17ß-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17ß-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17ß-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17ß-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

Pro-tumoral functions of tumor-associated macrophage EV-miRNA.

MicroRNAs (miRNAs) are central players in cancer biology. Their relevance in cancer development, progression and resistance to therapy has been further emphasized by the discovery that they are important cargo component of extracellular vesicles (EVs), which represent a prominent means of inter-cellular communication within the tumor microenvironment (TME). This review article focuses on the interaction between cancer cells and tumor-associated macrophages (TAMs) and in particular on the pro-tumoral phenotype elicited by EV-contained miRNAs released by TAMs and transferred to cancer cells. All main hallmarks of the malignant phenotype are affected by TAM-derived vesicular miRNAs, paving the road to the identification of such miRNAs as promising upcoming novel anti-cancer agents.

Diverse roles of EV-RNA in cancer progression.

Extracellular vesicles (EVs) have emerged as important players in all aspects of cancer biology. Their function is mediated by their cargo and surface molecules including proteins, lipids, sugars and nucleic acids. RNA in particular is a key mediator of EV function both in normal and cancer cells. This statement is supported by several lines of evidence. First, cells do not always randomly load RNA in EVs, there seems to be a specific manner in which cells populate their EVs with certain RNA molecules. Moreover, cellular uptake of EV-RNA and the secondary compartmentalization of EV-RNA in recipient cells is widely reported, and these RNAs have an impact on all aspects of cancer growth and the anti-tumoral immune response. Additionally, EV-RNA seems to work through various mechanisms of action, highlighting the intricacies of EVs and their RNA cargo as prominent means of inter-cellular communication.

Decrypting noncoding RNA interactions, structures, and functional networks.

The world of noncoding RNAs (ncRNAs) is composed of an enormous and growing number of transcripts, ranging in length from tens of bases to tens of kilobases, involved in all biological processes and altered in expression and/or function in many types of human disorders. The premise of this review is the concept that ncRNAs, like many large proteins, have a multidomain architecture that organizes them spatially and functionally. As ncRNAs are beginning to be imprecisely classified into functional families, we review here how their structural properties might inform their functions with focus on structural architecture-function relationships. We will describe the properties of "interactor elements" (IEs) involved in direct physical interaction with nucleic acids, proteins, or lipids and of "structural elements" (SEs) directing their wiring within the "ncRNA interactor networks" through the emergence of secondary and/or tertiary structures. We suggest that spectrums of "letters" (ncRNA elements) are assembled into "words" (ncRNA domains) that are further organized into "phrases" (complete ncRNA structures) with functional meaning (signaling output) through complex "sentences" (the ncRNA interactor networks). This semiotic analogy can guide the exploitation of ncRNAs as new therapeutic targets through the development of IE-blockers and/or SE-lockers that will change the interactor partners' spectrum of proteins, RNAs, DNAs, or lipids and consequently influence disease phenotypes.

Cancer-associated rs6983267 SNP and its accompanying long noncoding RNA CCAT2 induce myeloid malignancies via unique SNP-specific RNA mutations.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

MicroRNA-16 Restores Sensitivity to Tyrosine Kinase Inhibitors and Outperforms MEK Inhibitors in KRAS-Mutated Non-Small Cell Lung Cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Chemotherapy, the treatment of choice in non-operable cases, achieves a dismal success rate, raising the need for new therapeutic options. In about 25% of NSCLC, the activating mutations of the KRAS oncogene define a subclass that cannot benefit from tyrosine kinase inhibitors (TKIs). The tumor suppressor miR-16 is downregulated in many human cancers, including NSCLC. The main objectives of this study were to evaluate miR-16 treatment to restore the TKI sensitivity and compare its efficacy to MEK inhibitors in KRAS-mutated NSCLC. METHODS: We performed in vitro and in vivo studies to investigate whether miR-16 could be exploited to overcome TKI resistance in KRAS-mutated NSCLC. We had three goals: first, to identify the KRAS downstream effectors targeted by mir-16, second, to study the effects of miR-16 restoration on TKI resistance in KRAS-mutated NSCLC both in vitro and in vivo, and finally, to compare miR-16 and the MEK inhibitor selumetinib in reducing KRAS-mutated NSCLC growth in vitro and in vivo. RESULTS: We demonstrated that miR-16 directly targets the three KRAS downstream effectors MAPK3, MAP2K1, and CRAF in NSCLC, restoring the sensitivity to erlotinib in KRAS-mutated NSCLC both in vitro and in vivo. We also provided evidence that the miR-16-erlotinib regimen is more effective than the selumetinib-erlotinib combination in KRAS-mutated NSCLC. CONCLUSIONS: Our findings support the biological preclinical rationale for using miR-16 in combination with erlotinib in the treatment of NSCLC with KRAS-activating mutations.

mRNA and miRNA Profiles of Exosomes from Cultured Tumor Cells Reveal Biomarkers Specific for HPV16-Positive and HPV16-Negative Head and Neck Cancer.

Human papillomavirus (HPV)(+) and HPV(-) head and neck cancer (HNC) cells' interactions with the host immune system are poorly understood. Recently, we identified molecular and functional differences in exosomes produced by HPV(+) vs. HPV(-) cells, suggesting that genetic cargos of exosomes might identify novel biomarkers in HPV-related HNCs. Exosomes were isolated by size exclusion chromatography from supernatants of three HPV(+) and two HPV(-) HNC cell lines. Paired cell lysates and exosomes were analyzed for messenger RNA (mRNA) by qRT-PCR and microRNA (miR) contents by nanostring analysis. The mRNA profiles of HPV(+) vs. HPV(-) cells were distinct, with EGFR, TP53 and HSPA1A/B overexpressed in HPV(+) cells and IL6, FAS and DPP4 in HPV(-) cells. The mRNA profiles of HPV(+) or HPV(-) exosomes resembled the cargo of their parent cells. miR expression profiles in cell lysates identified 8 miRs expressed in HPV(-) cells vs. 14 miRs in HPV(+) cells. miR-205-5p was exclusively expressed in HPV(+) exosomes, and miR-1972 was only detected in HPV(-) exosomes. We showed that HPV(+) and HPV(-) exosomes recapitulated the mRNA expression profiles of their parent cells. Expression of miRs was dependent on the HPV status, and miR-205-5p in HPV(+) and miR-1972 in HPV(-) exosomes emerge as potential discriminating HPV-associated biomarkers.

Cancer-derived exosomic microRNAs shape the immune system within the tumor microenvironment: State of the art.

In recent years there has been an increasing interest of the scientific community on exosome research, with particular emphasis on the mechanisms by which tumor-derived exosomes can promote tumor growth. Particularly, exosome-mediated immune-escape is under deep investigation and still represents a quite controversial issue. Tumor-derived exosomes are carriers of information able to reprogram functions of immune target cells, influencing their development, maturation, and antitumor activities. They deliver proteins similar to those of the parent cancer cells, but also genetic messages like genomic DNA, mRNA, and microRNAs (miRNAs) that ultimately share the so called "tumor microenvironment" in a pro-tumoral fashion. The content of tumor-derived exosomes could be implicated in several signaling pathways operating in the tumor microenvironment, providing a further modality of dys-regulation of antitumor immunity. The aim of this review is to provide a state-of-the-art highlight of to the most recent discoveries in the field of interaction between tumor-derived exosomic miRNAs and the cells of immune system.

Serum miR-29a Is Upregulated in Acute Graft-versus-Host Disease and Activates Dendritic Cells through TLR Binding.

Acute graft-versus-host disease (aGVHD) continues to be a frequent and devastating complication of allogeneic hematopoietic stem cell transplantation (HSCT), posing as a significant barrier against the widespread use of HSCTs as a curative modality. Recent studies suggested serum/plasma microRNAs (miRs) may predict aGVHD onset. However, little is known about the functional role of circulating miRs in aGVHD. In this article, we show in two independent cohorts that miR-29a expression is significantly upregulated in the serum of allogeneic HSCT patients at aGVHD onset compared with non-aGVHD patients. Serum miR-29a is also elevated as early as 2 wk before time of diagnosis of aGVHD compared with time-matched control subjects. We demonstrate novel functional significance of serum miR-29a by showing that miR-29a binds and activates dendritic cells via TLR7 and TLR8, resulting in the activation of the NF-κB pathway and secretion of proinflammatory cytokines TNF-α and IL-6. Treatment with locked nucleic acid anti-miR-29a significantly improved survival in a mouse model of aGVHD while retaining graft-versus-leukemia effects, unveiling a novel therapeutic target in aGVHD treatment or prevention.

Mechanisms of Drug Resistance in Cancer: The Role of Extracellular Vesicles.

Drug resistance remains a major barrier to the successful treatment of cancer. The mechanisms by which therapeutic resistance arises are multifactorial. Recent evidence has shown that extracellular vesicles (EVs) play a role in mediating drug resistance. EVs are small vesicles carrying a variety of macromolecular cargo released by cells into the extracellular space and can be taken up into recipient cells, resulting in transfer of cellular material. EVs can mediate drug resistance by several mechanisms. They can serve as a pathway for sequestration of cytotoxic drugs, reducing the effective concentration at target sites. They can act as decoys carrying membrane proteins and capturing monoclonal antibodies intended to target receptors at the cell surface. EVs from resistant tumor cells can deliver mRNA, miRNA, long noncoding RNA, and protein inducing resistance in sensitive cells. This provides a new model for how resistance that arises can then spread through a heterogeneous tumor. EVs also mediate cross-talk between cancer cells and stromal cells in the tumor microenvironment, leading to tumor progression and acquisition of therapeutic resistance. In this review, we will describe what is known about how EVs can induce drug resistance, and discuss the ways in which EVs could be used as therapeutic targets or diagnostic markers for managing cancer treatment. While further characterization of the vesiculome and the mechanisms of EV function are still required, EVs offer an exciting opportunity in the fight against cancer.

The clinical and biological significance of MIR-224 expression in colorectal cancer metastasis.

OBJECTIVE: MicroRNA (miRNA) expression profile can be used as prognostic marker for human cancers. We aim to explore the significance of miRNAs in colorectal cancer (CRC) metastasis. DESIGN: We performed miRNA microarrays using primary CRC tissues from patients with and without metastasis, and validated selected candidates in 85 CRC samples by quantitative real-time PCR (qRT-PCR). We tested metastatic activity of selected miRNAs and identified miRNA targets by prediction algorithms, qRT-PCR, western blot and luciferase assays. Clinical outcomes were analysed in six sets of CRC cases (n=449), including The Cancer Genome Atlas (TCGA) consortium and correlated with miR-224 status. We used the Kaplan-Meier method and log-rank test to assess the difference in survival between patients with low or high levels of miR-224 expression. RESULTS: MiR-224 expression increases consistently with tumour burden and microsatellite stable status, and miR-224 enhances CRC metastasis in vitro and in vivo. We identified SMAD4 as a miR-224 target and observed negative correlation (Spearman Rs=-0.44, p<0.0001) between SMAD4 and miR-224 expression in clinical samples. Patients with high miR-224 levels display shorter overall survival in multiple CRC cohorts (p=0.0259, 0.0137, 0.0207, 0.0181, 0.0331 and 0.0037, respectively), and shorter metastasis-free survival (HR 6.51, 95% CI 1.97 to 21.51, p=0.0008). In the TCGA set, combined analysis of miR-224 with SMAD4 expression enhanced correlation with survival (HR 4.12, 95% CI 1.1 to 15.41, p=0.0175). CONCLUSIONS: MiR-224 promotes CRC metastasis, at least in part, through the regulation of SMAD4. MiR-224 expression in primary CRC, alone or combined with its targets, may have prognostic value for survival of patients with CRC.

Essential role of miRNAs in orchestrating the biology of the tumor microenvironment.

MicroRNAs (miRNAs) are emerging as central players in shaping the biology of the Tumor Microenvironment (TME). They do so both by modulating their expression levels within the different cells of the TME and by being shuttled among different cell populations within exosomes and other extracellular vesicles. This review focuses on the state-of-the-art knowledge of the role of miRNAs in the complexity of the TME and highlights limitations and challenges in the field. A better understanding of the mechanisms of action of these fascinating micro molecules will lead to the development of new therapeutic weapons and most importantly, to an improvement in the clinical outcome of cancer patients.

Toll-like receptor 3 (TLR3) activation induces microRNA-dependent reexpression of functional RARß and tumor regression.

Toll-like receptor 3 (TLR3) is a key effector of the innate immune system against viruses. Activation of TLR3 exerts an antitumoral effect through a mechanism of action still poorly understood. Here we show that TLR3 activation by polyinosinic:polycytidylic acid induces up-regulation of microRNA-29b, -29c, -148b, and -152 in tumor-derived cell lines and primary tumors. In turn, these microRNAs induce reexpression of epigenetically silenced genes by targeting DNA methyltransferases. In DU145 and TRAMP-C1 prostate and MDA-MB-231 breast cancer cells, we demonstrated that polyinosinic:polycytidylic acid-mediated activation of TLR3 induces microRNAs targeting DNA methyltransferases, leading to demethylation and reexpression of the oncosuppressor retinoic acid receptor beta (RARß). As a result, cancer cells become sensitive to retinoic acid and undergo apoptosis both in vitro and in vivo. This study provides evidence of an antitumoral mechanism of action upon TLR3 activation and the biological rationale for a combined TLR3 agonist/retinoic acid treatment of prostate and breast cancer.

Heat shock protein 70 regulates Tcl1 expression in leukemia and lymphomas.

T-cell leukemia/lymphoma 1 (TCL1) is an oncogene overexpressed in T-cell prolymphocytic leukemia and in B-cell malignancies including B-cell chronic lymphocytic leukemia and lymphomas. To date, only a limited number of Tcl1-interacting proteins that regulate its oncogenic function have been identified. Prior studies used a proteomic approach to identify a novel interaction between Tcl1 with Ataxia Telangiectasia Mutated. The association of Tcl1 and Ataxia Telangiectasia Mutated leads to activation of the NF-κB pathway. Here, we demonstrate that Tcl1 also interacts with heat shock protein (Hsp) 70. The Tcl1-Hsp70 complex was validated by coimmunoprecipitation experiments. In addition, we report that Hsp70, a protein that plays a critical role in the folding and maturation of several oncogenic proteins, associates with Tcl1 protein and stabilizes its expression. The inhibition of the ATPase activity of Hsp70 results in ubiquitination and proteasome-dependent degradation of Tcl1. The inhibition of Hsp70 significantly reduced the growth of lymphoma xenografts in vivo and down-regulated the expression of Tcl1 protein. Our findings reveal a functional interaction between Tcl1 and Hsp70 and identify Tcl1 as a novel Hsp70 client protein. These findings suggest that inhibition of Hsp70 may represent an alternative effective therapy for chronic lymphocytic leukemia and lymphomas via its ability to inhibit the oncogenic functions of Tcl1.

Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas.

Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.

MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response.

MicroRNAs (miRNAs) are small noncoding RNAs, 19-24 nucleotides in length, that regulate gene expression and are expressed aberrantly in most types of cancer. MiRNAs also have been detected in the blood of cancer patients and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor-secreted miR-21 and miR-29a also can function by another mechanism, by binding as ligands to receptors of the Toll-like receptor (TLR) family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately may lead to tumor growth and metastasis. Thus, by acting as paracrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in tumor-immune system communication and is important in tumor growth and spread, thus representing a possible target for cancer treatment.

Strand-specific miR-28-5p and miR-28-3p have distinct effects in colorectal cancer cells.

BACKGROUND & AIMS: MicroRNAs (miRNAs) can promote or inhibit tumor growth and are therefore being developed as targets for cancer therapies. They are diverse not only in the messenger RNAs (mRNA) they target, but in their production; the same hairpin RNA structure can generate mature products from each strand, termed 5p and 3p, that can bind different mRNAs. We analyzed the expression, functions, and mechanisms of miR-28-5p and miR-28-3p in colorectal cancer (CRC) cells. METHODS: We measured levels of miR-28-5p and miR-28-3p expression in 108 CRC and 49 normal colorectal samples (47 paired) by reverse transcription, quantitative real-time polymerase chain reaction. The roles of miR-28 in CRC development were studied using cultured HCT116, RKO, and SW480 cells and tumor xenograft analyses in immunodeficient mice; their mRNA targets were also investigated. RESULTS: miR-28-5p and miR-28-3p were down-regulated in CRC samples compared with normal colon samples. Overexpression of miRNAs in CRC cells had different effects and the miRNAs interacted with different mRNAs: miR-28-5p altered expression of CCND1 and HOXB3, whereas miR-28-3p bound NM23-H1. Overexpression of miR-28-5p reduced CRC cell proliferation, migration, and invasion in vitro, whereas miR-28-3p increased CRC cell migration and invasion in vitro. CRC cells overexpressing miR-28 developed tumors more slowly in mice compared with control cells, but miR-28 promoted tumor metastasis in mice. CONCLUSION: miR-28-5p and miR-28-3p are transcribed from the same RNA hairpin and are down-regulated in CRC cells. Overexpression of each has different effects on CRC cell proliferation and migration. Such information has a direct application for the design of miRNA gene therapy trials.

Reprogramming of miRNA networks in cancer and leukemia.

We studied miRNA profiles in 4419 human samples (3312 neoplastic, 1107 nonmalignant), corresponding to 50 normal tissues and 51 cancer types. The complexity of our database enabled us to perform a detailed analysis of microRNA (miRNA) activities. We inferred genetic networks from miRNA expression in normal tissues and cancer. We also built, for the first time, specialized miRNA networks for solid tumors and leukemias. Nonmalignant tissues and cancer networks displayed a change in hubs, the most connected miRNAs. hsa-miR-103/106 were downgraded in cancer, whereas hsa-miR-30 became most prominent. Cancer networks appeared as built from disjointed subnetworks, as opposed to normal tissues. A comparison of these nets allowed us to identify key miRNA cliques in cancer. We also investigated miRNA copy number alterations in 744 cancer samples, at a resolution of 150 kb. Members of miRNA families should be similarly deleted or amplified, since they repress the same cellular targets and are thus expected to have similar impacts on oncogenesis. We correctly identified hsa-miR-17/92 family as amplified and the hsa-miR-143/145 cluster as deleted. Other miRNAs, such as hsa-miR-30 and hsa-miR-204, were found to be physically altered at the DNA copy number level as well. By combining differential expression, genetic networks, and DNA copy number alterations, we confirmed, or discovered, miRNAs with comprehensive roles in cancer. Finally, we experimentally validated the miRNA network with acute lymphocytic leukemia originated in Mir155 transgenic mice. Most of miRNAs deregulated in these transgenic mice were located close to hsa-miR-155 in the cancer network.

A new role for microRNAs, as ligands of Toll-like receptors.

Tumor microenvironment plays a central role in the development and dissemination of cancer cells. In addition to study each specific cellular component of the microenvironment, it has become clear that it is the type and amount of information that cells exchange that ultimately affects cancer phenotype. Recently, it has been discovered that intercellular communication occurs through the release of microvesicles and exosomes, whose cargo represents the information released by one cell to a recipient cell. A key component of this cargo is represented by microRNAs (miRNAs), small non-coding RNAs with gene regulatory functions. We discovered that miRNAs released by cancer cells within microvesicles can reach and bind to Toll-like receptors (TLRs) in surrounding immune cells, and activate them in a paracrine loop. As a result, immune cells produce cytokines that increase cell proliferation and metastatic potential. This discovery provides the rationale for the development of new drugs that might be used in the treatment of cancer as well as other inflammation-related diseases.