-130a overexpression in human breast cancer cells promoted Akt phosphorylation, cell survival, and tumor growth. In clinical specimens of multiple human cancers, expression of miR-130 family members correlated inversely with PTEN expression. Overall, our results defined miR-130a as an oncogenic miRNA that targets PTEN to drive malignant cell survival and tumor growth. Cancer Res; 77(22); 6168-78. ©2017 AACR.

MicroRNA dysregulation to identify therapeutic target combinations for chronic lymphocytic leukemia.

Abstract: Loss of miR-15/16 is the most common genetic lesion in chronic lymphocytic leukemia (CLL), promoting overexpression of BCL2, which factors in leukemia pathogenesis. Indeed, an inhibitor of Bcl2, venetoclcax, is highly active in the treatment of patients with CLL. However, single-agent venetoclcax fails to eradicate minimal residual disease in most patients. Accordingly, we were interested in other genes that may be regulated by miR-15/16, which may target other drivers in CLL. We found that miR-15/16 targets ROR1, which encodes an onco-embryonic surface protein expressed on the CLL cells of over 90% of patients, but not on virtually all normal postpartum tissues. CLL with high-level expression of ROR1 also have high-level expression of Bcl2, but low-to-negligible miR-15/16. Moreover, CLL cases with high-level ROR1 have deletion(s) at the chromosomal location of the genes encoding miR-15/16 (13q14) more frequently than cases with low-to-negligible ROR1, implying that deletion of miR-15/16 may promote overexpression of ROR1, in addition to BCL2. ROR1 is a receptor for Wnt5a, which can promote leukemia-cell proliferation and survival, and can be targeted by cirmtuzumab, a humanized anti-ROR1 mAb. We find that this mAb can enhance the in vitro cytotoxic activity of venetoclcax for CLL cells with high-level expression of ROR1, indicating that combining these agents, which target ROR1 and Bcl2, may have additive, if not synergistic, activity in patients with this disease.

Immunotherapy Bridge 2016 and Melanoma Bridge 2016: meeting abstracts

Abstract: © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Immunotherapy Bridge 2016: Keynote speaker presentations Immunotherapy beyond melanoma

ROR1 expression as a biomarker for predicting prognosis in patients with colorectal cancer

Abstract: // Jian-Kang Zhou 1, * , Yu-Zhu Zheng 2, 3, * , Xue-Sha Liu 1 , Qiheng Gou 1 , Rui Ma 1 , Cheng-Lin Guo 1 , Carlo M. Croce 4 , Lunxu Liu 1 and Yong Peng 1 1 Department of Thoracic Surgery and Lab of Non-coding RNAs in Diseases, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan, China 2 Department of Thoracic Oncology, Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan, China 3 Department of Oncology, The Third People’s Hospital of Chengdu, Chengdu, Sichuan, China 4 Department of Cancer Biology and Genetics, The Ohio State University, Columbus, OH, USA * These authors have contributed equally to this work Correspondence to: Yong Peng, email: yongpeng@scu.edu.cn Lunxu Liu, email: lunxu_liu@aliyun.com Keywords: receptor-tyrosine-kinase-like orphan receptor 1, colorectal cancer, prognostic factor, tissue microarray, immunohistochemistry Received: November 30, 2016 Accepted: February 07, 2017 Published: March 02, 2017 ABSTRACT There is a lack of reliable prognosis biomarker in the current treatment of colorectal cancer. The receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is overexpressed and associated with poor prognosis in certain tumors. This study aimed to explore the prognostic significance of ROR1 in colorectal cancer. Western blot analysis and immunohistochemistry showed that the expression of ROR1 in colorectal cancer was significantly higher than that in the adjacent normal tissues. ROR1 expression was positively associated with the clinical stage and lymph-node metastasis ( p < 0.01). Kaplan-Meier survival analysis revealed that patients with higher ROR1 expression had a significantly shorter overall survival ( p < 0.01). Multivariate Cox regression analysis confirmed that ROR1 is an independent prognostic marker in colorectal cancer (p = 0.002, HR = 2.08, 95% CI: 1.314–3.292). Thus, our study demonstrated that ROR1 expression is correlated with malignant attributes and may serve as a novel prognostic marker and therapeutic target for colorectal cancer.

Selective targeting of point-mutated KRAS through artificial microRNAs

Abstract: Mutated protein-coding genes drive the molecular pathogenesis of many diseases, including cancer. Specifically, mutated KRAS is a documented driver for malignant transformation, occurring early during the pathogenesis of cancers such as lung and pancreatic adenocarcinomas. Therapeutically, the indiscriminate targeting of wild-type and point-mutated transcripts represents an important limitation. Here, we leveraged on the design of miRNA-like artificial molecules (amiRNAs) to specifically target point-mutated genes, such as KRAS, without affecting their wild-type counterparts. Compared with an siRNA-like approach, the requirement of perfect complementarity of the microRNA seed region to a given target sequence in the microRNA/target model has proven to be a more efficient strategy, accomplishing the selective targeting of point-mutated KRAS in vitro and in vivo.

Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia.

Abstract: Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated Here, we report that levels of both EGFL7 mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells High EGFL7 mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age <60 y) patients with cytogenetically normal AML We further show that AML blasts secrete EGFL7 protein and that higher levels of EGFL7 protein are found in the sera from AML patients than in sera from healthy controls Treatment of patient AML blasts with recombinant EGFL7 in vitro leads to increases in leukemic blast cell growth and levels of phosphorylated AKT EGFL7 blockade with an anti-EGFL7 antibody reduced the growth potential and viability of AML cells Our findings demonstrate that increased EGFL7 expression and secretion is an autocrine mechanism supporting growth of leukemic blasts in patients with AML

An integrated approach identifies mediators of local recurrence in head and neck squamous carcinoma

Abstract: Purpose: Head and neck squamous cell carcinomas (HNSCCs) cause more than 300,000 deaths worldwide each year. Locoregional and distant recurrences represent worse prognostic events and accepted surrogate markers of patients' overall survival. No valid biomarker and salvage therapy exist to identify and treat patients at high-risk of recurrence. We aimed to verify if selected miRNAs could be used as biomarkers of recurrence in HNSCC.Experimental Design: A NanoString array was used to identify miRNAs associated with locoregional recurrence in 44 patients with HNSCC. Bioinformatic approaches validated the signature and identified potential miRNA targets. Validation experiments were performed using an independent cohort of primary HNSCC samples and a panel of HNSCC cell lines. In vivo experiments validated the in vitro results.Results: Our data identified a four-miRNA signature that classified HNSCC patients at high- or low-risk of recurrence. These miRNAs collectively impinge on the epithelial-mesenchymal transition process. In silico and wet lab approaches showed that miR-9, expressed at high levels in recurrent HNSCC, targets SASH1 and KRT13, whereas miR-1, miR-133, and miR-150, expressed at low levels in recurrent HNSCC, collectively target SP1 and TGFβ pathways. A six-gene signature comprising these targets identified patients at high risk of recurrences, as well. Combined pharmacological inhibition of SP1 and TGFβ pathways induced HNSCC cell death and, when timely administered, prevented recurrence formation in a preclinical model of HNSCC recurrence.Conclusions: By integrating different experimental approaches and competences, we identified critical mediators of recurrence formation in HNSCC that may merit to be considered for future clinical development. Clin Cancer Res; 23(14); 3769-80. ©2017 AACR.

Extracellular Vesicle Biology in the Pathogenesis of Lung Disease.

Abstract: As our understanding of diseases of the lung continues to evolve, we recognize that the mechanisms that drive cell/environment communication leading to disease development and progression are complex. In 2008, investigators first identified intact cell free microRNAs in circulation with some localized within lipid-encapsulated microvesicles termed extracellular vesicles (EVs). Prior to and following this discovery, investigators have determined that EVs may drive biological processes fundamental to human disease and homeostasis. The cargo in EVs which includes, proteins, miRNAs and mRNAs is protected from degradation, often reflects the pathogenesis of the cell of origin and may serve as a conduit for communication between cells subsequently reprogramming recipient cell function. Furthermore, EVs have been detected in bodily fluids including blood urine, sputum and bronchoalveolar lavage. The release and shuttling of EVs between cells following both environmental exposure and during disease development and progression has added a new layer of complexity to cell-cell communication in lung disease. While the majority of studies implicating EVs in human biology have been conducted in cancer, we have witnessed an emergence of EV based studies in other physiopathological conditions. EV mediated transfer of genetic content may link environmental stressors to the lung immune response through intercellular communication within the lung and development of disease. This perspective will focus on the growing field of EV biology in human lung diseases and its application to both the development and progression of lung disease and in biomarker discovery.

Anti-leukemic activity of microRNA-26a in a chronic lymphocytic leukemia mouse model.

Abstract: Dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). The Eμ-TCL1 transgenic mouse develops a form of leukemia that is similar to the aggressive type of human B-CLL, and this valuable model has been widely used for testing novel therapeutic approaches. Here, we adopted this model to investigate the potential effects of miR-26a, miR-130an and antimiR-155 in CLL therapy. Improved delivery of miRNA molecules into CLL cells was obtained by developing a novel system based on lipid nanoparticles conjugated with an anti-CD38 monoclonal antibody. This methodology has proven to be highly effective in delivering miRNA molecules into leukemic cells. Short- and long-term experiments showed that miR-26a, miR-130a and anti-miR-155 increased apoptosis after in vitro and in vivo treatment. Of this miRNA panel, miR-26a was the most effective in reducing leukemic cell expansion. Following long-term treatment, apoptosis was readily detectable by analyzing cleavage of PARP and caspase-7. These effects could be directly attributed to miR-26a, as confirmed by significant downregulation of its proven targets, namely cyclin-dependent kinase 6 and Mcl1. The results of this study are relevant to two distinct areas. The first is related to the design of a technical strategy and to the selection of CD38 as a molecular target on CLL cells, both consenting efficient and specific intracellular transfer of miRNA. The original scientific finding inferred from the above approach is that miR-26a can elicit in vivo anti-leukemic activities mediated by increased apoptosis.

Discovery and characterization of the feline miRNAome.

Abstract: The domestic cat is an important human companion animal that can also serve as a relevant model for ~250 genetic diseases, many metabolic and degenerative conditions, and forms of cancer that are analogous to human disorders. MicroRNAs (miRNAs) play a crucial role in many biological processes and their dysregulation has a significant impact on important cellular pathways and is linked to a variety of diseases. While many species already have a well-defined and characterized miRNAome, miRNAs have not been carefully studied in cats. As a result, there are no feline miRNAs present in the reference miRNA databases, diminishing the usefulness of medical research on spontaneous disease in cats for applicability to both feline and human disease. This study was undertaken to define and characterize the cat miRNAome in normal feline tissues. High-throughput sequencing was performed on 12 different normal cat tissues. 271 candidate feline miRNA precursors, encoding a total of 475 mature sequences, were identified, including several novel cat-specific miRNAs. Several analyses were performed to characterize the discovered miRNAs, including tissue distribution of the precursors and mature sequences, genomic distribution of miRNA genes and identification of clusters, and isomiR characterization. Many of the miRNAs were regulated in a tissue/organ-specific manner.

Integration of metabolomics, transcriptomics, and microRNA expression profiling reveals a miR-143-HK2-glucose network underlying zinc-deficiency-associated esophageal neoplasia.

Abstract: // Louise Y. Fong 1,2,3 , Ruiyan Jing 1 , Karl J. Smalley 2 , Cristian Taccioli 4 , Johannes Fahrmann 5 , Dinesh K. Barupal 5 , Hansjuerg Alder 7 , John L. Farber 1 , Oliver Fiehn 5,6 and Carlo M. Croce 7 1 Department of Pathology, Anatomy & Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA 2 Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 3 Center for Molecular Carcinogenesis, Thomas Jefferson University, Philadelphia, PA, USA 4 Animal Medicine, Production and Health Department, University of Padua, Padua, Italy 5 University of California, Davis, West Coast Metabolomics Center, Davis, CA, USA 6 Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia 7 Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA Correspondence to: Louise Y. Fong, email: // Carlo M. Croce, email: // Keywords : metabolomic profiling, transcriptomics and microRNA profiling integration, esophageal neoplasia, dietary zinc-deficiency, miR-143 - Hk2 - glucose signaling Received : April 27, 2017 Accepted : May 29, 2017 Published : June 09, 2017 Abstract Esophageal squamous cell carcinoma (ESCC) in humans is a deadly disease associated with dietary zinc (Zn)-deficiency. In the rat esophagus, Zn-deficiency induces cell proliferation, alters mRNA and microRNA gene expression, and promotes ESCC. We investigated whether Zn-deficiency alters cell metabolism by evaluating metabolomic profiles of esophageal epithelia from Zn-deficient and replenished rats vs sufficient rats, using untargeted gas chromatography time-of-flight mass spectrometry ( n = 8/group). The Zn-deficient proliferative esophagus exhibits a distinct metabolic profile with glucose down 153-fold and lactic acid up 1.7-fold ( P < 0.0001), indicating aerobic glycolysis (the “Warburg effect”), a hallmark of cancer cells. Zn-replenishment rapidly increases glucose content, restores deregulated metabolites to control levels, and reverses the hyperplastic phenotype. Integration of metabolomics and our reported transcriptomic data for this tissue unveils a link between glucose down-regulation and overexpression of HK2, an enzyme that catalyzes the first step of glycolysis and is overexpressed in cancer cells. Searching our published microRNA profile, we find that the tumor-suppressor miR-143, a negative regulator of HK2, is down-regulated in Zn-deficient esophagus. Using in situ hybridization and immunohistochemical analysis, the inverse correlation between miR-143 down-regulation and HK2 overexpression is documented in hyperplastic Zn-deficient esophagus, archived ESCC-bearing Zn-deficient esophagus, and human ESCC tissues. Thus, to sustain uncontrolled cell proliferation, Zn-deficiency reprograms glucose metabolism by modulating expression of miR-143 and its target HK2. Our work provides new insight into critical roles of Zn in ESCC development and prevention.

Determination of absolute expression profiles using multiplexed miRNA analysis.

Abstract: Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR's ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes.

Comparative expression profiling of testis-enriched genes regulated during the development of spermatogonial cells

Abstract: The testis has been identified as the organ in which a large number of tissue-enriched genes are present. However, a large portion of transcripts related to each stage or cell type in the testis still remains unknown. In this study, databases combined with confirmatory measurements were used to investigate testis-enriched genes, localization in the testis, developmental regulation, gene expression profiles of testicular disease, and signaling pathways. Our comparative analysis of GEO DataSets showed that 24 genes are predominantly expressed in testis. Cellular locations of 15 testis-enriched proteins in human testis have been identified and most of them were located in spermatocytes and round spermatids. Real-time PCR revealed that expressions of these 15 genes are significantly increased during testis development. Also, an analysis of GEO DataSets indicated that expressions of these 15 genes were significantly decreased in teratozoospermic patients and polyubiquitin knockout mice, suggesting their involvement in normal testis development. Pathway analysis revealed that most of those 15 genes are implicated in various sperm-related cell processes and disease conditions. This approach provides effective strategies for discovering novel testis-enriched genes and their expression patterns, paving the way for future characterization of their functions regarding infertility and providing new biomarkers for specific stages of spematogenesis.

Identification of microRNAs implicated in the late differentiation stages of normal B cells suggests a central role for miRNA targets ZEB1 and TP53

Abstract: // Giorgio Malpeli 1, 2 , Stefano Barbi 2 , Simonetta Zupo 3 , Gabriele Tosadori 4 , Giovanni Scardoni 4 , Anna Bertolaso 2 , Silvia Sartoris 5 , Stefano Ugel 5 , Caterina Vicentini 2, 10 , Matteo Fassan 6 , Annalisa Adamo 7 , Mauro Krampera 7 , Maria Teresa Scupoli 8 , Carlo Maria Croce 9 , Aldo Scarpa 2, 10 1 Department of Surgical Sciences, Dentistry, Gynecology and Pediatrics, Section of Surgery, University of Verona, Verona, Italy 2 Department of Diagnostics and Public Health, Section of Pathological Anatomy, University of Verona, Verona, Italy 3 Laboratory of Molecular Diagnostics, IRCCS-AOU San Martino-IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 4 Center for BioMedical Computing (CBMC), University of Verona, Verona, Italy 5 Department of Medicine, Section of Immunology, University of Verona, Verona, Italy 6 Department of Medicine, Surgical Pathology and Cytopathology Unit, University of Padua, Padua, Italy 7 Department of Medicine, Section of Hematology, Stem Cell Research Laboratory, University of Verona, Italy 8 Department of Medicine, Section of Hematology, University of Verona, Italy 9 Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 10 Applied Research on Cancer-Network (ARC-NET), University of Verona, Verona, Italy Correspondence to: Giorgio Malpeli, email: giorgio.malpeli@univr.it Keywords: B cell development, follicle, germinal centre, microRNAs, network analysis Received: June 17, 2016 Accepted: December 12, 2016 Published: January 17, 2017 ABSTRACT In the late B cell differentiation stages, miRNAs expression modifications promoting or inhibiting key pathways are only partially defined. We isolated 29 CD19 + human B cell samples at different stages of differentiation: B cells from peripheral blood; naive, germinal center (GC) and subepithelial (SE) B cells from tonsils. SE cells were further split in activated and resting B cell. The miRNA expression profile of these B cells was assessed by microarray analysis and selected miRNAs were validated by quantitative RT-PCR and in situ hybridization on normal tonsils. The comparison of all samples showed changes in 107 miRNAs in total. Among 48 miRNAs differentially expressed in naive, GC and SE cells, we identified 8 miRNAs: mir-323, mir-138, mir-9*, mir-211, mir-149, mir-373, mir-135a and mir-184 , strictly specific to follicular cells that had never been implicated before in late stages of B cell development. Moreover, we unveiled 34 miRNAs able to discriminate between CD5 − activated B cells and resting B cells. The miRNAs profile of CD5 − resting B cells showed a higher similarity to naive CD5 + than CD5 − activated B cells. Finally, network analysis on shortest paths connecting gene targets suggested ZEB1 and TP53 as key miRNA targets during the follicular differentiation pathway. These data confirm and extend our knowledge on the miRNAs-related regulatory pathways involved in the late B cell maturation.

Mutation of TGFβ-RII eliminates NSAID cancer chemoprevention.

Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) exhibit anti-neoplastic (chemoprevention) activity for sporadic cancers and the hereditary cancer predisposition Lynch syndrome (LS/HNPCC). However, the mechanism of NSAID tumor suppression has remained enigmatic. Defects in the core mismatch repair (MMR) genes MSH2 and MLH1 are the principal drivers of LS/HNPCC. Previous work has demonstrated that the villin-Cre+/-Msh2flox/flox (VpC-Msh2) mouse is a reliable model for LS/HNPCC intestinal tumorigenesis, which is significantly suppressed by treatment with the NSAID aspirin (ASA) similar to human chemoprevention. Here we show that including a TGFβ receptor type-II (Tgfβ-RII) mutation in the VpC-Msh2 mouse (villin-Cre+/-Msh2flox/floxTgfβ-RIIflox/flox ) completely eliminates NSAID tumor suppression. These results provide strong genetic evidence that TGFβ signaling and/or effectors participate in NSAID-dependent anti-neoplastic processes and provide fresh avenues for understanding NSAID chemoprevention and resistance.

Correction: In vivo NCL targeting affects breast cancer aggressiveness through miRNA regulation.

Abstract: Vol. 210, No. 5, May 2013. Pages [951–968][1]. The authors regret that in their original paper, the RNU6 loading control panels shown in Fig. 1 (E and F) were incorrect as a result of an error in figure presentation during the final production process. The conclusions of the experiments shown in

MicroRNA Dysregulation to Identify Novel Therapeutic Targets.

Abstract: This paper describes how we discovered the juxtaposition of the MYC gene to the human immunoglobulin loci and how that finding was extended to characterize molecularly the t(14;18) chromosome translocation of follicular lymphoma and to clone the BCL2 gene. BCL2 is also overexpressed in CLL, the most common human leukemia. We discovered that most of human CLLs have a deletion of two microRNAs residing in the same polycistronic RNA, miR-15a and miR-16-1, and that these two microRNAs are negative regulators of BCL2. Thus, loss of miR-15/16 leads to overexpression of BCL2 that can be targeted by the new drug, venetoclax, that was recently approved by the FDA for the treatment of aggressive CLLs.