RNA-Based Applications in Diagnostic and Therapeutics for Cancer

CHAPTE R 2

RNA-Based Applications in Diagnostic and Therapeutics for Cancer Chapter Outline 2.1 Introduction  34 2.2 miRNA Working Model and Targets  35 2.3 miRNAs as Diagnostic Markers  38 2.4 RNA in Various Cancers  38

2.4.1 mRNA Stability in Gastric Cancer  38 2.4.2 miRNA in Medulloblastoma  39 2.4.2.1 Medulloblastoma Metastasis  39 2.4.3 miRNA in Prostate Cancer  40 2.4.3.1 PCA3 Diagnostic Assay Using lncRNA as Diagnostic Markers  40 2.4.4 Salivary Transcriptome Diagnostics for Oral Cancer  41

2.5 miRNA-Mediated Pathways in Cancer  41 2.6 RNA Detection Methods  42

2.6.1 Microarray Approach  43 2.6.2 Real-Time PCR–Based Detection  44 2.6.3 Northern Blot Analysis  44 2.6.4 In Situ Hybridization  45 2.6.5 Differential Display Method  45 2.6.6 Detection by High-Throughput Next-Generation Sequencing Platforms  45 2.6.7 Circulating miRNAs—Experimental Techniques and Normalization  45

2.7 RNA as Biomarkers in Cancer Diagnosis  46

2.7.1 miRNAs in Exosomes From Cancer Cells  47 2.7.2 Exosome Transported Role of miRNAs in Metastasis  47 2.7.3 Exosome-Derived miRNAs as Potential Biomarkers  48 2.7.4 Circulating miRNA Carried in Particles  49 2.7.4.1 Body Fluid Particles and Their Roles in Recipient Cells  49

2.8 Therapeutic Applications of RNA  50

2.8.1 Therapeutic Potential of miRNAs  50 2.8.2 Therapeutic Gene Silencing—RNA Interference Technology  50 2.8.3 Applications of RNAi  51 2.8.4 lncRNA-Based Therapies and Modular Assembly of Small Molecules  52 2.8.5 Therapeutic Potential of Exosome-Derived miRNAs in Cancer  52 2.8.6 mRNA-Dendritic Cells in Anticancer Vaccination  53

2.9 Conclusion  53 References  54 Advances in Cell and Molecular Diagnostics. http://dx.doi.org/10.1016/B978-0-12-813679-9.00002-6 Copyright © 2018 Elsevier Inc. All rights reserved.

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34  Chapter 2

2.1 Introduction For many years RNA was believed to have only three major roles in the cell: as a DNA photocopy (messenger RNA [mRNA]), as a coupler between the genetic code and the protein building blocks (transfer RNA), and as a structural component of ribosomes (ribosomal RNA). However in recent years, researchers have deciphered that the roles adopted by RNA are much broader and interesting. RNA plays an important role in regulating cellular processes–from cell division, differentiation, and growth to cell aging and death. Defects in certain RNAs or the regulation of RNAs have been implicated in a number of important human diseases, including heart disease, some cancers, stroke, and many others. Noncoding RNAs (ncRNAs) are RNA transcripts that do not encode for a protein. Their identity, function, and dysregulation in cancer are only beginning to be understood. General convention divide ncRNAs into two main categories: small ncRNAs <200bp and long ncRNAs (lncRNAs) >200bp. Depending on the type of ncRNA, transcription can occur by any of the three RNA polymerases (RNA Pol I, RNA Pol II, or RNA Pol III). The functions of ncRNAs include epigenetic transcriptional regulation of target genes that results in transcriptional repression. Some ncRNAs contribute to gene regulation by influencing the activity of gene enhancers, e.g., HOTTIP, eRNAs. lncRNAs are involved in modulating the activity of tumor suppressor genes like p53, CDKN2A, and CDKN2B. ncRNAs are involved in regulation of mRNA processing and translation. RNA-RNA interactions between ncRNAs and mRNA are conceptually akin to microRNAs (miRNA) regulation of mRNAs. miRNAs are small regulatory RNA that modulate the expression of their target genes and plays vital role in a variety of physiological processes, such as development, differentiation, cell proliferation, and apoptosis. miRNA synthesis requires several post-transcriptional processing steps to yield the functional mature miRNA. They play a key role in maintaining the balance among genes regulating cell’s fate, and their deregulation leads to different human malignancies that ultimately results in the cancer development and its progression. miRNA genes represent approximately 1% of the genome: it has been estimated that one MicroRNA can regulate up to 30% of the genes. To date, there are 940 mature human miRNA sequences listed in the miRNA registry (Sanger miRBaserelease 15). The deregulation of the expression of miRNAs has been shown to pave a way to cancer development, which includes mechanisms like deletions, amplifications, or mutations involving miRNA loci, or the inhibition of processing. Currently, miRNA expression profiling has rising importance as a useful diagnostic and prognostic tool, and many studies have predicted and shown that miRNAs act as either an oncogene or a tumor suppressor (Iorio and Croce, 2009). The discovery of miRNAs as potential biomarkers in serum or plasma represented a new approach for diagnostic screening in blood. As the approaches to cancer screening are

RNA-Based Applications in Diagnostic and Therapeutics for Cancer  35 invasive and difficult in detecting cancer in its early stages, it is important to understand the characteristics of secretory miRNAs and their usefulness in cancer detection. Proper control of miRNA expression is an essential process for maintaining a steady state of the cellular machinery. The existence of circulating miRNAs in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic markers (Kosaka et al., 2010). Recently, mRNA has become a tool of interest in anticancer vaccination approaches as it induces strong immune responses against cancer. The growing numbers of preclinical trials and as a consequence the increasing clinical application of mRNA as an off-the-shelf anticancer vaccine signifies a renaissance for transcript-based antitumor therapy (Van Lint et al., 2014). However, due to its rapid degradation in situ, direct application of mRNA faced many challenges during its early use. Consequently, mRNA was mainly used for the ex vivo transfection of dendritic cells, professional antigen-presenting cells known to trigger immunity. The direct application of mRNA attracted researcher’s interest as they became aware of the many advantages mRNA offers. As a direct source of gene products, mRNA has several advantages, including a lack of requirement for nuclear entry (Van Driessche et al., 2005), which poses a significant barrier to pDNA delivery, especially in nondividing cells. mRNA also has a negligible chance of integrating into the host genome, avoiding aberrant transcription and expression of oncogenes caused by insertional mutagenesis. mRNA is not being used as a conventional tool for gene therapy due its vulnerability, ubiquitously expressed nuclease, and the immune stimulation that substantially suppresses expression efficiency and elicits an immune toxicity in the host (Ponsaerts et al., 2003).

2.2 miRNA Working Model and Targets Biogenesis of miRNA represents multiple stages when there are potential points of intervention for antagomiRs (anti-miRs). As represented in the above figure (Fig. 2.1), anti-miRs can be designed to inhibit either the mature miRNA in the active RISC complex or any of its precursor. First step in miRNA production is transcription into long primary RNA transcripts known as pri-miRNAs. The pri-miRNAs are cleaved by Drosha in the nucleus into a 70 base pair pre-miRNA hairpin intermediate. Pre-miRNA are then exported to the cytoplasm and processed by Dicer ribonucleases into mature, double-stranded miRNA that are between 18 and 25 nucleotides in length. The mature miRNA interacts with the proteins that comprise the RISC, which separates the guide strand of the mature miRNA from the passenger strand, retaining the guide strand to form an active RISC. The miRNA guide strand then binds to complementary mRNA and enables target mRNA cleavage by the RISC-associated endonuclease Argonaut2 (Ago2). Most miRNA inhibitors are designed to bind to and inhibit the activity of the mature miRNA guide strand once it is loaded into the RISC (Fig. 2.1D–F). However, there has also been proof of successful inhibition of the mature miRNA precursors. Targeting pri- and pre-miRNAs can be advantageous because they contain sequences that are not

36  Chapter 2

(H)

(B) (A)

(C)

(G)

(D)

(F)

(E)

Figure 2.1 Sites of intervention for different anti-miRs. (A) miRNA biogenesis pathway. Anti-miRNA oligos (AMOs) are typically single-stranded oligos that are introduced exogenously into the cell and can bind to (B) pri-miRNA to inhibit Drosha activity or (C) pre-miRNA to inhibit Dicer cleavage. (D) miRNA sponges are expressed as transgenes that contain multiple miRNA-binding sites for competitive inhibition of binding to mRNA. (E) AMOs are most commonly designed to bind to and inhibit mature miRNA. (G) Blockmirs are oligonucleotides that block miRNA activity by specifically masking the 3′ UTR of target mRNA. Small molecule miRNA inhibitors act by either (F) inhibiting the formation of active RNA-induced silencing complex (RISC), or (H) preventing expression of miRNA genes into pri-miRNA. Courtesy: Beavers, K.R., Nelson, C.E., Duvall, C.L., 2015. MiRNA inhibition in tissue engineering and regenerative medicine. Adv. Drug Deliv. Rev. 88, 123–137.

present in mature miRNA; these sequences are typically not conserved among different miRNAs (even from the same family). Targeting miRNA precursors therefore enables better discrimination among miRNAs that possess similar mature sequences. miRNA activity can be inhibited by targeting the pri-miRNA or the pre-miRNA (Fig. 2.1B and C). Anti-miRs complementary to the pri-miRNA Drosha cleavage site inhibited processing

RNA-Based Applications in Diagnostic and Therapeutics for Cancer  37

Figure 2.2 Potential anti-miR targets for regenerative medicine. Courtesy: Beavers, K.R., Nelson, C.E., Duvall, C.L., 2015. MiRNA inhibition in tissue engineering and regenerative medicine. Adv. Drug Deliv. Rev. 88, 123–137.

into pre-miRNA, while those complementary to the Dicer-cleavage site on the stem of pre-miRNA inhibited Dicer processing into mature miRNA. The potential disadvantages in targeting miRNA precursors are that they are relatively transient species during processing to mature miRNA and that not all miRNAs are equally susceptible to inhibition at the level of pre- or pri-RNA. In addition, pri-miRNAs are especially difficult targets because they require inhibitor access to the nucleus. Another alternative approach to targeting mature miRNA known as “blockmir” technology has also shown promise. Blockmirs are approximately 15-mer antisense oligonucleotides that are instead targeted to the mRNA and function to target and block miRNA-binding sites (Fig. 2.1G). These molecules bind to untranslated regions of mRNA where miRNA bind, thus blocking miRNA-induced mRNA degradation while retaining the ability of the mRNA to be translated into protein. Because blockmirs target individual mRNAs, they may provide a means to reduce off-target effects and to achieve more predictable pharmacodynamics than anti-miRs that block all miR activities. This better specificity may aid clinical translation of anti-miR therapeutics for regenerative medicine, but it also abrogates the ability to develop diagnostic or therapeutics that simultaneously detect and regulate multiple genes.

38  Chapter 2 Basic scientists have and continue to elucidate a plethora of miRNAs and their mRNA targets. These discoveries provide abundant new opportunities for development of interventions to enhance tissue regeneration (Fig. 2.2). Anti-miRs may prove to be important both for development of translatable therapeutics and for mechanistic, loss-of-function studies intended to better understand miRNA function in tissue development and healing. Both the potential advantage and the potential downfall of targeting miRNAs in tissue regeneration is the robust, and often incompletely understood, effects that miRNAs can have on networks of multiple genes. It is, however, enticing that a single therapeutic could orchestrate a more comprehensive response that better recapitulates the complex biological control of tissue regeneration.

2.3 miRNAs as Diagnostic Markers miRNA expression profiles differ between disease states and normal tissue. Multiple studies have used miRNAs as diagnostics, either alone or in combination with other known biomarkers. Initial studies examining miRNA expression used tissues to determine functional and diagnostic roles of miRNAs. However, bodily fluids are more readily available and less invasive (in some instances) than biopsies. miRNAs are secreted by cells through exosomes and extracellular vesicles, and secreted miRNAs remain stable in bodily fluids. miRNAs have been isolated from blood (serum and plasma), saliva, urine, feces, follicular fluid, synovial fluid, pancreatic juice, bile, gastric juice, and other bodily fluids, and are being examined for utility as biomarkers for related diseases. A relevant example is miRNA profiling of bile to identify cholangiocarcinoma at an early stage. Bile from cholangiocarcinoma and control patients was assayed for the presence of miRNAs. They discovered a 5-miRNA panel that predicted early tumors better than carbohydrate antigen (CA19-9), with an overall sensitivity of 67% and specificity of 96%. Combining the miRNA panel with CA19-9, sensitivity increased to 89.7%. This panel was also able to identify patients without metastatic lymph nodes better than CA19-9, indicating the ability to detect tumors at an early stage. miRNAs also have the ability to indicate the cell type being analyzed. The most well-known example of this is the liver-specific miRNA, miR-122. miR-122 plays a role in cholesterol metabolism, hepatocellular carcinoma, and hepatitis C virus infection. Other tissue-specific miRNAs include miR-134 and miR-124a in the brain and miR-1 and miR-133 in the muscle.

2.4 RNA in Various Cancers 2.4.1 mRNA Stability in Gastric Cancer Cyclooxygenase-2 (COX-2) promotes carcinogenesis and its expression is linked to the clinicopathologic characteristics in gastric cancer. HuR is an RNA-binding protein that controls the stability of certain transcripts including COX-2 (Sengupta et al., 2003).

RNA-Based Applications in Diagnostic and Therapeutics for Cancer  39 The cytoplasmic HuR expression has been shown to associate with high grade and reduced survival in colon (Dixon et al., 2001), ovarian (Erkinheimo et al., 2003; Denkert et al., 2004) and breast cancers (Heinonen et al., 2005). Cytoplasmic HuR expression was associated with high COX-2 expression and with reduced survival, whereas nuclear positivity for HuR was not. Expressions of HuR and COX-2 were reduced when TMK-1 cells were treated with HuR small interfering RNA. It was observed that cytoplasmic HuR expression correlates to COX-2 expression and poor survival in gastric cancer and that reduction in HuR expression leads to inhibition of COX-2 expression. These results suggest that HuR regulates expression of COX-2, which is an independent prognostic marker for poor outcome in gastric cancer and that COX-2 may be one factor that facilitates carcinogenic properties of HuR. Hence they concluded that the downregulation of the mRNA stability factor HuR inhibits COX-2 expression in TMK-1 gastric cancer cells.

2.4.2 miRNA in Medulloblastoma miR-124a was amongst the first to be characterized as a tumor suppressor miRNA in MB (Pierson et al., 2008). miR-124 is a brain-enriched miRNA found to be expressed in the external granule cells of the cerebellum, reported to be cells of origin of MBs (Cheng et al., 2009). miR-124a was found to be decreased in MB cells compared to normal cerebellum, and restoration of its function inhibits medulloblastoma (MB) cell proliferation (Pierson et al., 2008). MB patient samples with aggressive vs. nonaggressive expression of a set of four upregulated miRNAs (let-7g, miR-19a, miR-106b, and miR-191) were previously identified in other brain tumors such as glioblastomas (Northcott et al., 2009) and impaired expression of specific miRNAs that are known to be expressed during neuronal development such as (miR-9, miR-125a, miR-128a, miR128b, and miR-181b), and some of these miRNAs might be involved in MB tumorigenesis (Pang et al., 2009). In a recent survey of miRNA expression in pediatric brain tumors including MB, miR-216, miR-135b, miR-217, miR-592, and miR-340 were found to be upregulated, whereas miR-92b, miR-23a, miR-27a, miR-146b, and miR-22 were found to be downregulated, compared to normal brain tissue (Birks et al., 2011). Overexpression of other oncogenic miRNA clusters such as miR-183-96–182 was reported in MB subgroups characterized by genetic amplification of MYC, and not in sonic hedgehog MBs and promotes metastasis, a hallmark of MYC aggressive MBs. 2.4.2.1 Medulloblastoma Metastasis miRNAs play a key role as suppressors or promoters of metastasis according to their mRNA targets. miR-21 upregulation is associated with metastasis and cell migration in a variety of solid tumors including breast, lung, colon, prostate, pancreas and stomach cancers, as well as brain tumors such as glioblastoma.

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2.4.3 miRNA in Prostate Cancer A few lncRNAs are biomarkers for prostate cancer claim that small non-coding RNA (sncRNA) are associated with the development and progression of this malignancy. The analysis states that the composition of the entire small transcriptome by Illumina/Solexa deep sequencing, checked the miRNA expression signatures of patient samples during prostate cancer progression by Agilent microarrays, and they obtained the differential miRNA expression profiles strongly associated with prostate cancer diagnosis and clinical outcome. They also used quantitative real-time PCR (Q-PCR) to cross validate both the platforms using a selected subset of miRNA. 2.4.3.1 PCA3 Diagnostic Assay Using lncRNA as Diagnostic Markers Tissue-specific expression distinguishes lncRNAs from miRNAs and protein-coding mRNAs. Given this specificity ncRNAs may be better biomarkers to many current protein-coding biomarkers, both of tissue-of-origin tests and for cancer diagnostics. A prominent example is development of PCA3 diagnostic assay for prostate cancer, which is clinically used. PCA3 is an lncRNA that is prostate specific gene and markedly overexpressed in prostate cancer. In this assay, PCA3 transcript is detected in urine samples from patients with prostate cancer. This sample contains prostate cancer cells that are released into and passed through the urethra. Thus, monitoring PCA3 does not require invasive procedures. In this test, urine sample is collected from patient, nucleic acid from the urine sediment is isolated, and the PCA3 expression is quantified. Illumina/Solexa deep sequencing method was used by Martens-Uzunova et al. (2012) to examine the diversity and abundance of sncRNA in two small prostate cancer RNA libraries: organ-confined prostate cancer and metastatic lymph node prostate cancer. miRNA, small nucleolar RNA (snoRNA), small nuclear RNA, ribosomal RNA, tRNA, fragments of large ncRNA, genomic repeats, mRNA, and other hairpins were observed in both the samples’ total RNA pool. Furthermore, to assess if the relative downregulation of miRNAs in the metastatic lymph node prostate cancer sample is mediated by alterations in the miRNA processing pathways, a Q-PCR was performed to check the expression levels of miRNA processing enzymes Drosha and Dicer. No significant change in the expression levels of the enzymes was found between the two samples. To evaluate whether the miRNAs have a potential clinical diagnostic or prognostic value a diagnostic miR-classifier and a prognostic miR-predictor were used. miRNA predictor forecasts prostate cancer recurrence after radical prostatectomy and demonstrates enhanced accuracy and suggests novel directions into prognostic prediction of prostate cancer. All in all, the findings provide miRNA expression signatures that may serve as an accurate tool for the diagnosis and prognosis of prostate cancer.

RNA-Based Applications in Diagnostic and Therapeutics for Cancer  41

2.4.4 Salivary Transcriptome Diagnostics for Oral Cancer The large panel of human mRNA in saliva (Li et al., 2004) has led to a novel clinical approach for applications in disease diagnostics as well as for normal health surveillance. It is a high-throughput, robust, and reproducible approach to retrieve RNA from saliva. Moreover, using saliva as a diagnostic fluid proves to be an inexpensive, noninvasive, and accessible diagnostic methodology. In the proposed hypothesis of Li et al. (2004), discrete mRNA expression patterns can be identified in saliva from cancer patients, and the differentially expressed transcripts can aid as biomarkers for cancer detection. The proof-of-principle disease in this study was oral squamous cell carcinoma (OSCC) and the rationale that oral cancer cells are immersed in the salivary environment, which showed genetic heterogeneity among the patients with OSCC (El-Naggar et al., 2001). RNA isolation was done from the saliva supernatant, followed by two-round linear amplification with T7 RNA polymerase. Human Genome U133A microarrays were applied for profiling human salivary transcriptome. Microarray analysis showed there are 1679 genes that exhibited significantly different expression level in saliva between cancer patients and controls. One of the most important applications of the salivary transcriptome diagnostics approach is to detect the cancer conversion of oral premalignant lesions in oral cancer. When fully explored, this innovative approach, “salivary transcriptome diagnostics,” will provide new opportunities for early diagnostics of oral cancer and other human diseases.

2.5 miRNA-Mediated Pathways in Cancer miRNA expressed in the tumor tissue, circulating miRNAs have been found highly stable and are both detectable and quantifiable in a range of accessible bio fluids; therefore, miRNA has the potential to be useful diagnostic, prognostic, and predictive biomarker. Along with being an important molecule in modulation of caner progression, miRNA have certain limitations such as lack of stable expression of multiple target genes and often disrupt entire signaling networks of cellular metabolic pathways. The miRNAs may act on different pathways in various cancers. miRNAs are being linked to breast cancers. The important miRNAs associated with breast cancers include miR-21, miR-155, miR-27a, miR-205, miR-145, and miR-320a. PI3K-Akt pathway is one of the most commonly dysregulated cancer-associated signaling pathways. In luminal breast cancer cells miR-181 family involves Akt hyperactivation. miR-181a and miR-181d post-transcriptionally suppress the expression of PHLPP2 and INPP4B phosphatases, resulting in elevated growth factor–induced Akt phosphorylation. miR-181a and miR-181d ectopic expression promoted S-phase entry and cell proliferation, which was reversed by pharmacological Akt inhibition. Expression of miR-181 family members and PHLPP2/INPP2B are inversely correlated in primary human estrogen receptor-positive breast cancers. miRNA (miR)-210 that regulates hypoxia-inducible factors (HIFs), inhibiting production of the mature

42  Chapter 2 miRNA, derepresses glycerol-3-phosphate dehydrogenase 1-like enzyme (GPD1L), a hypoxiaassociated protein negatively regulated by miR-210, decreases HIF-1α, and triggers apoptosis of triple negative breast cancer cells only under hypoxic conditions. miR-375 studies show a promising diagnostic marker and a therapeutic drug for colorectal cancer. miRNA-375 inhibits colorectal cancer cells proliferation by downregulating JAK2/STAT3 (Wei et al., 2017) and MAP3K8/ERK signaling pathways. miR-130b-3p could inhibit breast carcinoma cell invasion and migration by directly targeting the Notch ligand Delta-like 1 (DLL1). Also, MMP-9, MMP-13, and VEGF were regulated by miR-130b-3p and may be involved in the inhibition of cell invasion and migration in breast carcinoma. This new regulatory mechanism of miR-130b-3p suggest that miR-130b-3p may be a potential marker or target against human breast cancer metastasis (Shui et al., 2017). Neuroblastoma is a complex form of cancer with highly heterogeneous clinical behavior that arises during childhood from precursor cells of the sympathetic nervous system. In patients with neuroblastoma, mortality often occurs as a result of metastasis. The disease predominantly spreads to bone marrow, with a survival rate of approximately 40%. miRNA (miR)-506 directly targets and downregulates Rho-associated, coiled-coil–containing protein kinase 1 (ROCK1) in transforming growth factor (TGF)-β noncanonical pathways. ROCK1 contributes to the invasion and migration of neuroblastoma cells by directly downregulating miR-506, thus leading to the upregulation of ROCK1, which promotes cell invasion and migration. miR-506 directly regulates TGF-β noncanonical signaling (Li et al., 2017). miR-196a acts as a potential tumor marker for diagnosis for esophageal squamous cell carcinoma (ESCC). Salivary miR-196a may be a suitable non-invasive biomarker for diagnosis of ESCC. In addition, molecular pathway enrichment analysis (miR)-196a determined focal adhesion, spliceosome, and p53 signaling pathways as the most relevant pathways with miR-196a targetome (Fendereski et al., 2017). E-selectin influences the metastatic potential of breast, bladder, gastric, pancreatic, and colorectal carcinoma, as well as of leukemia and lymphoma. Also E-selectin expression induced by the proinflammatory cytokine IL-1β is directly and negatively regulated by miR-31. The transcription of miR-31 is activated by IL-1β. This activation depends on p38 and JNK MAP kinases, and their downstream transcription factors GATA2, c-Fos, and c-Jun. miR-31-mediated repression of E-selectin impairs the metastatic potential of colon cancer cells by decreasing their adhesion to, and migration through, the endothelium. miRNA mediates E-selectin–dependent extravasation of colon cancer cells and might be a potent marker for colorectal carcinoma (Zhong et al., 2017).

2.6 RNA Detection Methods Some of the molecular techniques used to detect RNA in oncology are transcriptional profiling for blood and bone marrow; Northern blotting for fresh tissues; RT PCR for

RNA-Based Applications in Diagnostic and Therapeutics for Cancer  43 blood, bone marrow, and fresh tissue sample; in situ hybridization (ISH); fluorescent in situ hybridization; and chromogenic in situ hybridization for blood (after cell capture assays), bone marrow, and fresh tissues. The expression of miRNAs in MB can be revealed by technologies that accurately assess changes in the content of miRNAs. It also will determine their abundance, including microarray-based and PCR-based approaches, Northern blot analysis with radiolabeled probes, ISH and high-throughput sequencing depending on the chosen sample (Fig. 2.3).

2.6.1 Microarray Approach Tissue microarrays are facilitating drug discovery and development by industrializing the assessment of mRNA and protein target expression in large numbers of clinical-defined human and animal samples. Microarray technology is based on nucleic acid hybridization between target miRNA molecules and their corresponding complementary probes. It is usually used to conduct a genome-wide analysis of miRNA expression of normal and/or disease samples, including

Figure 2.3 miRNA expression and profiling methods.

44  Chapter 2 cancer, and to distinguish expression signatures associated with diagnosis, prognosis, and therapeutic interventions (Li and Ruan, 2009). The technique involves oligonucleotide probes with the same sequence as the target miRNAs, which are immobilized on glass slides forming a ready-to-use miRNA microarray. The isolated miRNAs are converted to cDNA by reverse transcription, labeled with fluorescent dye, and then hybridized on the microarray. After a series of washing steps to remove any unbound cDNAs, the hybridized miRNAs are detected by a microarray scanner to determine the fluorescence intensity on each probe spot, which represents the level of expression of each target miRNA from the initial RNA sample.

2.6.2 Real-Time PCR–Based Detection In multiplex RT-PCR, housekeeping and gene-specific oligonucleotide primers are added along with the dye-conjugated probes to cDNA produced from RNA isolated from clinical samples, and a quantitative level of mRNA expression is obtained by normalizing amplification cycle time for the target gene against that of a housekeeping gene. The most commonly used method to detect specific miRNAs is the real-time PCR analysis. This approach relies on reverse transcription of miRNA to cDNA, followed by quantitative PCR with real-time monitoring of reaction product accumulation. Commercially available customizable plates and microfluidic cards can be designed either to examine a small set of miRNAs or to provide more comprehensive coverage by large-scale profiling of hundreds of miRNAs (Pritchard et al., 2012).

2.6.3 Northern Blot Analysis Northern blotting was the first mRNA detection method used to test gene expression patterns in human cancer. Currently Northern analysis is limited to research and is not widely used for clinical assessment of human samples since the technique is slow, tiresome, and less sensitive as a result of dilution of the malignant cell mRNA levels (target) by surrounding nonneoplastic tissues. Northern blotting was used to identify the very first miRNAs and still remains a gold standard for miRNA expression analysis. After isolation of total RNA from cells or tissue, the small RNAs are fractionated by electrophoresis on a high percentage gel. After transferring these small RNAs from the gel onto a nitrocellulose membrane, to allow detection by hybridization with fluorescent or radiolabeled probes that are complementary to the target miRNA, the RNA is fixed onto the membrane by UV cross-linking and/or baking the membrane. Because of the small size and the low abundance of miRNA molecules, the use of an oligonucleotide probe with high sensitivity is essential for the detection of a given miRNA. Northern blotting technique allows the validation of predicted miRNAs by the examination of their expression levels and the determination of their sizes.

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2.6.4 In Situ Hybridization ISH techniques have been applied to detect cell-specific mRNA levels in tissue sections, aspirates, and smears of various human malignancies. It has also been used to detect kappa and lambda light-chain mRNA expression in lymphoid infiltrates. Restriction of expression of light chain of mRNA has been demonstrated by the measurements of post-translational modification, up-/downregulation of proteins and absence of expression. ISH is an important tool used to detect miRNA accumulation in tissue sections or fixed cells. The technique uses labeled complementary nucleic acid sequences to detect a single strand of DNA or RNA. ISH is the ability to monitor specific miRNA expression at the cellular or even at subcellular level.

2.6.5 Differential Display Method Prior to the development of high-throughput microarray technologies, the differential display of mRNA was used. It is a technique in which mRNA expression levels in a cell population are reverse transcribed and are amplified by many separate PCR reactions. This robust and relatively simple procedure allows identification of genes that are differentially expressed in different cell populations and is particularly useful for the discovery of biomarkers. It is not currently used for direct clinical testing.

2.6.6 Detection by High-Throughput Next-Generation Sequencing Platforms This sequence-based method for miRNA profiling determine the nucleotide sequence of miRNAs and involve RNA isolation, ligation of linkers to both 3′ and 5′ ends, reverse transcription, and PCR amplification, followed by the “massively parallel” sequencing of millions of individual cDNA molecules. Bioinformatic analysis of the sequence reads identifies both known and novel miRNAs in the data sets and provides relative quantification using a digital approach. High-throughput sequencing methods permit high-resolution views of expressed miRNAs over a wide dynamic range of expression levels and the discrimination of miRNA family members that differ in only a nucleotide. In addition high-throughput sequencing of miRNAs have successfully revealed the differential expression of miRNAs in several cancerogenic and precarcinogenic lesions, suggesting that they can be used in diagnostics for early detection and for assessment of tumor aggressiveness (De Planell-Saguer and Rodicio, 2011).

2.6.7 Circulating miRNAs—Experimental Techniques and Normalization An optimal biomarker should be sensitive, specific, noninvasive, and comparable to the disease conditions. In the new decade, many studies clearly proved that cell-free, miRNAs,

46  Chapter 2 and tissue miRNAs fulfill these expectations. Accurate measurement of tissue and body fluid miRNAs has clinical importance for diagnosis and prognosis of several types of disease. miRNAs detection is a very challenging task for their native properties, e.g., small size, scarce concentration, high risk of cross-hybridization, and tissue/stage-specific expression. Therefore, quantification of cell-free miRNAs needs powerful tools. The rapid improvement of diagnostic device platforms is one of the leading experimental goals in the world of life science research and medical diagnostics. High-throughput miRNA profiling techniques including qRT-PCR, microarray, deep sequencing or next generation sequencing, and nanopores are effective tools for obtaining expression profiles of extracellular miRNAs. These techniques highly smoothen the process of free cell miRNA expression profiling. In this view, high-throughput techniques are better to the existing low-throughput techniques such as northern bolting and ISH. Currently, qRT-PCR is the most common, reliable and available, inexpensive method used for quantifying the small amount of miRNAs with the high sensitivity and specificity and the subsequent validation of clinical samples. However, some limitations of qRT-PCR, such as normalization of data and primer design, can highly influence the results. The lack of standardized housekeeping RNA for normalization, especially in body fluid, is a critical issue. Many miRNAs can be used as control reference such as miR-16, miR-142-3p, miR-30b, miR-145, miR-93, U6, U6B, SNORD68, RNU48, RNU43, RNU62, and 5sRNA. miR-16 is highly expressed in red blood cells and its levels in the extracellular content of blood fluids can differ significantly due to hemolysis. Several endogenous genes might be disease-specific. Therefore, some miRNAs, such as miR-16 and miR-93 and SNORD68, might be considered satisfactory reference gene for miRNA serum analysis of gastric and urologic cancers, respectively. At present, there is no unique housekeeping RNA that can be used as the standard endogenous reference gene in normalizing circulating miRNAs. Currently, many studies suggest the use of more than one reference gene or better a combination for normalizing circulating miRNA concentrations.

2.7 RNA as Biomarkers in Cancer Diagnosis miRNAs are nucleic acids of about 20 nucleotides that regulate about one-third of the genome at the post-transcriptional level. With their different forms of transport, miRNAs are stable and can be detected in biological fluids such as blood, urine, cerebrospinal fluid, or saliva. In addition, the profile of circulating miRNAs is a specific part of the cells in which it is secreted and is modified according to the physiological or pathological conditions of these cells. miRNAs therefore appear as biomarkers of interest for many diseases. Current use of miRNAs as biomarkers mainly in biological fluids addresses the perspectives that emerge from the fact that their vesicular circulating forms could be used to assess the state of the cells and the tissues that produce them. Along with them, exosomes are small membrane-bound vesicles secreted by most cell types. Exosomes contain various functional proteins, mRNAs, and miRNAs (miRNAs) that could be used for diagnostic and therapeutic purposes.

RNA-Based Applications in Diagnostic and Therapeutics for Cancer  47

2.7.1 miRNAs in Exosomes From Cancer Cells First evidence implicating miRNA transport by exosomes as a communication process among cancer cells came from Skog et al. (2008), who demonstrated that the messengers encoding for EGFRvIII protein and miR-21 are transported in glioblastoma-derived exosomes and that these molecules could be taken up by normal host cells and transformed into functional signals, stimulating proliferation of cancer cells. Mature Epstein–Barr virus (EBV)-encoded miRNAs are secreted by EBV-infected B-cells through exosomes, and these miRNAs accumulate in neighboring noninfected monocyte-derived dendritic cells (MoDCs). Furthermore, these EBV-miRNAs are functional because after internalization by MoDCs, investigators observed a dose-dependent miRNA-mediated repression of known EBV target genes, including CXCL11/ITAC, an immunoregulatory gene downregulated in primary EBV-associated lymphomas. This may represent some of the first evidence consistent with the idea that miRNAs delivered by exosomes can act as regulators of gene expression in distant cells. Let-7 miRNA family is abundant in both intracellular vesicles and exosomes from AZ-P7a cells, a metastatic gastric cancer cell line, while nonmetastatic AZ-521, the parent cell line of AZ-P7a, showed no such high levels. This suggests that the exosomal mechanism may be used by tumor cells to launch out miRNAs, which predominately have tumor suppressive functions, preventing them from acting in their parent cell and thus maintaining its oncologic potential. In recent years, the idea that exosomes can affect and modulate the gene expression programs of recipient cells by intercellular signaling has come to be accepted. Unique set of 11 miRNAs expressed exclusively in exosomes derived from Hep3B human hepatocellular carcinoma cells and this set of miRNAs targets genes predominantly involved in TGF-β activated kinase-1 (TAK1) signaling. Strikingly, these tumor-released (TR) exosomes can modulate the constitutive expression of TAK1 and modulate downstream signaling associated with TAK1 in recipient Hep3B cells. EGFR regulates the maturation of miRNAs in a trafficking-associated mechanism; this is thought-provoking concerning the functional relevance miRNAs secreted by TR exosomes hold in cancer. Exosomes in cancer have multiple functions including promotion of certain local and systemic processes that lead to cell growth and dissemination, or impairment of the immune system response. In fact, there is growing evidence that TR exosomes might act as a vehicle for suppressive signals and have suppressive effects on antitumor immune responses. Macrophages also regulate invasiveness of cancer cells through exosome-mediated delivery of oncogenic miRNAs.

2.7.2 Exosome Transported Role of miRNAs in Metastasis The metastatic process involves the manipulation of the cellular microenvironment to optimize conditions for deposition and growth both locally and at a distance. The discovery that miRNAs can function as hormones, entering the circulatory system and traveling to distant organs to deliver their message, where they are actively taken up by recipient cells located

48  Chapter 2 there, has pointed to the potential of these regulatory molecules as signals involved in preparing the distant site for tumor colonization. On the other hand, it is well established that TR exosomes are contributors to both formation of primary tumors and metastases. In this section, we present examples on how miRNAs secreted in TR exosomes participate in preparing and promoting the metastatic process in cancer. In one such example, it was shown that exosomes released by CD105 cancer stem cells from renal carcinomas may modify the tumor microenvironment by triggering angiogenesis and may promote formation of a premetastatic niche. Interestingly, these vesicles were enriched in miRNAs, which may modulate several biological functions relevant in tumor invasion and metastasis. To support the role of miRNAs secreted in TR exosomes in modulating the tumor microenvironment, THP-1–derived (the acute monocytic leukemia cell line) exosomes are incorporated into human HMEC-1 (the human mammary epithelial cell line) cells and deliver miR-150. The elevated exogenous miR-150 effectively reduces c-Myb expression enhancing cell migration. Exosomes released from dendritic cells can fuse with target dendritic cells and release their contents into the target cells, leading to mRNA silencing. A collection of studies from different groups provides evidence that miRNAs delivered by TR exosomes into recipient cells can regulate target gene expression and recipient cell function, modulating the tumor microenvironment to adapt it for tumor promotion and progression. This role of miRNAs acting on neighboring cells to transmit a message produced by a donor cell and taken up by a recipient cell resembles a paracrine mechanism of intercellular communication.

2.7.3 Exosome-Derived miRNAs as Potential Biomarkers Characterization of miRNAs derived from TR exosomes as surrogate biomarkers of cancer. Different groups have detected miRNAs from TR exosomes in patient plasma of different tumors such as lung cancer, glioblastoma multiforme (GBM), malignant glioma, gastric cancer, breast cancer, prostate cancer, ovarian carcinoma, and cervical cancer. It is widely accepted that resistance to therapy and relapse of cancer remains a central problem in detection, cancer treatment, and this process accounts for much cancer mortality. There is increasing evidence that miRNAs are potential biomarkers for diagnosis and prognosis, and also to monitor treatment response. Due to the fact that exosomes are released in accessible body fluids such as blood and urine, and can be isolated by minimally invasive methods, miRNAs in exosomes represent a promising biomedical tool, useful as biomarkers in the diagnosis and prognosis of malignant tumors. Serum levels of miR-141, an miRNA expressed in prostate cancer, can distinguish patients with prostate cancer from healthy subjects. By this means, proof is of the principle that TR miRNAs enter the circulation where their measurement in plasma can serve as a means for cancer detection. Significant upregulation of miRNAs derived from TR exosomes in metastatic prostate cancer patients compared with nonrecurrent cancer patients. The association of miRNAs with circulating tumorderived exosomes found that although epithelial cell adhesion molecule–positive exosomes were detectable in patients with both benign ovarian disease and ovarian cancer, exosomal miRNA from

RNA-Based Applications in Diagnostic and Therapeutics for Cancer  49 ovarian cancer patients had similar profiles, which were significantly different from profiles observed in benign disease. In breast cancer, it has been shown that the release of miRNAs from cells into blood, milk, and ductal fluids is selective, and that the selection of released miRNAs may correlate with malignancy. Serum exosomes isolated from patients with ESCC induce the proliferation of ESCC cells in vitro. Moreover, it was found that exosomal miR-21 expression is upregulated in serum from patients with ESCC, and this alteration is positively correlated with tumor progression and aggressiveness. Exposure of A549 lung cancer cells to cisplatin could cause cells to release more exosomes than in normal conditions, and that the interaction of these exosomes with other A549 cells could increase the resistance of these cells to cisplatin. miRNAs derived from TR exosomes after irradiation could be used as indicators of resistance to radiotherapy in GBM. In lung cancer, circulating miRNA profiles in patients with lung squamous cell carcinoma before and after tumor removal, assuming that the levels of all tumor-relevant miRNAs would drop after the surgery discovered a specific panel of the miRNAs whose levels decreased strikingly in the blood of patients after lung squamous cell carcinoma surgery. Interestingly some of these miRNAs were selectively secreted in exosomes. In light of these and other findings, it is plausible that the miRNAs enriched in different body fluids are derived from exosomes released by tumor cells under a specific set of stimuli. Therefore, the miRNA content in body fluids may represent the result of a highly refined process of cargo selection, uploading, and secretion in exosomes, and provides reliable information about the identity and status of the cell type from which they are derived and perhaps about the recipient cell, in the setting of the cellular heterogeneity that is characteristic of malignancies.

2.7.4 Circulating miRNA Carried in Particles Extracellular miRNAs circulate in the blood of both healthy and diseased patients, even if the ribonuclease is present in both plasma and serum. The circulating miRNAs are found embedded in lipid or lipoprotein complexes, like apoptotic bodies, microvesicles, or exosomes, and hence they are considered as highly stable structures. 2.7.4.1 Body Fluid Particles and Their Roles in Recipient Cells The presence of miRNA in exosomes was first studied by Valadi et al. (2007) who reported that exosomes that are released from human and murine mast cell lines contain mRNAs and miRNAs. miRNAs are present within exosomes that are isolated from human saliva. miR125a and miR-200a are present in significantly lower levels in the saliva of oral SCC patients than in control subjects. Recently, it was found that the miRNAs are also found in human breast milk. During the first 6 months of lactation period, high expression levels of immunerelated miRNAs are detected. miRNA molecules are found to be very stable even in very acidic conditions, indicating that breast milk allows the dietary intake of miRNAs by infants. Thus, the circulating miRNAs could be used as noninvasive diagnostic markers. Secreted miRNAs contained in exosomes potentially influence micro environmental cells, including

50  Chapter 2 immune cells, endothelial cells, and fibroblast cells. Soluble factors, such as cytokines and chemokines, have been shown to be intercellular communication tools between cancer and micro environmental cells.

2.8 Therapeutic Applications of RNA 2.8.1 Therapeutic Potential of miRNAs Generally there are two approaches to develop miRNA-based therapeutics: miRNA antagonists (Si et al., 2007) and miRNA mimics/replacement (Gandellini et al., 2011). miRNA antagonists inhibit endogenous miRNAs that show a gain-of-function in cancer tissues. Introduction of anti-miR that bind with high affinity to the active miRNA strand results in its degradation. On the other hand, miRNA mimics/replacements are used to restore a loss of function. This approach aims to reintroduce miRNAs into diseased cells that are normally expressed in healthy cells.   1. miRNA-based therapy in cancer is now being exploited, with the attempt to modulate their expression, reintroducing miRNAs lost in cancer, or inhibiting oncogenic miRNAs by using anti- miRNA oligonucleotides. 2. Moreover, miRNAs influence the response to targeted therapies or to chemotherapy: inhibition of miR-21 and miR-200b enhances sensitivity to gemcitabine in cholangiocytes, probably by modulation of CLOCK, PTEN, and PTPN12. 3. Reintroduction of miR-205 in breast cancer cells can improve the responsiveness to tyrosine kinase inhibitors through HER-3 silencing, besides targeted therapies and chemotherapy. 4. miRNAs could also alter the sensitivity to radiotherapy, as reported in lung cancer cells. The let-7 family of miRNAs can suppress the resistance to anticancer radiation therapy, probably through RAS regulation. 5. MicroRNA molecules with longer half-lives and efficiency have been developed, such as anti-miRNA oligonucleotides, 100 locked nucleic acid–modified oligonucleotides, and 101 cholesterol-conjugated antagomirs. Interestingly, Ebert et al. (2007) have recently described a new approach to inhibit miRNAs function: synthetic mRNAs containing multiple binding site for a specific miRNA, called miRNA sponges, are able to bind up the miRNA, preventing its association with endogenous target.

2.8.2 Therapeutic Gene Silencing—RNA Interference Technology RNA interference (RNAi) is a post-transcriptional process activated by the introduction of double-stranded RNA (dsRNA), which leads to gene silencing in a sequence-specific manner. RNAi is becoming an important method for analyzing gene functions in eukaryotes and holds promise for the development of therapeutic gene silencing. The first evidence that dsRNA

RNA-Based Applications in Diagnostic and Therapeutics for Cancer  51 could achieve efficient gene silencing through RNAi came from studying the nematode Caenorhabditis elegans. Further analyses in the fruit fly Drosophila melanogaster have contributed a great deal in understanding the biochemical nature of the RNAi pathway. RNAi pathway is based on two steps, each involving ribonuclease enzyme. In the first step, referred to as RNAi initiating step, the trigger RNA (either dsRNA or miRNA primary transcript) is cleaved into distinct ≈21- to ≈25-nucleotide RNA fragments (siRNA) by the RNase II enzymes Dicer and Drosha. In the second step, the siRNAs are introduced into the effector complex RNA-induced silencing complex (RISC) where it is unwound during RISC assembly and the single-stranded RNA combines with mRNA target. The nucleolytic degradation of the targeted mRNA by the RNase H enzyme Argonaute (Slicer) led to gene silencing. The mRNA is not cleaved if the siRNA/mRNA duplex contains mismatches, rather, gene silencing will be a result of translational inhibition.

2.8.3 Applications of RNAi The RNAi process holds the key to future technological applications, besides being an area of intense basic research. RNAi technology is proving to be useful to analyze quickly the functions of a number of genes in a wide variety of organisms. RNAi has been adapted with high-throughput screening formats in C. elegans (Fraser et al., 2000), for which the recombination-based gene knockout technique has not been established. Chromosomes I and III of C. elegans have been screened by RNAi to identify the genes involved in cell division and embryonic development. Recently, a large-scale functional analysis of ≈19,427 predicted genes of C. elegans was carried out with RNA interference. This study identified mutant phenotypes for 1722 genes (Kamath et al., 2003). Similarly, in Drosophila melanogaster, RNAi technology has been successfully applied to identify genes with essential roles in biochemical signaling cascades, embryonic development, and other basic cellular process. Gene knockdown–related functional studies are being carried out efficiently when transgenes are present in the form of hairpin (or RNAi) constructs. Associated endotoxins could also be removed if the toxin biosynthesis genes are targeted with the RNAi constructs. Recently, the theobromine synthase was knocked down with the hairpin construct of the transgene, leading to the production of specific loss of gene function. Virus-induced gene silencing has also been proven to be a successful approach for genetic studies. RNAi can be triggered experimentally by exogenous introduction of dsRNA or constructs that express short hairpin RNAs. The high degrees of efficiency and specificity are the main advantages of RNAi. Consequently, RNAi is used in functional genomics (systematic analysis of loss-of-function phenotypes induced by RNAi triggers) and in developing therapies for the treatment of viral infection, dominant disorders, neurological disorders, and many types of cancers (in vivo inactivation of gene products linked to human disease progression and pathology).

52  Chapter 2

2.8.4 lncRNA-Based Therapies and Modular Assembly of Small Molecules Currently, ongoing clinical trials are further evaluating the safety and efficacy of RNAi therapeutics in patients with a variety of diseases, including cancer, and these approaches could be adapted to target lncRNA transcripts. Another therapeutic approach employed was modular assembly of small molecules to adapt to aberrant RNA secondary structure motifs in disease. This approach could potentially target aberrant ncRNAs, mutant mRNAs, as well as nucleotide triplet-repeat expansions seen in several neurologic diseases like Huntington disease. Clinically, the use of GWAS data [In genetic epidemiology, a genome-wide association study (GWAS) or whole genome association study (WGAS) is an examination of many common genetic variants in different individuals to see if any variant is associated with a trait.] may identify patient populations at risk of cancer and may stratify patient disease phenotypes, such as aggressive versus indolent cancer, and patient outcomes. Single nucleotide polymorphism profiling may also be used to predict a patient’s response to a given therapy (Prensner and Chinnaiyan, 2011).

2.8.5 Therapeutic Potential of Exosome-Derived miRNAs in Cancer Because of their small size and ability to cross biological membranes and protect their mRNA, miRNA, and protein cargo from degradation, exosomes are ideal delivery systems that can be manipulated for the transfer of specific molecules such as miRNAs or anti-miRNAs. In an attempt to block miR-9, which is overexpressed in GBM cells and impairs the response to temozolomide, a method is refined for the delivery of an anti-miR-9 molecule from mesenchymal stem cells (MSCs) via release of exosome-type microvesicles, anti-miR-9 was transferred from MSCs to GBM cells. The delivery of anti-miR-9 to the resistant GBM cells reversed the expression of the multidrug transporter and sensitized the GBM cells to temozolomide, showed increased cell death and caspase activity, thus illustrating a potential role for MSCs in the functional delivery of synthetic anti-miR-9 to reverse chemoresistance of GBM cells. To reinforce the evidence that TR exosomes can serve as vehicles for the delivery of synthetic miRNAs or miRNA inhibitors, a proof of concept study tested whether MSC exosomes could be used as a vehicle for delivery of antitumor miRNAs. An miR-146b expression plasmid was transfected into MSCs, and exosomes released by the transfected MSCs were harvested. The study found that intratumoral injection of exosomes derived from miR-146–expressing MSCs significantly reduced glioma xenograft growth in a rat model of a primary brain tumor. Various approaches have made use of TR exosomes as immune modulators. However, to our knowledge none of these works have specifically demonstrated the participation of the miRNAs contained in the TR exosomes as modulator molecules in the communication process that influences the immune cell response (Katakowski et al., 2013). Nevertheless, it is highly

RNA-Based Applications in Diagnostic and Therapeutics for Cancer  53 plausible that miRNA cargo from TR exosomes somehow regulates and helps to reprogram immune response in cancer.

2.8.6 mRNA-Dendritic Cells in Anticancer Vaccination Today, mRNA is considered to be an ideal vehicle for the induction of strong immune responses against cancer. The growing numbers of preclinical trials and as a consequence the increasing clinical application of mRNA as an off-the-shelf anticancer vaccine signifies a renaissance for transcript-based antitumor therapy. The first description study was shown that DCs pulsed with mRNA encoding for tumor antigens are potent antigen-presenting cells (Boczkowski et al., 1996). The successes of these preclinical studies have resulted in several clinical trials in which the mRNA-based vaccination strategy has demonstrated its first promising results. In view of anticancer therapy, several advantages are coupled with the use of mRNA to deliver proteins to cells. The use of mRNA meets the requirement of favorable safety profile, as it lacks genomic integration, and its use results in transient expression of the encoded protein. Moreover, there is a certain degree of flexibility coupled with the use of mRNA as all proteins of interest can be generated. In addition, enhanced protein expression and coupled with enhanced presentation of antigenic peptides can be obtained by structural modifications, such as codon optimization, of the mRNA molecule. In view of cancer vaccination, the mRNA molecule can encode the entire tumor antigen. As a consequence, a maximal number of epitopes can be presented in class I MHC molecules. Moreover, when coupled to class II signaling sequences, antigenic epitopes can be presented in the context of class II MHC molecules. As such, both CD8+ T cells and CD4+ T cells can be stimulated respectively. This is essential in order to generate long-lasting and effective antitumor immunity. In addition, its in-depth chemical description facilitates its quality control. As such, the use of mRNA as an off-the- shelf vaccine is not limited by the amount of vaccine doses that can be prepared. Thus, from a pharmaceutical point of view, mRNA is an ideal tool for the induction of strong immune responses against cancer. Recently, the intradermal, subcutaneous, and intranodal delivery of mRNA-based vaccines are being studied and demonstrated in clinical trials.

2.9 Conclusion In cancer, dysregulated ncRNA expression characterizes the entire spectrum of disease and aberrant ncRNA function leads to cancer through destruction of normal cell processes, typically by facilitating epigenetic repression of downstream target genes. Studies conducted at the laboratory level have revealed the tremendous power of siRNAs as therapeutics and have shown the capability of miRNA s to reverse cellular developmental aberrations. The deregulation of miRNAs is highly responsible for many cancer incidences.

54  Chapter 2 Thus, some miRNAs are closely associated with clinical prognosis. It is estimated that circulating miRNAs in body fluids like serum and blood may become novel methods to reduce both false-positive and false-negative results in conventional diagnostic methods. Although the analysis of circulating miRNAs is gradually evolving, this indicates that they might have a biological role and may be involved in development of cancer cells. Hence, these miRNAs have a potential as diagnostic, prognostic, and predictive biomarkers and may also be considered as therapeutic targets. Increased research on RNAs will lead to greater understanding of functions of cancer cell and also to novel clinical applications in oncology.

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