Rational engineering of microRNA-regulated viruses for cancer gene therapy

MicroRNAs (miRNAs) are small noncoding RNA molecules that have important regulatory roles in a wide range of biological processes. miRNAs are often expressed in a tissue- and/or differentiation state-specific patterns, and it is estimated that miRNAs can regulate the expression of more than 50% of all human genes. We have exploited these tissue-specific miRNA expression patterns in the modification of viral replicative tropism. In order to engineer the replicative tropism of oncolytic adenoviruses, we developed a recombinant adenovirus that in the 3 UTR of the critical E1A gene contains sequences complementary to the liver-specific miRNA miR122. This allowed us to generate a novel recombinant adenovirus that was severely attenuated in human liver, but replicated to high titres in colorectal cancer. Systemic injection of miR122-targeted adenovirus into mice did not induce liver toxicity. In a human lung cancer xenograft mouse model this miR122-targeted adenovirus showed potent antitumour activity. We also studied the possibility to exploit neuron-specific miRNA expression patterns in the modification of tissue tropism of an alphavirus Semliki Forest virus (SFV). We engineered SFV genome to contain sequences complementary to the neuron-specific miRNA miR124. In vitro characterization of this novel virus showed that the modification of the SFV genome per se did not affect polyprotein processing or oncolytic potency. Intraperitoneally administered miR124-targeted SFV displayed an attenuated spread into the central nervous system (CNS) and increased survival of infected mice. Also, mice pre-infected with miR124-targeted SFV elicited strong protective immunity against otherwise lethal challenge with a highly virulent wild-type SFV strain. In conclusion, these results show that miRNA-targeting is a potent new strategy to engineer viral tropism in development of safer and more efficient reagents for virotherapy applications.MikroRNA:t (miRNA) ovat pieniä ei-koodaavia RNA molekyylejä joilla on tärkeä tehtävä useiden erilaisten biologisten prosessien säätelyssä. MiRNA:t ekpressoituvat usein kudos- ja/tai kehitysvaihespesifisesti sekä säätelevät jopa yli 50 prosenttia kaikista ihmisen geeneistä. Tässä väitöskirjatutkimuksessa pyrimme käyttämään hyväksi miRNA:iden kudosspesifistä ekpressiota virusten kudostropismin muokkaamisessa vähentääksemme virusvektoreiden haitallista kudostoksisuutta. Muokataksemme adenovirusvektoreiden kudostropismia, kehitimme uudentyyppisen adenoviruksen jonka E1A-geenin 3 ei-koodaavalle alueelle lisäsimme ihmisen maksaspesifisen miRNA miR122:n tunnistussekvenssejä. Tunnistussekvenssien lisäyksellä saimme aikaan adenoviruksen (miR122-targetoitu adenovirus) jonka replikaatiokyky oli huomattavasti heikentynyt ihmisen maksassa, mutta pystyi replikoitumaan voimakkaasti perä- ja paksusuolisyöpäkudoksessa. Hiireen systeemisesti injisoitu miR122-targetoitu adenovirus ei aiheuttanut maksatoksisuutta. Ihmisen keuhkosyöpähiirimallissa miR122-targetoitu virus tappoi tehokkaasti syöpäsoluja. Tässä väitöskirjatutkimuksessa tutkimme myös hermosoluspesifisen miRNA miR124:n hyväksikäyttöä Semliki Forest-viruksen (SFV) kudostropismin muokkauksessa. Kehitimme SFV:n jonka genomiin oli sisällytetty miR124:n tunnistussekvenssejä. In vitro-kokeilla osoitimme tämän miR124-targetoidun SFV:n proteiinien prosessoituvan normaalisti sekä onkolyyttisen tehon säilyneen villityypin viruksen kaltaisena. Vatsaonteloon injisoitu miR124-targetoitu SFV levisi hyvin heikosti keskushermostossa joka johti vähentyneeseen neurotoksisuuteen. Osoitimme myös miR124-targetoidun viruksen toimivan tehokkaana rokotteena erittäin patogeeniselle L10 SFV-kannalle. Tässä väitöskirjatutkimuksessa pystyimme osoittamaan miRNA-targetoinnin olevan tehokas uusi tapa muokata virusten kudostropismia ja parantaa virusvektoreiden turvallisuutta

MicroRNA Dysregulation in Colon Cancer Microenvironment Interactions: The Importance of Small Things in Metastases

The influence of the microenvironment through the various steps of cancer progression is signed by different cytokines and growth factors, that could directly affect cell proliferation and survival, either in cancer and stromal cells. In colon cancer progression, the cooperation between hypoxia, IL-6 and VEGF-A165 could regulate the DNA repair capacity of the cell, whose impairment is the first step of colon cancer development. This cooperation redirects the activity of proteins involved in the metabolic shift and cell death, affecting the cell fate. The pathways triggered by micro environmental factors could modulate cancer-related gene transcription, affecting also small non coding mRNA, microRNAs. MicroRNAs have emerged as key post-transcriptional regulators of gene expression, directly involved in human cancers. The present review will focus first on the intertwined connection between cancer microenvironment and aberrant expression of microRNAs which contribute to carcinogenesis. In particular, the epigenetic mechanisms triggered by tissue microenvironment will be discussed, in view of the recent identification of miRNAs able to directly or indirectly modulate the epigenetic machinery (epi-miRNAs) and that are involved in the epithelial to mesenchimal transition and metastases development

From single gene analysis to single cell profiling: a new era for precision medicine

Molecular profiling of DNA and RNA has provided valuable new insights into the genetic basis of non-malignant and malignant disorders, as well as an increased understanding of basic mechanisms that regulate human disease. Recent technological advances have enabled the analyses of alterations in gene-based structure or function in a comprehensive, high-throughput fashion showing that each tumor type typically exhibits distinct constellations of genetic alterations targeting one or more key cellular pathways that regulate cell growth and proliferation, evasion of the immune system, and other aspects of cancer behavior. These advances have important implications for future research and clinical practice in areas as molecular diagnostics, the implementation of gene or pathway-directed targeted therapy, and the use of such information to drive drug discovery. The 1st international and 32nd Annual Conference of Italian Association of Cell Cultures (AICC) conference wanted to offer the opportunity to match technological solutions and clinical needs in the era of precision medicine

MiRNAs in Cervical Cancer Radio- and Chemotherapy Response

Cervical cancer (CC) is a very frequent women disease with high mortality and morbidity incidence worldwide, being the developing countries the most affected. Persistent infection with an oncogenic high-risk human papillomavirus (HPV) type is the primary cause of cervical cancer, but other etiologies are needed for complete malignancy such as patient immune response, genetic, and cellular factors, and/or environment. Radiotherapy in combination with cisplatinum is the standard treatment for invasive cervical cancer. Nevertheless, this conventional treatment is restricted due to eventual development of drug resistance and systemic toxicity. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of protein-coding genes involved in various cellular processes including cancer where they play a very important role in the development and progression of malignancy. As part of this complex disease, miRNAs have been implicated in the process of drug and radiation resistance and sensitivity. Recent studies have been directed to understand how miRNAs under or over-expressed are determinants of clinical response, and other studies have focused to clarify how the process of radio and/or chemotherapy affects miRNA expression. These works could lead to the design of safer and more effective therapy approaches based on miRNA expression and their target regulation

The non-coding RNA landscape of plasma cell dyscrasias

Despite substantial advancements have been done in the understanding of the pathogenesis of plasma cell (PC) disorders, these malignancies remain hard-to-treat. The discovery and subsequent characterization of non-coding transcripts, which include several members with diverse length and mode of action, has unraveled novel mechanisms of gene expression regulation often malfunctioning in cancer. Increasing evidence indicates that such non-coding molecules also feature in the pathobiology of PC dyscrasias, where they are endowed with strong therapeutic and/or prognostic potential. In this review, we aim to summarize the most relevant findings on the biological and clinical features of the non-coding RNA landscape of malignant PCs, with major focus on multiple myeloma. The most relevant classes of non-coding RNAs will be examined, along with the mechanisms accounting for their dysregulation and the recent strategies used for their targeting in PC dyscrasias. It is hoped these insights may lead to clinical applications of non-coding RNA molecules as biomarkers or therapeutic targets/agents in the near future

The Nefarious Nexus of Noncoding RNAs in Cancer

The past decade has witnessed enormous progress, which has seen the noncoding RNAs (ncRNAs) turn from the so called dark matter RNA to critical functional molecules, influencing most physiological processes in development and disease contexts. Many ncRNAs interact with each other and are part of networks that influence the cell transcriptome and proteome and consequently the outcome of biological processes. The regulatory circuits controlled by ncRNAs have become increasingly more relevant in cancer. Further understanding of these complex network interactions and how ncRNAs are regulated, is paving the way for the identification of better therapeutic strategies in cancer

New Prognostic and Predictive Markers in Cancer Progression

Biomarkers are of critical medical importance for oncologists, allowing them to predict and detect disease and to determine the best course of action for cancer patient care. Prognostic markers are used to evaluate a patient’s outcome and cancer recurrence probability after initial interventions such as surgery or drug treatments and, hence, to select follow-up and further treatment strategies. On the other hand, predictive markers are increasingly being used to evaluate the probability of benefit from clinical intervention(s), driving personalized medicine. Evolving technologies and the increasing availability of “multiomics” data are leading to the selection of numerous potential biomarkers, based on DNA, RNA, miRNA, protein, and metabolic alterations within cancer cells or tumor microenvironment, that may be combined with clinical and pathological data to greatly improve the prediction of both cancer progression and therapeutic treatment responses. However, in recent years, few biomarkers have progressed from discovery to become validated tools to be used in clinical practice. This Special Issue comprises eight review articles and five original studies on novel potential prognostic and predictive markers for different cancer types

Dissecting the Role of microRNAs in the Radiation Response of Human Prostate Cancer

Radiotherapy is one of the gold standard treatments for non-metastatic prostate cancer (PCa), although development of radioresistance limits its effectiveness. Mounting evidence supports the ability of microRNAs to interfere with different radioresistance-associated pathways, suggesting their potential as radiosensitizers. To identify miRNAs likely to be involved in influencing PCa radiation response, we first performed miRNA profiling analysis of PCa cell lines exposed to photon irradiation. Based on their modulation upon radiation exposure, and their role in regulating pathways associated with radiation response we singled-out and characterized miR-23a-3p and miR-24-3p as potential radiosensitizer molecules in PCa, unrevealing a central role of miR- 23a-3p-IL-6 axis. In the framework of miRNAs downregulated in PCa and known to regulate genes relevant to radiation, miR-875-5p and miR-205 were identified. Reconstitution of miR-875-5p, the expression of which directly correlates with that of E-cadherin, was able to enhance radiation response in PCa cell lines and xenografts through EGFR direct targeting. Consistent with the established role of EGFR in sustaining epithelial-to-mesenchymal transition (EMT) and promoting DNA repair following radiation, we found that miR-875-5p reconstitution in PCa cells counteracted EMT and impaired DNA lesion clearance, mainly via impairment of EGFR-ZEB1 axis. Restoration of miR-205 was able to significantly enhance PCa sensitivity to radiation, in both cell and animal models. Specifically, we found that miR-205 radiosensitizing effect was imputable to the downregulation of PKC-ε. Consistent with PKC-ε well-established role in DNA damage repair, we found that pEGFR and, consequently, pDNA-PK levels were significantly reduced by miR-205 reconstitution, thus suggesting that miR-205 enhances PCa radiation response via disruption of PKC-ε−EGFR−DNA-PK axis. Overall, the results of this project support the clinical interest in developing novel therapeutic approaches based on miRNA reconstitution (miR-23a-3p, miR-875-5p and miR-205) to enhance PCa response to radiotherapy