- Email: [email protected]
Noncoding RNAs
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NONCODING RNAS: NOVEL TARGETS IN ANTICANCER DRUG DEVELOPMENT
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Ragini Kondetimmanahalli1, Kshipra M. Gharpure2, Sherry Y. Wu2, Gabriel Lopez-Berestein2, Anil K. Sood2 1The
University of Texas at Austin, Austin, TX, United States; 2The University of Texas MD Anderson Cancer Center, Houston, TX, United States
CHAPTER OUTLINE Introduction������������������������������������������������������������������������������������������������������������������������������������������ 448 Clinical Relevance of Noncoding RNAs�������������������������������������������������������������������������������������������������� 448 MicroRNAs.......................................................................................................................... 449 miR-34a������������������������������������������������������������������������������������������������������������������������������������� 449 miR-1258����������������������������������������������������������������������������������������������������������������������������������� 450 miR-15a and miR-16������������������������������������������������������������������������������������������������������������������ 450 miR-192������������������������������������������������������������������������������������������������������������������������������������� 450 let-7������������������������������������������������������������������������������������������������������������������������������������������� 451 miR-200������������������������������������������������������������������������������������������������������������������������������������� 451 miR-630������������������������������������������������������������������������������������������������������������������������������������� 451 Long Noncoding RNAs.......................................................................................................... 451 TRIM52-AS1������������������������������������������������������������������������������������������������������������������������������ 452 TUG1����������������������������������������������������������������������������������������������������������������������������������������� 452 NRCP����������������������������������������������������������������������������������������������������������������������������������������� 452 CCAT1���������������������������������������������������������������������������������������������������������������������������������������� 452 MALAT1������������������������������������������������������������������������������������������������������������������������������������� 452 PCGEM1������������������������������������������������������������������������������������������������������������������������������������ 452 Small Interfering RNAs......................................................................................................... 453 CALAA-01���������������������������������������������������������������������������������������������������������������������������������� 453 Atu027��������������������������������������������������������������������������������������������������������������������������������������� 453 EphA2���������������������������������������������������������������������������������������������������������������������������������������� 454 PIWI-Interacting RNAs.......................................................................................................... 454 Small Nucleolar RNAs.......................................................................................................... 454 Delivery Methods���������������������������������������������������������������������������������������������������������������������������������� 455 Future Implications������������������������������������������������������������������������������������������������������������������������������� 456 Cancer and Noncoding RNAs. http://dx.doi.org/10.1016/B978-0-12-811022-5.00024-3 Copyright © 2018 Elsevier Inc. All rights reserved.
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Conclusion������������������������������������������������������������������������������������������������������������������������������������������� 456 Glossary����������������������������������������������������������������������������������������������������������������������������������������������� 456 Grant Support��������������������������������������������������������������������������������������������������������������������������������������� 457 References������������������������������������������������������������������������������������������������������������������������������������������� 457
INTRODUCTION Advances in genome research have given scientists reason to believe that noncoding RNAs (ncRNAs) hold an influential role in cell biology. While protein-coding genes make up less than 2% of the human genome, ncRNAs make up 98% and offer new opportunities for biomarker and therapeutic applications in cancer and other diseases [1]. ncRNA is a term used to describe multiple classes of RNA that do not encode proteins but rather regulate transcription, stability, or translation of genes [2,3]. These families of ncRNAs have an important role in both gene regulation and normal development and disease [4]. Among the various types of ncRNAs, microRNAs (miRNAs or miRs) have been the most studied by researchers. However, potential for anticancer drug development can be found in the many other existing forms of ncRNAs described in Table 24.1. In this chapter, we will discuss the various classes of ncRNAs as well as their potential therapeutic applications.
CLINICAL RELEVANCE OF NONCODING RNAs Over the last decade, numerous studies have produced evidence supporting the assertion that ncRNAs are an effective tool in cancer therapy. The “druggability gap” has been a concern for small molecule inhibitors because many promising targets such as mutated RAS proteins are very difficult to target with small molecule drugs [5]. The excitement behind the use of ncRNAs in cancer therapy is due to the fact that ncRNAs have the ability to directly target any “undruggable” targets. The therapeutic potential of being able to specifically treat various cancers warrants careful investigation into the mechanistic and biological roles of ncRNAs in different types of cancer. Table 24.1 Various Types of Noncoding RNAs miRNAs lncRNAs siRNAs piRNAs snoRNAs sdRNAs
Single-stranded RNAs, approximately 19–24 nucleotides in length, that regulate transcription and translation of protein-coding genes [1]. Non-protein-coding RNA transcripts longer than 200 nucleotides with multiple functions and mechanisms [1]; may function as primary or spliced transcripts [4]. Small interfering RNAs of approximately 20 nucleotides in length; processed from several sources of endogenous double-stranded RNAs [1]. ∼21–35 nucleotides, PIWI-interacting RNAs, single-stranded. Play a role in germline transposon silencing and gametogenesis [1]. ∼60–300 nucleotides, small nucleolar RNAs. Help modify ribosomal RNAs [1]. sdRNAs derived from H/ACA snoRNAs are typically 20–24 nucleotides in length and originate from the 3′-end and those derived from C/D snoRNAs are predominantly 17–19 nucleotides long and originate from the 5′-end.
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MICRORNAS miRNAs are small, single-stranded, ncRNA molecules that bind to messenger RNAs (mRNAs) and prevent them from being translated into proteins [6]. miRNAs are present in nearly all biological pathways in multicellular organisms, which include many pathways that are cancer-relevant such as proliferation, cell cycle control, apoptosis, differentiation, migration, and metabolism. Due to the profound influence of miRNAs within the cell, abnormal miRNA expression may affect a wide array of transcripts and cancer-related signaling pathways [6]. An individual miRNA can target hundreds of genes depending on its function and context, giving miRNAs a vast array of silencing capabilities [6]. A compilation of research studies indicates that aberrant miRNA expression is commonly noted in cancer cells, illustrating the importance to better understand their nature and how they can be applied therapeutically for treating various cancers. RNase III proteins play a key role in miRNA biogenesis. The RNase III drosha cleaves primary miRNAs (pri-miRNAs) resulting in hairpin-shaped pre-miRNAs which are then cut by the RNase III Dicer to create mature miRNAs [7]. Lower dicer expression was associated with advanced tumor stage and suboptimal surgical cytoreduction [8]. From these results, we can conclude that interruptions in miRNA functioning are heavily associated with cancer. miRNAs are often downregulated in human cancers, leading to overexpression of downstream genes which may contribute to cancer progression [9]. Large-scale microarray analyses of miRNAs have demonstrated that a substantial proportion is downregulated in human solid tumor samples compared to normal controls [9]. For key downregulated miRNAs, delivery of synthetic miRNA mimics may hold potential for cancer treatment. On the other hand, miRNAs may also be upregulated in some cancers. In such settings, treatment with anti-miRs can be used to block oncogenic effects of these miRNAs. One of the main considerations of both mimics and anti-miRs is the delivery method, which will be discussed later in this chapter. Although there are many more miRNAs than the ones discussed in this section, we will discuss select miRNAs as therapeutic candidates by briefly discussing their identification as well their biological effects in preclinical testing.
miR-34a The miR-34 family has proven to have robust tumor-suppressive properties. It downregulates many proteins involved in cell cycle, differentiation and cell survival, and also antagonizes processes that are necessary for cancer cell progression, such as metastasis, chemoresistance, and cancer stemness [10]. miR-34a is downregulated in human breast cancer as well as multiple other types of cancer [11]. Several miR-34a targets and their biological relevance are discussed below. miR-34a was found to directly and effectively target NOTCH1 in MCF-7 breast cancer cells, negatively regulating cell proliferation, migration, invasion, and breast cancer stem cell p ropagation [11]. Deregulated Notch signaling has been linked to cancer, as Notch can act as either an oncogene or a tumor suppressor gene [12]. To determine the translational potential of miR-34a, it was incorporated into a targeting expression plasmid which can enhance cancer-specific promoter activity by 100-fold [10]. The plasmid, based on an engineered expression vector called T-VISA-miR-34a, induced persistent expression of miR-34a, significantly inhibiting tumor growth and prolonging survival [10]. miR-34a is not only negatively correlated with NOTCH1 expression in breast cancer tissues but also with tumor stages and metastasis, or the development of secondary malignant tumor growth [11].
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miR-34a has also been shown to directly bind to 3′-UTR of c-myc. C-Myc is known to suppress the transcription of genes involved in cell cycle regulation. It also positively regulates DNA synthesis that results in genomic instability [12]. C-Met is also a target of miR-34a and plays a role in tumor metastasis. miR-34a replacement in cancer cells downregulated the c-Met oncogene, inhibiting proliferation and metastasis of osteosarcoma, cervical carcinoma, choriocarcinoma, and hepatocellular carcinoma cells [10]. miR-34a therapy has shown promise in preclinical studies. Delivery of miR-34a at 1 mg/kg using MaxSuppressor in vivo RNALANCErII, (a lipid-based delivery reagent) inhibited the growth of established A549 non–small cell lung cancer (NSCLC) xenografts, demonstrating the anticancer activity of miR-34a [13]. The therapy resulted in increased apoptosis, decreased proliferation, and robust inhibition of tumor growth [14]. With successful preclinical studies, liposome-encapsulated miR-34a mimic was developed for clinical trials, and it became the first miRNA to reach phase 1 clinical trials. Although the clinical trial failed to have any positive impact on cancer patients, it represented an important step toward using miRNAs as anticancer drugs and shed light on the challenges associated with delivery systems and potential off-target effects [15]. With further development, application of miR-34 therapy in humans may become possible, leading the way for effective miRNA replacement therapy in human cancers.
miR-1258 Heparanase (HPSE) is a tumorigenic enzyme downregulated in brain metastatic breast cancer [16]. It participates in the degradation and remodeling of the extracellular matrix and also is involved in angiogenesis [17]. miR-1258 levels were found to be inversely correlated with HPSE expression, indicating that the miRNA may directly target the gene [16]. miR-1258 was generally downregulated in patient samples, although the correlation with HPSE expression was not evaluated in the same samples [16]. miR-1258 is currently being tested in a preclinical phase, having been injected into mice and tested for blocking metastasis [16]. One study found that mice with miR-1258-expressing HCCLM3 cells generated a significantly lower volume of tumors when compared to those generated by control-miR [17].
miR-15a and miR-16 Due to the difficulty in treating chemoresistant ovarian cancer, the need to identify novel therapeutic strategies exists [18]. miR-15a and miR-16 block several mechanisms that promote cancer progression, such as epithelial to mesenchymal transition, clonal growth, and the cisplatin efflux pump (ATP7B) [18]. These miRNAs serve as potential candidates for drug development due to their role in various aspects of cancer growth and progression [18]. Chronic lymphocytic leukemia (CLL) is characterized by overexpressed B cell lymphoma 2 (BCL2) protein and miR-15a, and miR-16-1 are downregulated in the majority of CLLs. These miRNAs are inversely correlated with BCL2 expression in CLL. Both miRNAs were found to negatively regulate BCL2 at a posttranscriptional level, resulting in induced apoptosis of the leukemia cell line model. Thus, miR-15a and miR-16 have the potential to be used as therapy in BCL2overexpressing tumors [19].
miR-192 miR-192 is a key regulator of tumor angiogenesis through regulation of tumor cell EGR1 and HOXB9 transcription factors. Angiogenesis is the development of new blood vessels. This is logically important to control because if the formation of blood vessels can be restricted, then cancer cells are no longer
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able to receive nutrients and oxygen from the blood stream and will not be able to survive and proliferate. Low expression of miR-192 is associated with poor clinical outcome in several cancer types, including renal and ovarian cancers. Mice treated with miR-192, delivered by a DOPC nanoliposome, showed an 85% reduction in tumor burden [20].
let-7 let-7 is underexpressed in multiple cancers, and restoration of its normal expression inhibited cancer growth by targeting oncogenes. let-7 proved to be an effective therapeutic against breast cancer and lung cancer in vivo in mouse-models of breast and lung cancers. let-7 was also found to regulate apoptosis and cancer stem cell differentiation, and for this reason, let-7 has a lot of potential as a cancer therapeutic [21].
miR-200 miR-200 has been seen to have different implications with different types of cancers. The expression of miR-200 actually has different impacts for various different types of cancer. For example, forced expression of miR-200 has been demonstrated to inhibit distant metastasis of lung cancer, but it can also enhance breast cancer metastasis. miR-200 is well known for its ability to inhibit the epithelialmesenchymal transition (EMT) through the downregulation of ZEB1 and ZEB2; however, it has been demonstrated to also be a regulator of several other oncogenes. Low miR-200 expression levels have been linked with increased chemotherapy and antiepidermal growth factor receptor therapy resistance. Additionally, miR-200 can induce apoptosis through upregulating CD95. All of these studies point to miR-200 having strong therapeutic potential as it plays an important role in cancer progression [22].
miR-630 Although miR-630 has multiple implications across different types of cancers, we will only touch on its implication in esophageal squamous cell carcinoma (ESCC), which is an extremely aggressive malignancy. miR-630 is significantly downregulated in ESCC tissues when compared to normal esophageal tissues. Within ESCC, it was determined that decreased miR-630 expression is directly associated with poor patient survival rates. It was also determined that ectopic miR-630 expression can inhibit invasion and metastasis while miR-630 knockdown induces invasion and metastasis. These findings point to miR-630 as a potential target for therapeutic applications in ESCC [23]. Collectively, many studies with miRNAs have shown encouraging results in preclinical studies, further establishing their role as promising candidates for anticancer therapeutics.
LONG NONCODING RNAs Long noncoding RNAs (lncRNAs) are important in many aspects of gene regulation. They interact with major pathways of cell growth, proliferation, differentiation, and survival and thus can regulate many aspects of tumor growth and progression. The roles of multiple lncRNAs in cancers have been characterized, and strategies to target them have been successful in inhibiting malignant cells in vivo and in vitro [24]. A majority of lncRNAs are located in the nucleus, which is consistent with their primary function as epigenetic regulators of gene expression. The therapeutic difficulty then arises from the fact that to target lncRNAs, effective delivery into the nucleus is necessary. Primary methods for targeting lncRNAs include antisense oligonucleotides and siRNAs. In this section, we will briefly describe the role of some of the lncRNAs in tumor pathogenesis.
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TRIM52-AS1 Expression of the lncRNA TRIM52-AS1 was found to be downregulated in renal cell carcinoma [25]. Increased reexpression of TRIM52-AS1 using a synthesized vector-mitigated cell migration and proliferation and even induced apoptosis of the renal cell carcinoma cells in vitro, and interference of its expression led to the opposite effects [25]. The study was among the first to demonstrate an lncRNA that functioned as a tumor suppressor in renal cell carcinoma.
TUG1 The lncRNA TUG1 and one of its transcript variants n377360 were found at significantly higher levels in osteosarcoma tissues when compared to nontumor tissues. The inhibition of TUG1 expression via TUG1 siRNA significantly increased osteosarcoma apoptosis. These results provided evidence that the use of siTUG1 may be a viable strategy in tumors that overexpress these factors [26].
NRCP The lncRNA ceruloplasmin (NRCP) was found to be highly upregulated in ovarian cancer cells and inhibition of NRCP resulted in increased cancer cell apoptosis, decreased cell proliferation, and decreased glycolysis. When siNRCP was delivered via a nanoliposomal method to a mouse model of ovarian cancer, there was significant reduction in tumor growth [27]. This study highlighted the potential of siRNA-mediated silencing of lncRNAs for anticancer drug development.
CCAT1 CCAT1 is a recently discovered oncogenic lncRNA that was found to be consistently upregulated in various cancer tissues. The aberrant expression of CCAT1 plays a role in cell apoptosis, proliferation, migration, and invasion by regulating various target genes and cellular pathways. CCAT1 is especially exciting due to the fact that it is significantly overexpressed in cancer tissues, but only seldom expressed in a variety of normal tissues, making it a potential therapeutic target [28].
MALAT1 The lncRNA-metastasis associated lung adeno carcinoma transcript 1 (MALAT1) is known to be misregulated in a significant number of cancer types. Specifically, MALAT1 has been seen to play an important role in colorectal cancer although it is also involved in hepatocellular carcinoma, bladder cancer, and lung cancer. The MALAT1 gene was analyzed in multiple fragments and the sequencing process was used to identify mutations in the gene in normal colorectal tissue, colorectal cancer cells (SW620, SW480), and primary colorectal cancer tissues. In SW620 and SW480 colorectal cancer cells as well as in primary colorectal cancer tissue, the fragment 5434 nt-6951 was mutated while SW480 cells and primary colorectal cancer tissues also had a mutation at fragment 6918 nt-8441. With the knowledge that there are specific mutations of the MALAT1 ncRNA in colorectal cancer cells, new therapeutic applications can be created that explore molecular mechanisms that occur during the invasion and metastasis of colorectal cancer cells [29].
PCGEM1 PCGEM1 (prostate cancer gene expression marker 1) is a prostate cancer–associated ncRNA gene specific to prostate tissue. There appears to be a significant association between elevated PCGEM1 expression levels in the prostate cancer cells of African American patients who also have the highest
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rate of mortality among prostate cancer patients. There is a higher expression level of PCGEM1 in prostate tumor specimens than in matched normal tissues. Overexpression of PCGEM1 in LNCaP cell culture models (LNCaP cells are androgen-sensitive human prostate adenocarcinoma cells) results in inhibition of doxorubicin induced apoptosis [30]. PCGEM1 is essentially induced through androgen signaling to enhance cell proliferation and colony formation. As a result, PCGEM1 is only prevalent in androgen-sensitive cell lines. This association between PCGEM1 and androgen signaling indicates the high specificity of PCGEM1 to prostate cancer. This provides vital insight into the potential of targeting PCGEM1 as a specific treatment for prostate cancer.
SMALL INTERFERING RNAs RNA interference (RNAi) is considered an effective research tool because it allows specific gene silencing to determine downstream biological effects. There are multiple ways to create RNAi-mediating molecules. They can either be synthesized as short hairpin (shRNA), which are then hewed into small interfering RNAs (siRNAs) by endogenous cellular dicer or they can be directly synthesized as siRNAs [2]. Chemical modification of siRNAs can minimize potential off-target effects, enhance nuclease stability, and modulate immune stimulation [31]. The ability to synthesize specific siRNAs for certain genes results in rapid and potentially more effective targeting of an oncogene. The systemic administration of siRNA enables the therapeutic to reach both localized and metastatic tumors, serving as an important drug for anticancer treatment [31]. Several phase 1 clinical trials in cancer patients have been completed using siRNAs as anticancer drugs [31].
CALAA-01 CALAA-01 is a four-component, polymer-based nanoparticle siRNA delivery system. The siRNA was designed to reduce the expression of RRM2 (ribonucleotide reductase regulatory subunit M2), a key enzyme in nucleic acid metabolism. RRM2 is upregulated in many cancer types, and its suppression is proposed to result in cell cycle arrest and cell death. CALAA-01 entered a phase 1 clinical trial and treatment was continued until the disease progressed or the treatment could no longer be tolerated by the patient. 21% of patients discontinued the trial due to adverse effects. No objective tumor responses were observed with the exception of one patient who had stable disease following 4 months of treatment at the dose of 30 mg/m2. However, fluorescence imaging showed that RRM2 protein and mRNA were inhibited in patients’ tumors posttreatment [31]. Overall, the results of this study showed that siRNAs have great potential as anticancer drugs and that off-target effects need to be more closely examined.
Atu027 Atu027 is a liposomal delivery vehicle containing siRNA that targets protein kinase N3 in endothelial cells. The purpose of targeting protein kinase N3 is to stabilize vessel integrity and attenuate inflammatory responses in the vasculature of tumors. Thus, by inhibiting protein kinase N3, the mobilization and engraftment of metastatic tumor cells are also inhibited. The clinical trial showed that in patients with advanced solid tumors, 52% had disease stabilization with Atu027 treatment. One patient had complete regression [31]. This study also highlighted some of the challenges of siRNA-mediated silencing, which includes the variety of off-target effects that may be experienced. Transient elevations in complement activation products were observed at all dosing levels, but no allergic reactions were observed and no antibodies against the siRNAs were detected [31].
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EphA2 The enrythropoietin-producing hepatocellular (Eph) receptors is the biggest subfamily of receptortyrosine kinases, which is a large family of transmembrane receptors involved in the coordination of diverse cellular functions including but not limited to proliferation and differentiation. Both are important cellular pathways in cancer. EphA2 is a member of the Eph subfamily and is usually expressed at low levels in normal epithelial cells. However, EphA2 overexpression has been shown to be strongly correlated with various solid tumors including glioma, non–small cell lung cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, and endometrial cancer [32]. EphA2 silencing has been shown to inhibit gastric cancer SGC-7901 cell proliferation in vitro and in vivo. It has also been shown to inhibit invasion and matrix metalloproteinase-9 expression [33]. Additionally, EphA2 overexpression was found to promote ovarian cancer cell growth by boosting extracellular matrix adhesion and augmenting anchorage dependent growth [32]. The specific mechanism of EphA2 is discussed to shed light on the mechanism behind how siRNA therapeutic “EPHARNA” has potential to be an extremely effective therapeutic for a multitude of cancers. The EphA2 can be targeted with EPHARNA (DOPC liposome containing siEphA2), which has been shown to be successful in murine and primate models [33]. As such, EPHARNA has various potential applications that target the receptor-tyrosine kinase subfamily of EphA2 in cancer-related pathways. EPHARNA is currently being tested in a Phase 1 clinical trial.
PIWI-INTERACTING RNAs PIWI-interacting RNAs (piRNAs) are endogenous small ncRNAs that protect the genome from invasive elements. In contrast to miRNAs, piRNAs interact with PIWI proteins which are members of the argonaute protein family and function mainly in the nucleus [34]. It has also been shown that animals that lack piRNA functions have defects in gametogenesis and tend to exhibit sterility. This warrants further investigation into exploring treatments for diseases that cause sterility as piRNAs may be intrinsically linked. Additionally, any descendants of animals that lacked piRNA were also more susceptible to genomic mutations in general. The piRNA pathway represses transposons posttranscriptionally and transcriptionally. PiRNAs are so complex and unique that they are considered the most enigmatic among noncoding silencing RNAs. As such, there is not much research to point to conclusive evidence with the application of piRNAs as a target for anticancer drug development [35]. However, there is some evidence that PIWI proteins and piRNAs may have important roles in epigenetic regulation of tumorigenesis [36]. An increasing number of studies have shown that abnormal piRNA expression is a notable trait across numerous tumor types, but their specific functions still remain unclear. Deep sequencing and RT-PCR of breast cancer tumors found that piR-4987, piR-20365, piR-20485, and piR-20582 were upregulated. PIWIL1 and PIWIL4 expression and other key players in the piRNA biogenesis pathway suggest that piRNAs have a driver role in breast cancer. One study associated piR932 with aberrant DNA methylation. In addition, many piRNAs have been found to be either upregulated or downregulated in cancer [34].
SMALL NUCLEOLAR RNAs Small nucleolar RNAs (snoRNAs), the most abundant group of intron-encoded ncRNAs, are small RNAs of 60–300 nucleotides in length and are predominantly found in the nucleolus [35]. Most snoRNAs function in posttranscriptional modification of ribosomal RNAs, which is important for the
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production of efficient RNAs [35,37]. Until recently, the role of snoRNAs dysfunction in cancer was not well understood or justified, but screens for cancer genes have uncovered unexpected roles of these molecules. Two main groups of snoRNAs have been described, box C/D snoRNAs and box H/ACA snoRNAs, which are defined by characteristic secondary structures and conserved signature-sequence elements [38]. The C/D and H/ACA RNAs are necessary for biological processes such as protein translation, mRNA splicing, and genome stability [38]. Most known C/D and H/ACA RNAs are responsible for the modification of other ncRNAs [38]. C/D RNAs direct 2′-O-ribose methylation, while H/ACA RNAs are responsible for pseudouridylation, or the conversion of uridine to pseudouridine. The box C/D snoRNA U50 was found to be downregulated in prostate cancer and has also been associated with breast cancer [35]. Normally, expression of U50 inhibits colony formation in prostate cancer, but when a homozygous two base-pair deletion is present, the effect on colony formation is abolished [35]. A recent study demonstrated that small nucleolar RNA 24 (SNORNA24), a box H/ACA snoRNA, was overexpressed in lung tumors [39]. Suppression of SNORNA24 via siRNA diminished the in vitro tumorigenicity of nonsmall cell lung cancer cells and inhibited cancer cell proliferation [39]. Many reports describe that snoRNAs are further processed to yield smaller fragments, sdRNAs or sno-derived RNAs, with miRNA-like functionality [40]. sdRNAs displayed stronger differential expression than miRNAs and were massively upregulated in prostate cancer [40]. The expression of SNORD78, a snoRNA, and its sdRNA was significantly higher in a subset of patients who developed metastatic disease, indicating that snoRNA and its sdRNA may represent novel diagnostic biomarkers for prostate cancer [40].
DELIVERY METHODS Various biotechnology companies are developing delivery technologies for miRNAs or siRNAs. Among these, liposomes are tiny vesicles composed of the same material as cell membranes, allowing them to easily pass into cells through the cell membrane [41]. They can be filled with drugs and used as delivery mechanisms to treat cancer and other diseases [41]. NOV340 technology, used to deliver miR-34a is a liposome containing amphoteric lipids, lipids which are capable of acting as an acid and a base [14]. When the miRNA mimics and lipids are mixed, a liposome forms and its slightly anionic character prevents disruption with the negative charges of cellular membranes in the target tissue [14]. Neutral liposomes are another effective method of siRNA or miRNA delivery. DOPC (1,2-dioleoylsn-glycero-3-phosphocholine) nanoliposomes are one such example. Because DOPC nanoliposomes are neutral, they are more effective in reaching their target as exemplified in the example of miR-630 [42]. In one study, the efficacy of aptamers and siRNA conjugation to streptavidin was evaluated as a potential delivery mechanism, and it was found that the siRNA-mediated inhibition of gene expression was as efficient as conventional lipid-based reagents. These results suggest new venues for the therapeutic delivery of siRNAs [43]. An alternative mechanism being researched involved nanotechnology and the use of hydrogels for local release of siRNA [44]. Hydrogels containing nanoparticles have been found to be successful in releasing siRNAs to a specific part of tissue in hopes of treating breast cancer and proved to be highly promising in combating cancer [44]. Furthermore, chitosan nanoparticles, another form of nanoparticles, decorated with RGD peptides localize to the tumor area and exert antiangiogenic effects [45].
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FUTURE IMPLICATIONS Recent advances in target screening technology have identified a multitude of ncRNA targets. 3D RNAi screening which produces phenotypes that are more relevant than those obtained in 2D cell cultures has now been adapted for use in large-scale screening. Improved approaches, such as Updated Targets of RNAi Reagents (UP-TORR) and Genome RNAi, make it possible to identify subsets of off-target effects that occur due to extended regions of complementarity between RNAi reagents and genes. UP-TORR stays up-to-date with gene annotation through daily synchronization and receives the most current information available [46]. Furthermore, advances in bioinformatics analyses allow for identification of potential off-target effects, producing the best results when large data sets are used [47]. Such advances allow for the identification of more ncRNAs that can be used for cancer treatment.
CONCLUSION Identifying targets for anticancer drug development holds great importance in the quest for improving patient outcomes. Cancer itself is very difficult to treat as it is a multiple pathway disease. Thus, identifying specific malfunctioning genes could allow for more effective cancer treatments. Given that the ncRNA part of the genome represent most of the genetic code, it is not surprising that ncRNAs play such a prominent role in many diseases, including cancer. The explosion in research related to unlocking the potential of ncRNAs as biomarkers and novel therapeutic strategies offers new hope for improving all aspects of cancer diagnosis and management.
GLOSSARY Antisense A sequence of nucleotides that are complimentary to a coding sequence. It could be either complimentary to a strand of a DNA double helix that is undergoing transcription or that of a messenger RNA. Downregulation The process by which a cell decreases the quantity of a cellular component such as an RNA or protein [25]. Doxorubicin Commonly used in cancer chemotherapy to treat a wide range of cancers including blood cancers and various types of carcinomas. A common adverse effect of doxorubicin includes hair loss. Epigenetics The study of inheritable alterations in gene expression that does not involve alterations in the gene sequence itself. It is believed that these changes occur for various reasons such as age, environment, or lifestyle [40]. Gametogenesis The process in which cells undergo meiosis to form gametes. Heterochromatin Chromosomes at a different density from its usual value (usually refers to a greater density), in which the activity of the genes is modified or suppressed. Liposomes Tiny vesicles composed of the same material as cell membranes, allowing them to easily pass into cells through the cell membrane [41]. LNCaP A cell line of human cells used commonly in oncological studies. They are androgen-sensitive human prostate adenocarcinoma cells derived from the left supraclavicular lymph node. They grow readily in vitro with a doubling time of 60 h. They are highly resistant to human fibroblast interferon and form clones in semisolid media [48].
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Long Noncoding RNA (lncRNA) Non-protein-coding RNA transcripts longer than 200 nucleotides with multiple functions and mechanisms [2]; may function as primary or spliced transcripts [4]. MicroRNA (miRNA) Single-stranded RNAs, approximately 19–24 nucleotides in length, which regulate transcription and translation of protein-coding genes [2]. NSCLC Non–small cell lung cancer. PIWI-Interacting RNAs (piRNAs) 21–35 nucleotides, PIWI-interacting RNAs, single-stranded. Play a role in germline transposon silencing and gametogenesis [2]. PIWI Proteins A subfamily of Argonaute proteins are known to bind to endogenous small RNAs. They are important in the cellular process of RNA interference [49]. Ribonucleoprotein A nucleoprotein that contains RNA. It combines ribonucleic acid and protein together. Ribosomal RNA A molecular component of a ribosome that makes polypeptides from amino acids that make up proteins. RNA-Induced Silencing Complexes (RISC) RISC is a multiprotein complex which incorporates siRNAs or miRNAs [50]. Small Interfering RNA (siRNAs) ∼20 nucleotides, small interfering RNAs; processed from several sources of endogenous double-stranded RNAs [2]. Small Nucleolar RNAs (snoRNAs) ∼60–300 nucleotides, small nucleolar RNAs. Help modify ribosomal RNAs [2]. Sno-derived RNAs (sdRNAs) sdRNAs derived from H/ACA snoRNAs are typically 20–24 nucleotides in length and originate from the 3′-end and those derived from C/D snoRNAs are predominantly 17–19 nucleotides long and originate from the 5′-end. TUG1 Taurine upregulated 1 (nonprotein coding). A long noncoding RNA [51]. Tumorigenesis The production or formation or tumors. Upregulation The increase of a cellular component.
GRANT SUPPORT This work is supported, in part, by the National Institutes of Health (CA177909, CA016672, CA109298, UH2TR000943, P50 CA083639, and P50 CA098258), Cancer Prevention and Research Institute of Texas (RP110595, RP120214), Ovarian Cancer Research Fund, Inc. (Program Project Development Grant), The RGK Foundation, The Gilder Foundation, The Judi A Rees ovarian cancer research fund, Mr. and Mrs. Daniel P. Gordon, The Blanton-Davis Ovarian Cancer Research Program and the Frank McGraw Memorial Chair in Cancer Research (A.K.S.). S.Y.W. is supported by Ovarian Cancer Research Fund Alliance, Foundation for Women’s Cancer, Colleen Dream Foundation, and CPRIT training grants (RP101502 and RP101489).
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NONCODING RNAS: NOVEL TARGETS IN ANTICANCER DRUG DEVELOPMENT
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Ragini Kondetimmanahalli1, Kshipra M. Gharpure2, Sherry Y. Wu2, Gabriel Lopez-Berestein2, Anil K. Sood2 1The
University of Texas at Austin, Austin, TX, United States; 2The University of Texas MD Anderson Cancer Center, Houston, TX, United States
CHAPTER OUTLINE Introduction������������������������������������������������������������������������������������������������������������������������������������������ 448 Clinical Relevance of Noncoding RNAs�������������������������������������������������������������������������������������������������� 448 MicroRNAs.......................................................................................................................... 449 miR-34a������������������������������������������������������������������������������������������������������������������������������������� 449 miR-1258����������������������������������������������������������������������������������������������������������������������������������� 450 miR-15a and miR-16������������������������������������������������������������������������������������������������������������������ 450 miR-192������������������������������������������������������������������������������������������������������������������������������������� 450 let-7������������������������������������������������������������������������������������������������������������������������������������������� 451 miR-200������������������������������������������������������������������������������������������������������������������������������������� 451 miR-630������������������������������������������������������������������������������������������������������������������������������������� 451 Long Noncoding RNAs.......................................................................................................... 451 TRIM52-AS1������������������������������������������������������������������������������������������������������������������������������ 452 TUG1����������������������������������������������������������������������������������������������������������������������������������������� 452 NRCP����������������������������������������������������������������������������������������������������������������������������������������� 452 CCAT1���������������������������������������������������������������������������������������������������������������������������������������� 452 MALAT1������������������������������������������������������������������������������������������������������������������������������������� 452 PCGEM1������������������������������������������������������������������������������������������������������������������������������������ 452 Small Interfering RNAs......................................................................................................... 453 CALAA-01���������������������������������������������������������������������������������������������������������������������������������� 453 Atu027��������������������������������������������������������������������������������������������������������������������������������������� 453 EphA2���������������������������������������������������������������������������������������������������������������������������������������� 454 PIWI-Interacting RNAs.......................................................................................................... 454 Small Nucleolar RNAs.......................................................................................................... 454 Delivery Methods���������������������������������������������������������������������������������������������������������������������������������� 455 Future Implications������������������������������������������������������������������������������������������������������������������������������� 456 Cancer and Noncoding RNAs. http://dx.doi.org/10.1016/B978-0-12-811022-5.00024-3 Copyright © 2018 Elsevier Inc. All rights reserved.
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Conclusion������������������������������������������������������������������������������������������������������������������������������������������� 456 Glossary����������������������������������������������������������������������������������������������������������������������������������������������� 456 Grant Support��������������������������������������������������������������������������������������������������������������������������������������� 457 References������������������������������������������������������������������������������������������������������������������������������������������� 457
INTRODUCTION Advances in genome research have given scientists reason to believe that noncoding RNAs (ncRNAs) hold an influential role in cell biology. While protein-coding genes make up less than 2% of the human genome, ncRNAs make up 98% and offer new opportunities for biomarker and therapeutic applications in cancer and other diseases [1]. ncRNA is a term used to describe multiple classes of RNA that do not encode proteins but rather regulate transcription, stability, or translation of genes [2,3]. These families of ncRNAs have an important role in both gene regulation and normal development and disease [4]. Among the various types of ncRNAs, microRNAs (miRNAs or miRs) have been the most studied by researchers. However, potential for anticancer drug development can be found in the many other existing forms of ncRNAs described in Table 24.1. In this chapter, we will discuss the various classes of ncRNAs as well as their potential therapeutic applications.
CLINICAL RELEVANCE OF NONCODING RNAs Over the last decade, numerous studies have produced evidence supporting the assertion that ncRNAs are an effective tool in cancer therapy. The “druggability gap” has been a concern for small molecule inhibitors because many promising targets such as mutated RAS proteins are very difficult to target with small molecule drugs [5]. The excitement behind the use of ncRNAs in cancer therapy is due to the fact that ncRNAs have the ability to directly target any “undruggable” targets. The therapeutic potential of being able to specifically treat various cancers warrants careful investigation into the mechanistic and biological roles of ncRNAs in different types of cancer. Table 24.1 Various Types of Noncoding RNAs miRNAs lncRNAs siRNAs piRNAs snoRNAs sdRNAs
Single-stranded RNAs, approximately 19–24 nucleotides in length, that regulate transcription and translation of protein-coding genes [1]. Non-protein-coding RNA transcripts longer than 200 nucleotides with multiple functions and mechanisms [1]; may function as primary or spliced transcripts [4]. Small interfering RNAs of approximately 20 nucleotides in length; processed from several sources of endogenous double-stranded RNAs [1]. ∼21–35 nucleotides, PIWI-interacting RNAs, single-stranded. Play a role in germline transposon silencing and gametogenesis [1]. ∼60–300 nucleotides, small nucleolar RNAs. Help modify ribosomal RNAs [1]. sdRNAs derived from H/ACA snoRNAs are typically 20–24 nucleotides in length and originate from the 3′-end and those derived from C/D snoRNAs are predominantly 17–19 nucleotides long and originate from the 5′-end.
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MICRORNAS miRNAs are small, single-stranded, ncRNA molecules that bind to messenger RNAs (mRNAs) and prevent them from being translated into proteins [6]. miRNAs are present in nearly all biological pathways in multicellular organisms, which include many pathways that are cancer-relevant such as proliferation, cell cycle control, apoptosis, differentiation, migration, and metabolism. Due to the profound influence of miRNAs within the cell, abnormal miRNA expression may affect a wide array of transcripts and cancer-related signaling pathways [6]. An individual miRNA can target hundreds of genes depending on its function and context, giving miRNAs a vast array of silencing capabilities [6]. A compilation of research studies indicates that aberrant miRNA expression is commonly noted in cancer cells, illustrating the importance to better understand their nature and how they can be applied therapeutically for treating various cancers. RNase III proteins play a key role in miRNA biogenesis. The RNase III drosha cleaves primary miRNAs (pri-miRNAs) resulting in hairpin-shaped pre-miRNAs which are then cut by the RNase III Dicer to create mature miRNAs [7]. Lower dicer expression was associated with advanced tumor stage and suboptimal surgical cytoreduction [8]. From these results, we can conclude that interruptions in miRNA functioning are heavily associated with cancer. miRNAs are often downregulated in human cancers, leading to overexpression of downstream genes which may contribute to cancer progression [9]. Large-scale microarray analyses of miRNAs have demonstrated that a substantial proportion is downregulated in human solid tumor samples compared to normal controls [9]. For key downregulated miRNAs, delivery of synthetic miRNA mimics may hold potential for cancer treatment. On the other hand, miRNAs may also be upregulated in some cancers. In such settings, treatment with anti-miRs can be used to block oncogenic effects of these miRNAs. One of the main considerations of both mimics and anti-miRs is the delivery method, which will be discussed later in this chapter. Although there are many more miRNAs than the ones discussed in this section, we will discuss select miRNAs as therapeutic candidates by briefly discussing their identification as well their biological effects in preclinical testing.
miR-34a The miR-34 family has proven to have robust tumor-suppressive properties. It downregulates many proteins involved in cell cycle, differentiation and cell survival, and also antagonizes processes that are necessary for cancer cell progression, such as metastasis, chemoresistance, and cancer stemness [10]. miR-34a is downregulated in human breast cancer as well as multiple other types of cancer [11]. Several miR-34a targets and their biological relevance are discussed below. miR-34a was found to directly and effectively target NOTCH1 in MCF-7 breast cancer cells, negatively regulating cell proliferation, migration, invasion, and breast cancer stem cell p ropagation [11]. Deregulated Notch signaling has been linked to cancer, as Notch can act as either an oncogene or a tumor suppressor gene [12]. To determine the translational potential of miR-34a, it was incorporated into a targeting expression plasmid which can enhance cancer-specific promoter activity by 100-fold [10]. The plasmid, based on an engineered expression vector called T-VISA-miR-34a, induced persistent expression of miR-34a, significantly inhibiting tumor growth and prolonging survival [10]. miR-34a is not only negatively correlated with NOTCH1 expression in breast cancer tissues but also with tumor stages and metastasis, or the development of secondary malignant tumor growth [11].
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miR-34a has also been shown to directly bind to 3′-UTR of c-myc. C-Myc is known to suppress the transcription of genes involved in cell cycle regulation. It also positively regulates DNA synthesis that results in genomic instability [12]. C-Met is also a target of miR-34a and plays a role in tumor metastasis. miR-34a replacement in cancer cells downregulated the c-Met oncogene, inhibiting proliferation and metastasis of osteosarcoma, cervical carcinoma, choriocarcinoma, and hepatocellular carcinoma cells [10]. miR-34a therapy has shown promise in preclinical studies. Delivery of miR-34a at 1 mg/kg using MaxSuppressor in vivo RNALANCErII, (a lipid-based delivery reagent) inhibited the growth of established A549 non–small cell lung cancer (NSCLC) xenografts, demonstrating the anticancer activity of miR-34a [13]. The therapy resulted in increased apoptosis, decreased proliferation, and robust inhibition of tumor growth [14]. With successful preclinical studies, liposome-encapsulated miR-34a mimic was developed for clinical trials, and it became the first miRNA to reach phase 1 clinical trials. Although the clinical trial failed to have any positive impact on cancer patients, it represented an important step toward using miRNAs as anticancer drugs and shed light on the challenges associated with delivery systems and potential off-target effects [15]. With further development, application of miR-34 therapy in humans may become possible, leading the way for effective miRNA replacement therapy in human cancers.
miR-1258 Heparanase (HPSE) is a tumorigenic enzyme downregulated in brain metastatic breast cancer [16]. It participates in the degradation and remodeling of the extracellular matrix and also is involved in angiogenesis [17]. miR-1258 levels were found to be inversely correlated with HPSE expression, indicating that the miRNA may directly target the gene [16]. miR-1258 was generally downregulated in patient samples, although the correlation with HPSE expression was not evaluated in the same samples [16]. miR-1258 is currently being tested in a preclinical phase, having been injected into mice and tested for blocking metastasis [16]. One study found that mice with miR-1258-expressing HCCLM3 cells generated a significantly lower volume of tumors when compared to those generated by control-miR [17].
miR-15a and miR-16 Due to the difficulty in treating chemoresistant ovarian cancer, the need to identify novel therapeutic strategies exists [18]. miR-15a and miR-16 block several mechanisms that promote cancer progression, such as epithelial to mesenchymal transition, clonal growth, and the cisplatin efflux pump (ATP7B) [18]. These miRNAs serve as potential candidates for drug development due to their role in various aspects of cancer growth and progression [18]. Chronic lymphocytic leukemia (CLL) is characterized by overexpressed B cell lymphoma 2 (BCL2) protein and miR-15a, and miR-16-1 are downregulated in the majority of CLLs. These miRNAs are inversely correlated with BCL2 expression in CLL. Both miRNAs were found to negatively regulate BCL2 at a posttranscriptional level, resulting in induced apoptosis of the leukemia cell line model. Thus, miR-15a and miR-16 have the potential to be used as therapy in BCL2overexpressing tumors [19].
miR-192 miR-192 is a key regulator of tumor angiogenesis through regulation of tumor cell EGR1 and HOXB9 transcription factors. Angiogenesis is the development of new blood vessels. This is logically important to control because if the formation of blood vessels can be restricted, then cancer cells are no longer
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able to receive nutrients and oxygen from the blood stream and will not be able to survive and proliferate. Low expression of miR-192 is associated with poor clinical outcome in several cancer types, including renal and ovarian cancers. Mice treated with miR-192, delivered by a DOPC nanoliposome, showed an 85% reduction in tumor burden [20].
let-7 let-7 is underexpressed in multiple cancers, and restoration of its normal expression inhibited cancer growth by targeting oncogenes. let-7 proved to be an effective therapeutic against breast cancer and lung cancer in vivo in mouse-models of breast and lung cancers. let-7 was also found to regulate apoptosis and cancer stem cell differentiation, and for this reason, let-7 has a lot of potential as a cancer therapeutic [21].
miR-200 miR-200 has been seen to have different implications with different types of cancers. The expression of miR-200 actually has different impacts for various different types of cancer. For example, forced expression of miR-200 has been demonstrated to inhibit distant metastasis of lung cancer, but it can also enhance breast cancer metastasis. miR-200 is well known for its ability to inhibit the epithelialmesenchymal transition (EMT) through the downregulation of ZEB1 and ZEB2; however, it has been demonstrated to also be a regulator of several other oncogenes. Low miR-200 expression levels have been linked with increased chemotherapy and antiepidermal growth factor receptor therapy resistance. Additionally, miR-200 can induce apoptosis through upregulating CD95. All of these studies point to miR-200 having strong therapeutic potential as it plays an important role in cancer progression [22].
miR-630 Although miR-630 has multiple implications across different types of cancers, we will only touch on its implication in esophageal squamous cell carcinoma (ESCC), which is an extremely aggressive malignancy. miR-630 is significantly downregulated in ESCC tissues when compared to normal esophageal tissues. Within ESCC, it was determined that decreased miR-630 expression is directly associated with poor patient survival rates. It was also determined that ectopic miR-630 expression can inhibit invasion and metastasis while miR-630 knockdown induces invasion and metastasis. These findings point to miR-630 as a potential target for therapeutic applications in ESCC [23]. Collectively, many studies with miRNAs have shown encouraging results in preclinical studies, further establishing their role as promising candidates for anticancer therapeutics.
LONG NONCODING RNAs Long noncoding RNAs (lncRNAs) are important in many aspects of gene regulation. They interact with major pathways of cell growth, proliferation, differentiation, and survival and thus can regulate many aspects of tumor growth and progression. The roles of multiple lncRNAs in cancers have been characterized, and strategies to target them have been successful in inhibiting malignant cells in vivo and in vitro [24]. A majority of lncRNAs are located in the nucleus, which is consistent with their primary function as epigenetic regulators of gene expression. The therapeutic difficulty then arises from the fact that to target lncRNAs, effective delivery into the nucleus is necessary. Primary methods for targeting lncRNAs include antisense oligonucleotides and siRNAs. In this section, we will briefly describe the role of some of the lncRNAs in tumor pathogenesis.
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TRIM52-AS1 Expression of the lncRNA TRIM52-AS1 was found to be downregulated in renal cell carcinoma [25]. Increased reexpression of TRIM52-AS1 using a synthesized vector-mitigated cell migration and proliferation and even induced apoptosis of the renal cell carcinoma cells in vitro, and interference of its expression led to the opposite effects [25]. The study was among the first to demonstrate an lncRNA that functioned as a tumor suppressor in renal cell carcinoma.
TUG1 The lncRNA TUG1 and one of its transcript variants n377360 were found at significantly higher levels in osteosarcoma tissues when compared to nontumor tissues. The inhibition of TUG1 expression via TUG1 siRNA significantly increased osteosarcoma apoptosis. These results provided evidence that the use of siTUG1 may be a viable strategy in tumors that overexpress these factors [26].
NRCP The lncRNA ceruloplasmin (NRCP) was found to be highly upregulated in ovarian cancer cells and inhibition of NRCP resulted in increased cancer cell apoptosis, decreased cell proliferation, and decreased glycolysis. When siNRCP was delivered via a nanoliposomal method to a mouse model of ovarian cancer, there was significant reduction in tumor growth [27]. This study highlighted the potential of siRNA-mediated silencing of lncRNAs for anticancer drug development.
CCAT1 CCAT1 is a recently discovered oncogenic lncRNA that was found to be consistently upregulated in various cancer tissues. The aberrant expression of CCAT1 plays a role in cell apoptosis, proliferation, migration, and invasion by regulating various target genes and cellular pathways. CCAT1 is especially exciting due to the fact that it is significantly overexpressed in cancer tissues, but only seldom expressed in a variety of normal tissues, making it a potential therapeutic target [28].
MALAT1 The lncRNA-metastasis associated lung adeno carcinoma transcript 1 (MALAT1) is known to be misregulated in a significant number of cancer types. Specifically, MALAT1 has been seen to play an important role in colorectal cancer although it is also involved in hepatocellular carcinoma, bladder cancer, and lung cancer. The MALAT1 gene was analyzed in multiple fragments and the sequencing process was used to identify mutations in the gene in normal colorectal tissue, colorectal cancer cells (SW620, SW480), and primary colorectal cancer tissues. In SW620 and SW480 colorectal cancer cells as well as in primary colorectal cancer tissue, the fragment 5434 nt-6951 was mutated while SW480 cells and primary colorectal cancer tissues also had a mutation at fragment 6918 nt-8441. With the knowledge that there are specific mutations of the MALAT1 ncRNA in colorectal cancer cells, new therapeutic applications can be created that explore molecular mechanisms that occur during the invasion and metastasis of colorectal cancer cells [29].
PCGEM1 PCGEM1 (prostate cancer gene expression marker 1) is a prostate cancer–associated ncRNA gene specific to prostate tissue. There appears to be a significant association between elevated PCGEM1 expression levels in the prostate cancer cells of African American patients who also have the highest
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rate of mortality among prostate cancer patients. There is a higher expression level of PCGEM1 in prostate tumor specimens than in matched normal tissues. Overexpression of PCGEM1 in LNCaP cell culture models (LNCaP cells are androgen-sensitive human prostate adenocarcinoma cells) results in inhibition of doxorubicin induced apoptosis [30]. PCGEM1 is essentially induced through androgen signaling to enhance cell proliferation and colony formation. As a result, PCGEM1 is only prevalent in androgen-sensitive cell lines. This association between PCGEM1 and androgen signaling indicates the high specificity of PCGEM1 to prostate cancer. This provides vital insight into the potential of targeting PCGEM1 as a specific treatment for prostate cancer.
SMALL INTERFERING RNAs RNA interference (RNAi) is considered an effective research tool because it allows specific gene silencing to determine downstream biological effects. There are multiple ways to create RNAi-mediating molecules. They can either be synthesized as short hairpin (shRNA), which are then hewed into small interfering RNAs (siRNAs) by endogenous cellular dicer or they can be directly synthesized as siRNAs [2]. Chemical modification of siRNAs can minimize potential off-target effects, enhance nuclease stability, and modulate immune stimulation [31]. The ability to synthesize specific siRNAs for certain genes results in rapid and potentially more effective targeting of an oncogene. The systemic administration of siRNA enables the therapeutic to reach both localized and metastatic tumors, serving as an important drug for anticancer treatment [31]. Several phase 1 clinical trials in cancer patients have been completed using siRNAs as anticancer drugs [31].
CALAA-01 CALAA-01 is a four-component, polymer-based nanoparticle siRNA delivery system. The siRNA was designed to reduce the expression of RRM2 (ribonucleotide reductase regulatory subunit M2), a key enzyme in nucleic acid metabolism. RRM2 is upregulated in many cancer types, and its suppression is proposed to result in cell cycle arrest and cell death. CALAA-01 entered a phase 1 clinical trial and treatment was continued until the disease progressed or the treatment could no longer be tolerated by the patient. 21% of patients discontinued the trial due to adverse effects. No objective tumor responses were observed with the exception of one patient who had stable disease following 4 months of treatment at the dose of 30 mg/m2. However, fluorescence imaging showed that RRM2 protein and mRNA were inhibited in patients’ tumors posttreatment [31]. Overall, the results of this study showed that siRNAs have great potential as anticancer drugs and that off-target effects need to be more closely examined.
Atu027 Atu027 is a liposomal delivery vehicle containing siRNA that targets protein kinase N3 in endothelial cells. The purpose of targeting protein kinase N3 is to stabilize vessel integrity and attenuate inflammatory responses in the vasculature of tumors. Thus, by inhibiting protein kinase N3, the mobilization and engraftment of metastatic tumor cells are also inhibited. The clinical trial showed that in patients with advanced solid tumors, 52% had disease stabilization with Atu027 treatment. One patient had complete regression [31]. This study also highlighted some of the challenges of siRNA-mediated silencing, which includes the variety of off-target effects that may be experienced. Transient elevations in complement activation products were observed at all dosing levels, but no allergic reactions were observed and no antibodies against the siRNAs were detected [31].
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EphA2 The enrythropoietin-producing hepatocellular (Eph) receptors is the biggest subfamily of receptortyrosine kinases, which is a large family of transmembrane receptors involved in the coordination of diverse cellular functions including but not limited to proliferation and differentiation. Both are important cellular pathways in cancer. EphA2 is a member of the Eph subfamily and is usually expressed at low levels in normal epithelial cells. However, EphA2 overexpression has been shown to be strongly correlated with various solid tumors including glioma, non–small cell lung cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, and endometrial cancer [32]. EphA2 silencing has been shown to inhibit gastric cancer SGC-7901 cell proliferation in vitro and in vivo. It has also been shown to inhibit invasion and matrix metalloproteinase-9 expression [33]. Additionally, EphA2 overexpression was found to promote ovarian cancer cell growth by boosting extracellular matrix adhesion and augmenting anchorage dependent growth [32]. The specific mechanism of EphA2 is discussed to shed light on the mechanism behind how siRNA therapeutic “EPHARNA” has potential to be an extremely effective therapeutic for a multitude of cancers. The EphA2 can be targeted with EPHARNA (DOPC liposome containing siEphA2), which has been shown to be successful in murine and primate models [33]. As such, EPHARNA has various potential applications that target the receptor-tyrosine kinase subfamily of EphA2 in cancer-related pathways. EPHARNA is currently being tested in a Phase 1 clinical trial.
PIWI-INTERACTING RNAs PIWI-interacting RNAs (piRNAs) are endogenous small ncRNAs that protect the genome from invasive elements. In contrast to miRNAs, piRNAs interact with PIWI proteins which are members of the argonaute protein family and function mainly in the nucleus [34]. It has also been shown that animals that lack piRNA functions have defects in gametogenesis and tend to exhibit sterility. This warrants further investigation into exploring treatments for diseases that cause sterility as piRNAs may be intrinsically linked. Additionally, any descendants of animals that lacked piRNA were also more susceptible to genomic mutations in general. The piRNA pathway represses transposons posttranscriptionally and transcriptionally. PiRNAs are so complex and unique that they are considered the most enigmatic among noncoding silencing RNAs. As such, there is not much research to point to conclusive evidence with the application of piRNAs as a target for anticancer drug development [35]. However, there is some evidence that PIWI proteins and piRNAs may have important roles in epigenetic regulation of tumorigenesis [36]. An increasing number of studies have shown that abnormal piRNA expression is a notable trait across numerous tumor types, but their specific functions still remain unclear. Deep sequencing and RT-PCR of breast cancer tumors found that piR-4987, piR-20365, piR-20485, and piR-20582 were upregulated. PIWIL1 and PIWIL4 expression and other key players in the piRNA biogenesis pathway suggest that piRNAs have a driver role in breast cancer. One study associated piR932 with aberrant DNA methylation. In addition, many piRNAs have been found to be either upregulated or downregulated in cancer [34].
SMALL NUCLEOLAR RNAs Small nucleolar RNAs (snoRNAs), the most abundant group of intron-encoded ncRNAs, are small RNAs of 60–300 nucleotides in length and are predominantly found in the nucleolus [35]. Most snoRNAs function in posttranscriptional modification of ribosomal RNAs, which is important for the
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production of efficient RNAs [35,37]. Until recently, the role of snoRNAs dysfunction in cancer was not well understood or justified, but screens for cancer genes have uncovered unexpected roles of these molecules. Two main groups of snoRNAs have been described, box C/D snoRNAs and box H/ACA snoRNAs, which are defined by characteristic secondary structures and conserved signature-sequence elements [38]. The C/D and H/ACA RNAs are necessary for biological processes such as protein translation, mRNA splicing, and genome stability [38]. Most known C/D and H/ACA RNAs are responsible for the modification of other ncRNAs [38]. C/D RNAs direct 2′-O-ribose methylation, while H/ACA RNAs are responsible for pseudouridylation, or the conversion of uridine to pseudouridine. The box C/D snoRNA U50 was found to be downregulated in prostate cancer and has also been associated with breast cancer [35]. Normally, expression of U50 inhibits colony formation in prostate cancer, but when a homozygous two base-pair deletion is present, the effect on colony formation is abolished [35]. A recent study demonstrated that small nucleolar RNA 24 (SNORNA24), a box H/ACA snoRNA, was overexpressed in lung tumors [39]. Suppression of SNORNA24 via siRNA diminished the in vitro tumorigenicity of nonsmall cell lung cancer cells and inhibited cancer cell proliferation [39]. Many reports describe that snoRNAs are further processed to yield smaller fragments, sdRNAs or sno-derived RNAs, with miRNA-like functionality [40]. sdRNAs displayed stronger differential expression than miRNAs and were massively upregulated in prostate cancer [40]. The expression of SNORD78, a snoRNA, and its sdRNA was significantly higher in a subset of patients who developed metastatic disease, indicating that snoRNA and its sdRNA may represent novel diagnostic biomarkers for prostate cancer [40].
DELIVERY METHODS Various biotechnology companies are developing delivery technologies for miRNAs or siRNAs. Among these, liposomes are tiny vesicles composed of the same material as cell membranes, allowing them to easily pass into cells through the cell membrane [41]. They can be filled with drugs and used as delivery mechanisms to treat cancer and other diseases [41]. NOV340 technology, used to deliver miR-34a is a liposome containing amphoteric lipids, lipids which are capable of acting as an acid and a base [14]. When the miRNA mimics and lipids are mixed, a liposome forms and its slightly anionic character prevents disruption with the negative charges of cellular membranes in the target tissue [14]. Neutral liposomes are another effective method of siRNA or miRNA delivery. DOPC (1,2-dioleoylsn-glycero-3-phosphocholine) nanoliposomes are one such example. Because DOPC nanoliposomes are neutral, they are more effective in reaching their target as exemplified in the example of miR-630 [42]. In one study, the efficacy of aptamers and siRNA conjugation to streptavidin was evaluated as a potential delivery mechanism, and it was found that the siRNA-mediated inhibition of gene expression was as efficient as conventional lipid-based reagents. These results suggest new venues for the therapeutic delivery of siRNAs [43]. An alternative mechanism being researched involved nanotechnology and the use of hydrogels for local release of siRNA [44]. Hydrogels containing nanoparticles have been found to be successful in releasing siRNAs to a specific part of tissue in hopes of treating breast cancer and proved to be highly promising in combating cancer [44]. Furthermore, chitosan nanoparticles, another form of nanoparticles, decorated with RGD peptides localize to the tumor area and exert antiangiogenic effects [45].
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FUTURE IMPLICATIONS Recent advances in target screening technology have identified a multitude of ncRNA targets. 3D RNAi screening which produces phenotypes that are more relevant than those obtained in 2D cell cultures has now been adapted for use in large-scale screening. Improved approaches, such as Updated Targets of RNAi Reagents (UP-TORR) and Genome RNAi, make it possible to identify subsets of off-target effects that occur due to extended regions of complementarity between RNAi reagents and genes. UP-TORR stays up-to-date with gene annotation through daily synchronization and receives the most current information available [46]. Furthermore, advances in bioinformatics analyses allow for identification of potential off-target effects, producing the best results when large data sets are used [47]. Such advances allow for the identification of more ncRNAs that can be used for cancer treatment.
CONCLUSION Identifying targets for anticancer drug development holds great importance in the quest for improving patient outcomes. Cancer itself is very difficult to treat as it is a multiple pathway disease. Thus, identifying specific malfunctioning genes could allow for more effective cancer treatments. Given that the ncRNA part of the genome represent most of the genetic code, it is not surprising that ncRNAs play such a prominent role in many diseases, including cancer. The explosion in research related to unlocking the potential of ncRNAs as biomarkers and novel therapeutic strategies offers new hope for improving all aspects of cancer diagnosis and management.
GLOSSARY Antisense A sequence of nucleotides that are complimentary to a coding sequence. It could be either complimentary to a strand of a DNA double helix that is undergoing transcription or that of a messenger RNA. Downregulation The process by which a cell decreases the quantity of a cellular component such as an RNA or protein [25]. Doxorubicin Commonly used in cancer chemotherapy to treat a wide range of cancers including blood cancers and various types of carcinomas. A common adverse effect of doxorubicin includes hair loss. Epigenetics The study of inheritable alterations in gene expression that does not involve alterations in the gene sequence itself. It is believed that these changes occur for various reasons such as age, environment, or lifestyle [40]. Gametogenesis The process in which cells undergo meiosis to form gametes. Heterochromatin Chromosomes at a different density from its usual value (usually refers to a greater density), in which the activity of the genes is modified or suppressed. Liposomes Tiny vesicles composed of the same material as cell membranes, allowing them to easily pass into cells through the cell membrane [41]. LNCaP A cell line of human cells used commonly in oncological studies. They are androgen-sensitive human prostate adenocarcinoma cells derived from the left supraclavicular lymph node. They grow readily in vitro with a doubling time of 60 h. They are highly resistant to human fibroblast interferon and form clones in semisolid media [48].
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Long Noncoding RNA (lncRNA) Non-protein-coding RNA transcripts longer than 200 nucleotides with multiple functions and mechanisms [2]; may function as primary or spliced transcripts [4]. MicroRNA (miRNA) Single-stranded RNAs, approximately 19–24 nucleotides in length, which regulate transcription and translation of protein-coding genes [2]. NSCLC Non–small cell lung cancer. PIWI-Interacting RNAs (piRNAs) 21–35 nucleotides, PIWI-interacting RNAs, single-stranded. Play a role in germline transposon silencing and gametogenesis [2]. PIWI Proteins A subfamily of Argonaute proteins are known to bind to endogenous small RNAs. They are important in the cellular process of RNA interference [49]. Ribonucleoprotein A nucleoprotein that contains RNA. It combines ribonucleic acid and protein together. Ribosomal RNA A molecular component of a ribosome that makes polypeptides from amino acids that make up proteins. RNA-Induced Silencing Complexes (RISC) RISC is a multiprotein complex which incorporates siRNAs or miRNAs [50]. Small Interfering RNA (siRNAs) ∼20 nucleotides, small interfering RNAs; processed from several sources of endogenous double-stranded RNAs [2]. Small Nucleolar RNAs (snoRNAs) ∼60–300 nucleotides, small nucleolar RNAs. Help modify ribosomal RNAs [2]. Sno-derived RNAs (sdRNAs) sdRNAs derived from H/ACA snoRNAs are typically 20–24 nucleotides in length and originate from the 3′-end and those derived from C/D snoRNAs are predominantly 17–19 nucleotides long and originate from the 5′-end. TUG1 Taurine upregulated 1 (nonprotein coding). A long noncoding RNA [51]. Tumorigenesis The production or formation or tumors. Upregulation The increase of a cellular component.
GRANT SUPPORT This work is supported, in part, by the National Institutes of Health (CA177909, CA016672, CA109298, UH2TR000943, P50 CA083639, and P50 CA098258), Cancer Prevention and Research Institute of Texas (RP110595, RP120214), Ovarian Cancer Research Fund, Inc. (Program Project Development Grant), The RGK Foundation, The Gilder Foundation, The Judi A Rees ovarian cancer research fund, Mr. and Mrs. Daniel P. Gordon, The Blanton-Davis Ovarian Cancer Research Program and the Frank McGraw Memorial Chair in Cancer Research (A.K.S.). S.Y.W. is supported by Ovarian Cancer Research Fund Alliance, Foundation for Women’s Cancer, Colleen Dream Foundation, and CPRIT training grants (RP101502 and RP101489).
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