MicroRNAs as novel targets and tools in cancer therapy

Accepted Manuscript Title: MicroRNAs as novel targets and tools in cancer therapy Author: Mohammed L. Abba, Nitin Patil, Jörg Hendrik Leupold, Marcin Moniuszko, Jochen Utikal, Jacek Niklinski, Heike Allgayer PII: DOI: Reference:

S0304-3835(16)30204-X http://dx.doi.org/doi: 10.1016/j.canlet.2016.03.043 CAN 12843

To appear in:

Cancer Letters

Please cite this article as: Mohammed L. Abba, Nitin Patil, Jörg Hendrik Leupold, Marcin Moniuszko, Jochen Utikal, Jacek Niklinski, Heike Allgayer, MicroRNAs as novel targets and tools in cancer therapy, Cancer Letters (2016), http://dx.doi.org/doi: 10.1016/j.canlet.2016.03.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

MicroRNAs as novel targets and tools in cancer therapy (Invited review for Special Issue “New developments and important issues on targeted cancer therapy”) Mohammed L. Abba#1, Nitin Patil#1, Jörg Hendrik Leupold#1, Marcin Moniuszko2, Jochen Utikal3, Jacek Niklinski 4, and Heike Allgayer1* 1

Department of Experimental Surgery, Medical Faculty Mannheim, and Center for

Biomedicine and Medical Technology Mannheim (CBTM), Ludolf-Krehl-Str. 6, 68135 Mannheim, Ruprecht-Karls University of Heidelberg, Germany. 2

Department of Regenerative Medicine and Immune Regulation, Medical University of

Bialystok, ul. Waszyngtona 13, 15-269 Bialystok, Poland. 3

Skin Cancer Unit, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280,

69121 Heidelberg, Germany; and Department of Dermatology, Venereology and Allergology, Medical Faculty Mannheim, Theodor Kutzer Ufer 1-3, 68135 Mannheim, Ruprecht-Karls University of Heidelberg, Germany. 4

Department of Clinical Molecular Biology, Medical University of Bialystok, Waszyngtona

13, Bialystok, 15-269, Poland.

Key words: microRNAs, cancer, therapy, stem cells, metastasis. Running title: microRNAs in cancer therapy # contributed equally and share first authorship *Corresponding author:

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Prof. Dr med. Heike Allgayer, MD, PhD, Head, Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, and Centre for Biomedicine and Medical Technology Mannheim (CBTM), Mannheim, Germany. Address: Ludolf-Krehl-Str. 13-17, 68167 Mannheim, Germany. Tel.: +49-621-383-6876, Fax: +49-621-383-6878 E-mail: [email protected]

Highlights 

MicroRNAs are both relevant molecular targets and therapeutic targets in cancer.

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Diverse synthetic antagonists have been developed and can be used to target miRNAs in vivo.

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The delivery of miRNA mimics and inhibitors to specific organs is still a therapeutic challenge.

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A liposome-based miR-34 mimic (MRX34), is the first cancer-targeted microRNA drug to enter Phase I clinical trials in human patients.

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Abstract MicroRNAs (miRNAs) are currently experiencing a renewed peak of attention not only as diagnostics, but especially as highly promising novel targets or tools for clinical therapy in several different malignant diseases. Moreover, the recent discovery of competing endogenous RNAs (ceRNAs) as novel miRNA-regulators has contributed exciting insights in this regard. Therefore, this review summarizes and discusses the latest findings on (1) how miRNAs have become therapeutic targets of diverse synthetic antagonists, (2) how novel endogenous regulators of miRNAs such as ceRNAs or pseudogenes could emerge as therapeutics scavenging oncogenic miRNAs and (3) how miRNAs themselves are already, and will increasingly be, used as therapeutics. Recent advances on the importance of miRNAtarget affinity and the subcellular localization of miRNAs are also discussed. The potential of these developments in different tumor entities and particular hallmarks of cancer such as metastasis, disease progression, interactions with the tumor microenvironment, or cancer stem cells are equally highlighted.

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1. Introduction Distorted microRNA (miRNA, miR) regulation and expression contributes to many diverse pathologies. However, nowhere is the perturbation of miRNA function more evident than in cancer. Distinct miRNA expression profiles have become pathognomonic of individual cancer entities, as cancer-specific miRNA fingerprints have now been identified in several cancer types [1-8]. Moreover, miRNA expression profiles are able to better classify tumors as compared with the mRNAs and recently, our group suggested a miRNA signature indicative of metastatic colorectal cancer [9, 10]. The recognition that miRNA dysregulation plays a pivotal role in cancer (other diseases as well), has led to an increased awareness of the diagnostic and therapeutic potential of miRNAs. Therefore, this review focuses on the recent development of (1) how miRs have become therapeutic targets of synthetic antagonists, (2) how novel endogenous regulators of miRs such as competing endogenous RNAs (ceRNAs) or pseudogenes, might emerge as tools to counter the activity of oncogenic miRNAs and (3) how miRNAs themselves are already, and will increasingly be, used as therapeutics themselves. A mature miRNA is a small molecule that is on average 22nt in length and exerts its activity by binding via its seed sequence to a complementary motif located in the untranslated region (UTR) of the target mRNA. The seed sequence of a miRNA is a short region, mostly situated at positions 2-7 from the miRNA 5´-end that is essential for the binding of the miRNA to the mRNA [11-15]. Consequently, methodologies that have been developed to target miRNAs involve one of the following approaches. The first involves the use of synthetic complementary oligonucleotides that bind to an endogenous miRNA via Watson-Crick base pairing and prevent the miRNA from being functional, e. g. binding to its natural targets [13, 16]. This is the antisense approach and makes use of the so-called antisense oligonucleotides [17, 18]. The second approach makes use of multiple miRNA-specific complementary sequences/binding sites that are stringed together and artificially expressed via a vector [19]. 4 Page 4 of 41

These constructs are called miRNA sponges and act as baits by ‘mopping up’ and sequestering endogenous miRNAs, preventing their interaction with their native targets. The third approach involves the use of compounds that either prevent the transcription [20], processing or activity of a miRNA transcript, thus in effect creating a scenario where a mature miRNA is either unavailable or is functionally incompetent [21, 22]. A schematic representation of the miRNA therapeutic pipeline including the appropriately employed strategies is shown in figure 1. 2. General strategies of using microRNAs as therapeutic targets or tools 2.1. Synthetic antagonists targeting miRNAs As with any other class of drugs, miRNA targeting therapies should exhibit favorable pharmacokinetics, have high target avidity, a long half-life coupled with low toxicity and high potency. Importantly, in order to target miRNAs, they must be relatively stable in and outside the cell and must be able to traverse the cell membrane. Antisense oligonucleotides (ASO) that mimic naturally occurring RNA/DNA are highly unstable and are very rapidly degraded by nucleases. An array of chemical modifications has been incorporated into ASOs in order to overcome this shortcoming. These modifications include changes to the phosphate backbone chemistry, the sugar moieties and, in RNA, the 2’ hydroxyl group. Starting from the first-generation ASOs where the non-bridging phosphoryl oxygen of DNA was substituted with sulphur (phosphorothioate modifications) in a bid to increase resistance to nuclease digestion and prolong half-life, to the second-generation ASOs that had yet further improved stability and affinity as a result of modifications made to the 2′-position of the ribose sugar (2′-O-methyl or 2′-O-methoxyethyl (MOE) groups), and even later versions of this modification where the 2’OH of the ribose sugar is locked into a C3’-endo conformation via a 2′-O,4’-C-methylene bridge (locked nucleic acid; LNA), ASOs have been transformed into stable and potent molecules targeting nucleic acids. ASOs that specifically target miRNAs are 5 Page 5 of 41

called anti-miRs regardless of the chemistry, however, those that include additional cholesterol modifications are called antagomiRs [18]. ASOs are administered via liposomal complexes, viral particles and nanoparticles. Small molecule inhibitors of miRNAs, now commonly referred to as “SMIR” [23, 24] represent the most recent attempt at targeting miRNAs. Their design and use is based on the premise of other small molecule inhibitors used in treating other ailments. However, limited knowledge about the crystallographic structures of miRNAs and their protein interacting partners has been a bottleneck in the advancement of this field of therapy [25, 26]. The SMIR approach aims to find compounds that bind and subsequently decrease the levels of mature miRNAs, however, such compounds can principally function to prevent transcription [27]. 2.2. miRNA scavenging synthetic and natural sponges MiRNA sponges are a dominant negative method of scavenging miRNA expression and are able to inhibit the activity of a family of miRNAs sharing a common seed sequence, an advantage they have over chemically modified antisense oligonucleotides. The efficacy of a miRNA sponge depends not just on the affinity and avidity of binding sites, but also on the concentration of the individual miRNA sponges relative to the concentration of the targeted miRNA [28]. Typical sponge constructs contain four to 10 binding sites separated by a few nucleotides each. The miRNA binding sites in these constructs are either perfectly antisense or contain mismatches in the middle positions, which, if perfectly base-paired, would be vulnerable to argonaute RNA-induced silencing complex (RISC) catalytic component 2 (Ago2)- mediated endonucleolytic cleavage. Sponges are expressed as transgenes often under the control of a strong promoter and can be tagged with a reporter gene for identification and monitoring of expression. In vitro, they can be delivered by transient plasmid transfections or viral transduction for long term silencing. In vivo, however, delivery is only possible with the use of viral vectors that are injected systemically [29, 30]. Nonetheless, intrinsic challenges 6 Page 6 of 41

associated with miRNA sponges have hindered their clinical translation. Quite recently, a completely new concept of miRNA regulation was put forward by Pierre Pandolfi’s group. They postulated and experimentally demonstrated that endogenous other RNA species, most prominently pseudogenes, function as ceRNAs. They compete with target mRNAs for miRNA binding, making them endogenous sponges [31]. The Phosphatase and tensin homolog (PTEN) pseudogene was the first demonstrated example, but since then, other pseudogenes and long non-coding RNAs have been shown to be valid ceRNAs [32-36]. The therapeutic exploitation of ceRNAs as a form of miRNA targeted therapy is still at best experimental. 2.3. General strategies to use miRNAs as therapeutic agents In order to maximize the true potential of miRNAs, either as therapeutic agents themselves or as targets of therapeutic inhibition, miRNAs must be delivered directly to target cells, or inhibited in organs where the suppression of their expression is necessary. Basically, two major approaches exist for delivery, viral and non-viral, and depending on whether antisense inhibition or replacement desired, the appropriate approach(es) is utilized. A generalized overview of these strategies is shown in Figure 1. Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells and can be engineered to deliver foreign DNA into cells. Certain viruses are better suited as delivery vehicles because of their capacities to carry foreign DNA and their ability to efficiently deliver these with efficient expression [37]. The majority of viral vectors in preclinical and clinical use are derived from retroviruses, adenovirus, adeno-associated virus and herpesvirus. Several examples exist in the literature, mostly in vitro, where viruses have been used to mediate the overexpression and/or replacement of a miRNA (Figure 2 and 4), but by far the most abundant usage in in vivo models has been as sponges to suppress miRNA

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expression (Figure 2) [38]. Examples of some of these instances are given in this review in the subchapter on sponges. The second and by far most abundantly used class of delivery molecules is non-viral. This approach is centered mainly on the incorporation of a synthetic miRNA (for replacement therapy) or an antisense-oligonucleotide/AntagomiR into lipid- or polymer-based nanocarriers (Figures 3 and 4). The vast majority of nonviral delivery systems for miRNAs to date are based on the formation of an electrostatic complex between the negatively charged miRNA and the positively charged vehicle, but other avenues include encapsulation inside biodegradable nanoparticles and conjugation to a nano-carrier. Specific illustrations of these delivery systems comprise lipid vesicles (complexation), polymeric nanoparticles (encapsulation), dendrimers (complexation), gold nanoparticles (complexation/conjugation), and cationic polymeric polyplexes [39]. Examples of these are given in specific subchapters of this review. To increase the specificity to target cells, or at least target organs, interesting advances have been made, e.g. very recently through the development of delivery peptides for miRNAs which are able to target miRNAs to specific organs, as suggested by Frank Slack and his group [40]. Certainly, as with all modern therapeutics, further developments need to be pushed forward to increase organ and cell-type specificity. Nevertheless, since many miRNAs exert their action in the cross-talk between tumor- and surrounding stromal cells, it might not even be necessary to achieve cancer cell specific targeting in the instance of particular miRNAs. 3. miRNAs as therapeutic tools and targets in specific tumor hallmarks and subtypes In this chapter we will specifically describe the use of miRNAs in the treatment of specific tumor diseases, as well as important hallmarks such as stemness and metastasis. 8 Page 8 of 41

3.1. MiRNAs as targets and tools in hematopoietic and leukemic malignant diseases To date, numerous studies demonstrated that targeting certain miRNAs can significantly alter the fate, proliferation, apoptosis and function of acute myeloid leukemia (AML) blasts [41]. Thus, it is not surprising that different miRNAs have been tested as potential novel therapeutic agents capable of improving anti-leukemic features of currently used chemotherapeutics. One of the first miRNAs tested in that context was miR-193a. MiR-193a expression was found to be repressed in AML cell lines and primary AML blasts, but not normal bone marrow cells [42]. Interestingly, either restoration or overexpression of miR193a expression in AML cells harboring c-kit mutations, either by synthetic miR-193a transfection or by DNA hypomethylating agents, resulted in a significant inhibition of leukemic cell growth. This study suggested that therapeutic upregulation of miR-193a expression could be of clinical benefit in c-kit-positive AML patients. Recently, miR-638 appeared as another important regulator of leukemogenesis. Lin and colleagues found that overexpression of miR-638 in leukemic cell lines and primary AML blasts by targeting (inhibiting) cyclin-dependent kinase 2, induced G1 cell cycle arrest and reduced colony formation [43]. Thus, miR-638 could be considered as another potential therapeutic tool capable of regulating cell proliferation and myeloid differentiation. Similarly, Emmrich and colleagues found that different subgroups of AML are associated with a suppression of miR-139-5p [44]. Based on that discovery these authors showed that the experimental restoration of miR-139-5p expression in AML cell lines resulted in arresting their cell cycle and caused their apoptosis, both in vitro and in xenograft mouse models. These experimental findings were further confirmed by a more favorable clinical outcome in pediatric AML patients who presented with elevated miR-139-5p expression. Previously, miR-29a and miR-29b were shown to play an active tumor-suppressing role in the pathogenesis of AML. In concert with this notion, Gong and colleagues demonstrated that the 9 Page 9 of 41

levels of miR-29a, -29b and -29c were significantly decreased in AML patients [45]. Corroborating this paradigm, the experimental overexpression of miR-29a, miR-29b, -miR29c in leukemic cell lines profoundly suppressed cell proliferation and augmented cell apoptosis. In addition, restoration of the expression of each of the miR-29 members in AML blasts inhibited their abnormal proliferation and corrected apoptosis repression and myeloid differentiation arrest. Moreover, ectopic administration of miR-29a, -29b and -29c in an experimental mouse model significantly improved the clinical outcome of AML mice. This study confirmed earlier findings of Huang and colleagues, who had found that targeted delivery of miR-29b by transferrin-conjugated anionic lipopolyplex nanoparticles resulted in a profound downregulation of genes playing crucial roles in the development of AML, namely DNA (cytosine-5)-methyltransferase 1 (DNMTs), cyclin-dependent kinase 6 (CDK6), specificity protein 1 (SP1), KIT, fms-related tyrosine kinase (FLT3), and a subsequent significant decrease in AML cell growth [46]. In concordance with these notions, AML mice that were treated with transferrin-conjugated anionic lipopolyplex nanoparticles bound to synthetic miR-29b presented with significantly longer survival. Moreover, pre-treating AML cells with functional miR-29b before administration of a chemotherapeutic agent resulted in a significant increase in the anti-leukemic activity of this drug. Altogether, these studies pointed to members of the miR-29 family as one of the most attractive candidates to be used in future anti-leukemic regimens. Lastly, also miR-181 became a target of novel experimental therapies tested in animal models of AML [47]. Inhibition of miR-181 family expression results in a loss of the engraftment of leukemic cells and released leukemic symptoms. In vitro, miR-181a acted through the inhibition of granulocytic and macrophage-like differentiation of leukemic cell lines and hematopoietic stem/progenitor cells by targeting and decreasing the expression of protein kinase C delta (PRKCD), carboxy-terminal domain small phosphatase like (CTDSPL) and 10 Page 10 of 41

calcium/calmodulin-dependent protein kinase kinase 1 alpha (CAMKK1), genes that were shown to be targets of miR-181b, miR-181c and miR-181d, respectively. Importantly, to date, a wealth of anti-leukemic therapies are targeting dividing blast populations but are not capable of efficiently targeting leukemic stem cells/leukemic initializing cells (LSC/LIC). Similarly to hematopoietic stem cells, LSC are capable of selfrenewing and further differentiating, albeit in an altered manner, towards leukemic blasts. Thus, a failure in their elimination results in resistance to applied therapy and in consequence, in disease maintenance and recurrence. It has been widely established that numerous miRNAs play crucial roles in regulating the function and fate of LSC [48]. To date, several experimental regimens were proposed to inhibit these miRNAs that are either crucial for LSC maintenance, or to augment actions of these miRNAs that are known to suppress leukemic growth and differentiation. Recently, Dorrance and collaborators found that a higher expression of miR-126 was associated with poorer clinical outcome in a large cohort of older cytogenetically normal AML patients treated with standard chemotherapy [49]. Moreover, they found that overexpression of miR-126 was specific for AML LSC-enriched cell subpopulations and accounted for LSC long-term maintenance and self-renewal. Next, these authors demonstrated that the use of antagomiR-126 nanoparticles targeting miR-126 resulted in an in vivo reduction of LSC. These findings indicate that targeting even single miRNAs can represent a promising therapeutic tool in AML. Interestingly, however, in another recent study, Li and colleagues showed that both experimental overexpression and knockout of miR126 resulted in enhanced leukemogenesis [50]. The overexpression of miR-126 targeted, and downregulated, ERBB receptor feedback inhibitor 1 (ERRFI1) and sprouty-related EVH1 domain containing 1 (SPRED1) and, in turn, activated genes characteristic for LSCs/LICs. On the other hand, a miR-126 knockout activated genes that are highly expressed in more mature hematopoietic progenitor cells. In addition, the miR-126 knockout significantly augmented 11 Page 11 of 41

susceptibility of leukemic cells to standard chemotherapy. From the clinical point of view, this last finding indicates that priming cells to the effects of chemotherapy by silencing miR126 could constitute a new promising tool in the therapy of AML. In some contrast, the role of microRNAs as potential therapeutic targets or agents seems to have been less studied in chronic myeloid leukemia (CML) or chronic lymphocytic leukemia (CLL) settings. In a few studies on CML experimental models, miR-30a enhanced imatinibinduced cytotoxicity and promoted intrinsic apoptosis of CML cells. Conversely, the knockdown of miR-30a by antagomiR-30a inhibited the cytotoxicity of imatinib and increased the expression of genes related to autophagy, namely Beclin 1 and autophagy related 5 (ATG5) [51]. More recently, Wang and collaborators demonstrated that the silencing of miR-21 sensitized CML stem/progenitor cells to imatinib-induced apoptosis by blocking the phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene homolog 1 (PI3K/AKT) pathway [52]. Both reports clearly indicated that the targeting of miR-30a, which also plays a pivotal role in migration and metastasis [53], and miR-21 could be used as potent modulators of CML cell sensitivity to imatinib-related effects. In primary CLL cells, ectopic expression of miR-15a and miR-16 was associated with declines in the levels of myeloid cell leukemia 1 (Mcl-1), but not B-cell CLL/lymphoma 2 (Bcl-2), and an induction of CLL cell death [54]. In a very recent study, Wang and collaborators showed that experimental restoration of miR-3151 which is usually suppressed in the course of CLL led to an inhibition of cell proliferation, repression of anti-apoptotic MCL1, and increased apoptosis [55]. Contrarily, the silencing of miR-3151 by DNA methylation protected CLL cells from apoptosis. In a last study conducted on CLL, miR-377 was shown to suppress the expression of anti-apoptotic molecule, BCL2-like 1 or Bcl-xL [56]. Moreover, this report revealed that the down-regulation of miR-377 increased BCL-xL expression, and, in consequence, promoted resistance to chemotherapy. 12 Page 12 of 41

A strongly oncogenic miRNA is miR-155. It is overexpressed in numerous cancers, particularly in hematopoietic malignancies where it regulates myeloid cell proliferation and survival by directly targeting SH2-containing inositol phosphatase (SHIP1). Overexpression of miR-155 in lymphoid tissues results in disseminated lymphoma characterized by clonal, pre-B-cell neoplastic lymphocytes. Systemic delivery of a miR-155 anti-miR encapsulated in a penetratin coated nanoparticle (ANTP-NP) at 1.5 mg/kg in miR-155-addicted tumors was able to cause a 5-fold decrease in tumor growth compared to the control group [57]. In an attempt to further enhance targeted delivery of anti-miRs, a peculiar property of the tumor microenvironment was exploited, low pH in this instance. Using a peptide with a low pHinduced transmembrane structure (pHLIP) coupled with a peptide nucleic acid anti-miR, Cheng and colleagues could effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. In addition to targeting the acidic tumor microenvironment, the pHLIP peptide is able to evade systemic clearance by the liver, and facilitate cell entry via a non-endocytic pathway [40]. 3.2. miRNAs as therapeutic targets and tools in stem cells of solid tumors In different solid tumor subtypes, cells within the tumor population exhibit functional heterogeneity. This functional heterogeneity of carcinoma cells has led to the creation of different models such as the cancer stem cell (CSC) model, which have been put forward to account for heterogeneity and differences in tumor-regenerative capacity. The CSC model derives from the fact that cancers are dysregulated tissue clones whose continued propagation is vested in a biologically distinct subset of cells that are typically rare. These rare CSCs possess characteristics associated with normal multipotent stem cells such as the ability to give rise to all cell types found in a particular cancer sample. CSCs are therefore tumorigenic (tumor-forming) in contrast to other non-tumorigenic cancer cells. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into different cell types 13 Page 13 of 41

found within the tumor [58]. The CSC model has been widely applied to interpreting tumor initiation, growth, metastasis, dormancy and relapse. However, its investigation has been hampered both by a lack of consistency in the terms used for these cells and by how they are defined [59]. Also, cancer cells in general exhibit several traits that are also characteristic for pluripotent stem cells such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) [60]. Notably, cancer cells as well as ESCs/ iPSCs give rise to tumors/teratomas after injection into immunocompromised mice. They show immortal cell growth with high proliferation rates as well as genomic instability, which leads to numerical and structural aberrations. Transcriptional and posttranscriptional regulations reveal several similarities in cancer and pluripotent stem cells [48, 60]. Since earlier work on iPSCs had shown that it was feasible to reprogram somatic cells with cocktails of miRNAs, it was only logical to hypothesize that miRNAs play a significant role in tumor cells with stem cell features. Also, key signaling cascades in CSCs including Hedgehog, Notch and Wnt are important in self-renewal and stemness and are affected by both up- and down regulated miRNAs, depending on context (reviewed in [48]). Thus, targeting signaling pathways in CSCs, e.g., via antagonizing activating miRNAs, is of therapeutic value in cancer cells [48, 61]. For example, miR-582-3p was recently reported to activate Wnt/β-catenin signaling and its expression correlated with the overall- and recurrence-free-survival of non-small cell lung carcinoma (NSCLC) patients. Antagonizing miR-582-3p was reported to inhibit tumor initiation and progression in xenografted animal models, suggesting that miR-582-3p might serve as a potential therapeutic target to overcome stem cells in NSCLC [62]. Another example is miR-744 that plays a vital role in promoting the stem cell-like phenotype of pancreatic cancer cells by activating the Wnt/β-catenin pathway. The expression of miR-744 was markedly upregulated in pancreatic cancer and positively correlated with poor patient survival. Thus it may represent a novel prognostic 14 Page 14 of 41

biomarker and therapeutic target which could help to target stem cells in pancreatic cancer [63]. On the other hand, activating specific miRNAs and/or applying them as therapeutics are of increasing therapeutic interest in the context of cancer stem cells. For example, miR-506 exerts an anti-proliferative function in cervical cancer cells by directly targeting Gli3, a member of the Hedgehog signaling pathway which is known to drive stemness. This newly identified miR-506/Gli3 axis provides further insight into the pathogenesis of cervical cancer and indicates a potential novel therapeutic agent for the treatment of this disease [64]. Another example is miR-134. Exogenous miR-134 overexpression was found to downregulate protein O-glucosyltransferase 1 (POGLUT1) and Notch pathway proteins in endometrial cancer stem cells. MiR-134 overexpression affects the G2/M phase of endometrial cancer stem cells and was reported to suppress the growth of xenograft tumors formed. Interestingly, the expression level of miR-134 was found to differ between human endometrial cancer stem cells and endometrial cancer cells. Thus, miR-134 might be of high interest in suppressing tumorigenesis in human endometrial carcinoma [65]. The POU-domain transcription factor Octamer binding transcription factor 4 (Oct4) and the homeobox A7 (HOX7) class gene Nanog facilitate self-renewal and are downregulated during differentiation in pluripotent stem cells such as ESCs and iPSCs. As a consequence, Oct4 and Nanog have become a focus of interest as potential regulators of self-renewal in cancer cells [60]. It was shown recently that Oct4 variants and pseudogenes are differentially expressed in normal urothelium and urothelial cancer [66]. Also, Oct4 has been reported to be controlled by pseudogene-expressed antisense RNAs (asRNAs) (summarized in [67]). Processed pseudogenes such as POU class 5 homeobox 1 B (POU5F1B) that are highly homologous to OCT4 were recently shown to be transcribed in cancer cells. The POU5F1B pseudogene is amplified and expressed at a high level in gastric cancer cells and confers an aggressive 15 Page 15 of 41

phenotype in gastric cancer patients [68]. In hepatocellular carcinoma, mechanistic analysis revealed that OCT4-pg4, a pseudogene (pg) of OCT4, functions as a natural micro RNA sponge to protect the OCT4 transcript from being inhibited by miR-145. It was also shown that OCT4-pg4 can promote the growth and tumorigenicity of hepatocellular carcinoma cells, thus exerting an oncogenic role in hepatocarcinogenesis. Survival analysis suggests that high OCT4-pg4 is significantly correlated with poor prognosis of hepatocellular cancer (HCC) patients [69]. Recently, it was reported that NANOG and its pseudogene NANOGP8 contribute to the high malignant potential of prostate cancer. Knockouts of NANOG and NANOGP8 significantly attenuated the malignant potential of prostate cancer cells, including sphere formation, anchorage-independent growth, migration and drug resistance [70]. Thus, targeting the expression of NANOG, but especially also its pseudogene in cancer cells might be of highly interesting future therapeutic value. It will be incomplete to address cancer stem cells and stemness without referring to epithelial to mesenchymal transition (EMT). EMT is tightly linked to the cancer hallmark of metastasis [71] and characterizes the process where cells dissociate from their epithelial junctions, morph into spindle-like mesenchymal shapes, and migrate [72]. In addition to fostering metastasis, EMT has been shown to also promote the generation of cells with stem cell-like or so-called stemness properties. The EMT process itself is primarily orchestrated by a set of transcription factors including, but not limited to SNAIL, SLUG, ZEB1, ZEB2 and TWIST1, that function in a large part to transcriptionally repress E-Cadherin. Interestingly, several miRNAs have been found to impact EMT by modulating the expression of these core transcription factors, but also by targeting cytoskeletal and associated proteins that are integral to the junctional integrity of epithelial cells. As a result, vital stemness features are either strengthened or repressed. Moreover, some miRNAs like the miR-200 family, miRs-34a/b/c and let-7 also

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directly regulate self-renewal factors that contribute to stemness. The association between EMT and stemness has been well described in several excellent reviews [73, 74]. 3.3 MiRNAs as therapeutic targets and tools in the proliferation, progression and metastasis of solid tumors Virtually, all miRNAs decoded to have a role in solid cancer have been investigated to some extent in vitro, however, only those found promising have been further evaluated in vivo. Consequently, a much smaller number of miRNAs have been found to have potential therapeutic value. Here, we elaborate on some of these miRNAs in important processes such as proliferation, progression and metastasis of solid tumors, identifying the strategies that have been employed to utilize them therapeutically. 3.3.1. MiRNA antisense-strategies in solid tumors Notable examples where miRNA expression has been counteracted include glioblastoma cells in xenograft models, where miR-124-3p was found to work in concert with gap junctions to inhibit proliferation. An intratumoral injection of a miR-124-3p antagomiR (AgomiR-124-3p) was effective in reducing tumor volume, especially in the presence of the gap junction protein, alpha 1 (Cx43) [75]. Oncogenic miR-21 is highly overexpressed in several solid tumors, including glioblastoma. Here, using poly[lactic-co-glycolic]acid (PLGA) nanoparticle encapsulated anti-miR -21 oligonucleotides as pre-treatment, the sensitivity of U87 MG glioblastoma cells to temozolomide could be enhanced significantly (Figure 2). The nanoparticle delivered anti-miR-21 was able to reduce cell viability, at the same time increasing cell cycle arrest in the G2/M phase [76]. Pancreatic ductal adenocarcinoma (PDAC) is another tumor entity characterized by the overexpression of several miRNAs, miR21 inclusive. Using a novel liposomal delivery combination comprising HSAEPOPC:Chol/AMOs (+/−) (4/1) in which anti-miRs were LNA based, a significant reduction 17 Page 17 of 41

of miR-21 in addition to other miRs (miR-10b, miR-221, and miR-222) was observed in Hs766T cells (Figure 2). This strategy resulted in a higher expression of examined targets of these miRNAs, especially of miR-21. Additionally, the combination of the anti-miRs with sunitinib resulted in a notable synergistic antitumor effect in the cell line investigated [77]. Similar results were obtained when another cationic polymer, poly (l-lysine)-modified polyethylenimine (PEI-PLL) was used in breast cancer cell lines, with miR-21 as the prime target [78]. 3.3.2. MiRNA sponges as therapeutics in solid tumors MiRNA sponges contain multiple miRNA antisense binding sites (MBS) and also function to repress miRNA expression (see chapter 1.2.). Several examples of their use exist in literature (Figure 3) and the strategy in most situations involves the identification of a strongly overexpressed miRNA family or cluster in a specific cancer entity and then designing a sponge that is able to sequester all the members of the group. Several instances exist, however, where a sponge also has been used to target only one miRNA. Delivery is almost always achieved via viral vectors, ranging from lentiviral to adeno- and adeno-associated viruses and even retroviruses. For example, in osteosarcoma, miRs-27a and -27a* are frequently overexpressed and the overexpression of these miRs was found to promote pulmonary metastasis formation in vivo. The sequestration of this miRNA duo with a sponge suppressed invasion and metastasis formation, in part by setting free the tumor suppressor core-binding factor runt domain alpha subunit 2 translocated to 3 (CBFA2T3) [79]. Likewise, vulvar carcinoma, a rather rare gynecological tumor, is characterized by the overexpression of the miR-17 family (miR-17, miR-20a/b, miR-93, and miR-106a/b), amongst others. A sponge composed of six in tandem seed-sequence complementary motifs was used to inhibit this family and with the exception of miR-20a, the sponge was able to cause a significant repression of all members of the miR-17 18 Page 18 of 41

family in SW954 cells. No functional validation of this construct was made, however, in this case [80]. The miR-183/-96/-182 cluster is highly expressed in most breast cancers. Li and colleagues targeted the mature members of this cluster, both individually with antagomiRs and with a sponge targeting all of the members. They found a significant suppression of miRNA-expression using both methods. Inhibition of the cluster decreased cell proliferation and induced cell death in both MCF-7 and T47D breast cancer cell lines [81]. Furthermore, miR-221 has been shown to be up-regulated in up to 70% of hepatocellular carcinomas (HCC), which is often associated with advanced tumor stages and metastasis. A miR-221 sponge administered via an adenoviral vector was able to reduce endogenous miR-221 levels, rescuing the expression of CDKN1B/p27, inducing cell apoptosis and reducing the viability of HCC cells [82]. Other valid examples of sponge use include miR-135b in NSCLC cells [83], miR-125b in invasive cervical cancer [84], miR-574-5p in lung cancer cells [85], miR-221/miR-222 in colorectal cancer (CRC) [86] and miR-9 in metastatic breast cancer [87]. The sponges have proven to be very valuable tools not only in suppressing oncogenic miRNAs but also in validating the function of miRNAs in general. 3.3.3. ceRNAs in solid tumors Pseudogenes, small non-coding, long non-coding and circular RNAs are all able to act as competing ceRNAs by titrating miRNA availability. The PTEN pseudogene, PTENP1, was found to regulate the expression of miR-17, miR-19, miR-21, miR-26 and miR-214 families, which resulted in reducing migration and invasion [88]. Since then, other examples have been put forward. The tumor suppressor protein programmed cell death 4 (PDCD4) is an independently validated target of miR-21 [89]. The embryonic lethal abnormal vision like RNA binding protein 1 (ELAVL1 or HuR) interacts with the PDCD4 3'-untranslated region (UTR), preventing miR-21-mediated repression of PDCD4 translation. Interestingly, HuR 19 Page 19 of 41

was also found to bind miR-21 directly, preventing its interaction with the PDCD4 3'-UTR, thereby acting as a ceRNA [90]. MiR-145 has been shown to function as a tumor suppressor in triple negative breast cancer (TNBC) by regulating tumor cell invasion. The long intergenic noncoding RNA regulator of reprogramming, lincRNA-RoR, was found to function as a competitive endogenous RNA in TNBC by significantly limiting the levels of freely expressed miR-145 [91]. Another long non-coding RNA, H19, harbors both canonical and non-canonical binding sites for the let-7 family of microRNAs. H19 modulates let-7 availability by acting as a molecular sponge, thus modulating its natural roles in development and cancer [92]. The prospect of using ceRNAs in cancer therapy is indeed very promising. 3.3.4. miRNA replacement therapy in solid tumors As a result of their rather unique predisposition of being able to act as both tumor suppressors and oncogenic modulators, several instances have been revealed where the expression of a miRNA is either lost or repressed in cancer. In this scenario, miRNAs need to be replaced to effect a suppression of the cancer phenotype. The strategy always involves the administration of a double stranded RNA mimic of the respective miRNA with an appropriate delivery tool. For instance, an Ala-Pro-Arg-Pro-Gly peptide-modified poly ethylene glycol conjugated modified lipoplex (APRPG-PEG) carrying miR-499 was injected via the tail vein into mice bearing subcutaneously implanted mouse colon cancer cell line (NL-17) tumors. This miR accumulated in significant amounts in angiogenic vessels and cancer cells and, in addition to suppressing miR-499-target proteins, was able to cause a significant decrease in tumor burden at doses as low as 0.5mg/kg [93]. In another study, the plasma levels of miR-101 were observed to be significantly lower in hepatocellular carcinoma patients harboring distant metastasis as compared to those with early stage disease. In a mouse model of this cancer, the systemic delivery of miR-101 via a 20 Page 20 of 41

lentivirus abrogated growth in the liver, and inhibited distant metastasis to the lung and to the mediastinum, significantly enhancing survival without toxic side effects. As with miR-200c, several targets were implicated in this response including enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), cytochrome c oxidase subunit II (COX2), stathmin 1 (STMN1) and Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) [94]. The miR-200 family is arguably one of the most significantly downregulated miRNA families in cancer, where several of its members have been found to play a role in the mediation of epithelial- to -mesenchymal transition (EMT) and metastatic dissemination. In lung cancer, for example, Cortez and colleagues demonstrated that the administration of miR-200c via NOV30 liposomal nanoparticles significantly sensitized lung cancer cells to radiation in a xenograft model. This was achieved by the ability of miR-200c to downmodulate oxidative response genes and inhibit DNA repair primarily via the direct regulation of peroxiredoxin 2 (PRDX2), nuclear factor, erythroid 2 like 2 (Nrf2), and SESN1 [95]. The miR-34 family comprising miRs-34a, -34b, and -34c functions downstream of p53 and is a key regulator of tumor suppression [96-98]. It regulates the expression of an assemblage of target proteins involved in cell cycle regulation, differentiation, apoptosis and metastasis. It inhibits multiple oncogenic pathways as well as stimulates anti-tumor immune response to induce cancer cell death. Some of its validated targets include v-myc avian myelocytomatosis viral oncogene homolog (c-MYC), E2F transcription factor 1 (E2F), cyclin-dependent kinase 4 and 6 (CDK4 and CDK6), Bcl2, sirtuin 1 (SIRT1), platelet-derived growth factor receptor, alpha polypeptide (PDGFR-ɑ), WNT1/3, CD44, Nanog, AXL and the MET proto-oncogene receptor tyrosine kinase (c-MET). The expression of the miR-34 family, especially miR-34a, is significantly suppressed in several cancer types (reviewed in [99]). It is therefore not surprising that several attempts have been made to replace miR-34a in different cancer types.

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In multiple myeloma, synthetic miR-34a mimics encapsulated in stable nucleic acid lipid particles (SNALPs) were evaluated therapeutically. The SNALP-miR-34a composites were highly efficient in inhibiting the growth of multiple myeloma cells vitro and in xenografts in SCID mice. No systemic toxicity was observed [100]. In hepatocellular carcinoma (HCC), miRNA-34a was co-expressed with IL-24 and delivered via an oncolytic AdCN205 adenoviral vector into tumor bearing mice. This combination induced strong antitumor activity, resulting in complete tumor regression without recurrence during the experimental period. Bcl-2 and SIRT1 were the prime targets [101]. Although several interventional strategies have been tried in mouse models, very few have transitioned to human subjects. An updated search in March, 2016, so far revealed four clinical trials utilizing miRNA mimics or antagonists as potential therapeutic tools, with two of these being cancer-related. The first cancer-targeted microRNA drug to enter a phase I clinical trial in human patients, MRX34, is a liposome-based miR-34 mimic advocated for patients with advanced hepatocellular carcinoma (NCT01829971). MRX34 is a doublestranded RNA mimic of miR-34, encapsulated in a liposomal nanoparticle formulation called SMARTICLES®. More recently, a miR-16-based microRNA mimic encapsulated in EnGeneIC Delivery Vehicle (EDV) nanoparticles designed to target EGFR-expressing cancer cells (TargomiR) also entered phase-1 trials as a prospective second- or third-line treatment for patients with recurrent malignant pleural mesothelioma and non-small cell lung cancer (NCT02369198) [102]. These trials are summarized in Table 1 and for completion, the other miRNA clinical trials are also included. It is important to mention, however, that several trials assessing the role of miRNAs as diagnostic/prognostic/predictive/surrogate biomarkers or in patient stratification for other therapeutic interventions are either in progress or have recently been completed.

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4. Conclusion It is very clear that microRNAs, and also especially recently discovered molecular regulators of these as, for example, ceRNAs or pseudogenes, currently gain a second peak of high attention as highly promising novel targets or tools for therapy in several different malignant diseases. For both instances, powerful and attractive (bio-) chemical or biological formulations have already been developed, as discussed in this review. In addition, first promising ideas on how to increase specificity of miRNA-based therapeutics to particular cells have been put forward [40]. Regarding miRNA-targeted therapeutics, it is exciting to speculate that they are quite likely to not only to target particular miRNAs, thus potentially modifying their mRNA-target affinity and activity, but that with their action they might also be able to change or modify particular subcellular localizations of miRNAs, for example in exosomes. It has been shown only recently that particular miRNAs localize in, and can be secreted via, exosomes, thus being able to prime metastatic niches for tumor cells [103]. Also, our own group just now has shown that particular miRNAs can be concentrated in exosomes especially in tumor cells with a high metastatic capacity, in contrast to tumor cells which are significantly less metastatic [104]. Furthermore, miRNA targeted agents and also miRNAs as therapeutics certainly will not only have an impact on the tumor cells, but most certainly on their interaction with their microenvironment, since numerous papers have shown that miRNAs are important molecules in the crosstalk between tumor cells and, e.g., macrophages, endothelial cells, cells of the immune system, or fibroblasts, in this context even being able to transform the latter into cancer-associated fibroblasts (CAF) [105-110]. Finally, it is very clear that miRNAs certainly not only are in the process of emerging as exciting therapeutic targets and tools themselves, as discussed in this review, but that they

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increasingly become attractive novel biomarkers in cancer diagnosis and in the stratification of classical and novel targeted therapies in tumor diseases [111-115]. “Great things are done by a series of small things put together” (Vincent Willem van Gogh; 30 March 1853– 29 July 1890) -----Let’s look forward to what little miRNAs can do within the next few years to make a difference in treating cancer. 5. Acknowledgements HA was supported by Alfried Krupp von Bohlen und Halbach Foundation, Essen, Dr HellaBuehler-Foundation, Heidelberg, Walter Schulz Foundation, Munich, the Deutsche Krebshilfe, Bonn (109558; together with MA), the DKFZ-MOST German Israel Cooperation, Heidelberg (CA149), the HIPO/POP-Initiative for Personalized Oncology, Heidelberg (H032 and H027; together with MA and NP). HA and JHL were supported by the Wilhelm Sander Foundation, Munich, Germany (2012.036.1). JU was supported from the German Research Council (DFG), the German Cancer Aid (Max-Eder Research Group), the BadenWürttemberg Foundation, the German Center for Cardiovascular Disease (DZHK) and the Hella Bühler Foundation (Heidelberg, Germany). JU, HA and MM were supported by funds from Leading National Research Centre (KNOW) in Bialystok, Poland.

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Figure legends Figure 1: Schematic overview of miRNA therapeutic pipelines. The figure illustrates the main strategies to either target oncogenic miRNAs or to increase the expression of tumor suppressor miRNAs, delivered by viral transfer, nanoparticles or liposomes (green arrow indicates up-regulation of the respective miRNAs by the therapeutic; red arrow indicates down-regulation; suppression of transcriptional regulation by small molecules is indicated by red truncated lines). Figure 2: Schematic overview of miRNAs used as sponge-based potential therapeutics. The figure illustrates miRNAs that have been targeted in various solid tumor entities by specific miRNA sponges. Oncogenic miRNAs are indicated in red. Figure 3: Schematic overview of miRNAs used as nanoparticle-based potential therapeutics. The figure illustrates various miRNAs delivered by nanoparticles that have recently been used in the treatment of various solid human carcinomas. Oncogenic miRNAs are indicated in red. Figure 4: Schematic overview of miRNAs used as potential virus- or liposome- based therapeutics. The figure illustrates miRNAs used in the treatment of various cancer types delivered either by viral transfer (left half) or by liposomal complexes (right half). The use of liposomal complexes applies to both miRNA mimics (black) and anti-miRNA oligomers (red). Oncogenic miRNAs are indicated in red letters.

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Table Legend Table 1: Overview of current clinical trials using miRNA based strategies.

Title

miRNA

Conditions

Drug Miravirsen (SPC3649), antisense microRNA antagonist, locked nucleic acid (LNAs) ribonucleotide TargomiRs, miR-16 Mimic

Clinical Phase Phase 1

Clinical Trial Number NCT00688012

Phase 1

NCT02369198

Safety study of SPC3649 in healthy men

miR-122

Hepatitis C

MesomiR 1: A phase I study of targomiRs as 2nd or 3rd line treatment for patients with recurrent MPM and NSCLC

miR-16

Malignant pleuralmesothelioma Non-small cell lung cancer

Effect of microvesicles and exosomes therapy on β-cell mass in type I diabetes mellitus (T1DM)

various

Diabetes mellitus type 1

MSC exosomes

Phase 2 Phase 3

NCT02138331

A multicenter phase I study of MRX34, microRNA miR-RX34 liposomal injection

miR-34

Primary liver cancer, SCLC, lymphoma, melanoma, multiple myeloma, renal cell carcinoma, NSCLC

MRX34, microRNA miR-RX34 liposomal injection

Phase 1

NCT01829971

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