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Transfer RNA in Cancer
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TRANSFER RNA IN CANCER
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Sanga Mitra1,2, Jayprokas Chakrabarti1,3,* 1Indian Association
for the Cultivation of Science, Kolkata, India; 2National Institute of Health, Bethesda, MD, United States; 3Gyanxet, Kolkata, India
CHAPTER OUTLINE Introduction������������������������������������������������������������������������������������������������������������������������������������������ 151 tRNA Mediates Translation of Proliferating Cancer Cells������������������������������������������������������������������������� 152 tRNA Functions Beyond Translation in Cancer����������������������������������������������������������������������������������������� 153 Role of tRNA-Derived Fragments in Cancer Predisposition����������������������������������������������������������������������� 155 tRNA Acts as an Anticancer Drug Target and Therapeutic Agent��������������������������������������������������������������� 158 Future Perspective�������������������������������������������������������������������������������������������������������������������������������� 159 References������������������������������������������������������������������������������������������������������������������������������������������� 159
INTRODUCTION Functions of transfer RNA (tRNA) in cellular pathways other than in translation are of recent interest. Alterations in the expression levels of tRNAs are pervasive in cancer. Studies indicated the roles of tRNA in the efficiency of translation in cancer and tRNA breakdown products were detected in the urine of cancer patients. In 1976, Mukerjee and Goldfeder determined that tRNAs play a role in cell growth, differentiation, and carcinogenesis in mouse [1]. Following that, in 1977, it was reported that some tRNAs have regulatory roles in mammalian cells and show high turnover rates in tumor tissues and produce tRNA-derived nucleotides in urine [2]. In continuation, in 1979, Speer et al. reported that the breakdown nucleotides, by-products of very high turnover of tRNAs in tumor tissues, might be used as reliable biomarkers of cancer [3]. After a gap, in 2009, Zhou et al. demonstrated that tRNA levels were drastically elevated in multiple myeloma (MM) cell lines compared with normal bone marrow cells. The differences in abundances in MM cell lines highlighted that the tRNA pools varied between cell lines [4]. The same year, Eternod et al., in cancer-derived versus non–cancer-derived breast cancer cell lines, reported that nuclear tRNAs showed a threefold and mitochondrial tRNAs (mt-tRNAs) a fivefold overexpression. In tumor versus *Senior Author. Cancer and Noncoding RNAs. http://dx.doi.org/10.1016/B978-0-12-811022-5.00009-7 Copyright © 2018 Elsevier Inc. All rights reserved.
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normal breast tissues, both the tRNAs showed a 10-fold overexpression. Expression levels of tRNA isoacceptors specific to housekeeping genes remained the same in normal and cancer tissue, but tRNA isoacceptors required to boost the translation of oncogenes showed a hike in their expression [5]. Thus, tRNAs augmented translation efficiency in proliferating cancer cells. tRNAs, particularly mt-tRNAs, act as scavengers of proapoptotic protein cytochrome c (cyt c), both inside and outside of the mitochondria, and help to inhibit cyt c-mediated apoptosis. This new finding of tRNA–protein interaction gives us a sign that tRNA can be used as potential target for anticancer drugs, making tumor cells more disposed to apoptosis induced by degrading tRNA or inhibiting tRNA synthesis [6]. As both cyt c and tRNA are ancient molecules, cyt c–tRNA interaction might be evolutionarily ancient [7]. Mutations and polymorphisms of tRNA genes in cancer cell lines/tumors are of growing interest. A novel point mutation, a G-to-A transition at position 4450 of mt-tRNAiMet, was involved in lymphoproliferative process [8]. In 2007, Datta et al., reported that the major allele, A, at 12,308 nucleotide pairs on mt-tRNALeu-CUN, increased the risk of oral cancer. In 2012, it was reported that mutations in mt-tRNA genes in breast cancer could affect the cell and cause its dysfunction [9]. Following that in 2015, a polymorphic mutation in V-loop of mt-tRNALeu-CUN, A12308G, was detected in colorectal tumors [10]. Polymorphism may alter the efficiency of mt-tRNALeu-CUN causing disruption in the mitochondrial protein synthesis leading to a decrease in oxidative phosphorylation and an increase in reactive oxygen species (ROS) production. tRNAs are heavily modified posttranscriptionally during maturation. Aberrant expression of tRNA modification enzymes are linked to cancer [11]. In 2007, using deep sequencing, Lui et al. determined the small RNA profiles for six human cervical carcinoma cell lines; out of more than 7000 small RNA clones, 8% were tRNA fragments [12]. Following intensive studies, it transpired that these fragments were the remnants of mature tRNAs and had unique roles to play. Lee and coworkers reported that tRF-1001, derived from the 3′-end of a precursor of tRNASer-TGA, was required for cell proliferation and was highly expressed in prostate cancer (PCa) cell lines [13]. Interestingly, anticancer drugs bind to multiple sites on tRNAs without changing tRNA conformation from A-family structure, rather inducing tRNA aggregation. tRNAs are one of the critical entities driving tumorigenesis, hence the rationale for targeting tRNA by anticancer drugs to treat cancer. The interaction of the potent anticancer agent cis-(Pt(NH3)2Cl2) to yeast tRNAPhe has been investigated using X-ray crystallography [14].
tRNA MEDIATES TRANSLATION OF PROLIFERATING CANCER CELLS A wide variety of biological processes, such as cell proliferation [15], differentiation [15,16], and apoptosis [7] have varied tRNA levels. Change of tRNA levels can greatly transform the cell state by various mechanisms. For example, codon usage is different between the genes serving cell-autonomous functions and the genes involved in multicellularity. tRNAs induced by proliferation and differentiation often carry anticodons that correspond to the codons enriched for these genes, suggesting coordination between tRNA production and messenger RNA (mRNA) translation [15,17,18]. Gingold et al. demonstrated that the tRNA pools are different between cancer and differentiated noncancer cells. tRNAs that are elevated in differentiated/arrested cells are repressed in proliferating cells. On the other hand, tRNAs whose levels are increased in proliferating cells become minimal in differentiated/arrested cells. Cancer cells regulate their tRNA repertoire to selectively boost translation
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of the mRNAs that are required for tumor progression [15,19]. Comparing tRNA expression in tumor versus normal breast tissues via tRNA-customized array, Pavon-Eternod et al. found that nuclear and mt-tRNAs exhibit distinct expression patterns, indicating the potential of using tRNAs as biomarkers for breast cancer [5]. Overexpression of tRNAiMet significantly fine-tunes the global tRNA expression profile and increases the cell metabolic activity and cell proliferation, whereas tRNAiMet remains repressed in differentiated and arrested cells [16]. Increased levels of the translation initiator tRNAiMet can destabilize the balance leading to transformation [20]. Elevated tRNAiMet in melanoma coerces migratory, invasive behavior and metastatic potential without affecting cell proliferation and primary tumor growth [21]. Further studies showed tRNAGlu-UUC and tRNAArg-CCG promote breast cancer metastasis by directly enhancing EXOSC2 (exosome complex component RRP4) and GRIPAP1 (GRIP1-associated protein 1) expression [22]. Using tRNA-specific microarrays, Zhou et al. revealed that tRNA levels are considerably high in MM cell lines compared with normal bone marrow cells. It was also established that the proteasome inhibitor, bortezomib (Velcade, PS-341), could decrease charging levels of tRNAs, specifically those coding for hydrophobic amino acids. These results suggested that amount of tRNA fluctuated in MM in accordance with the type of protein production, and that proteasome inhibition directly affected protein synthesis in MM through effects on tRNA charging [4]. Expression levels of telomerase reverse transcriptase (TERT) and tRNA are correlated in breast and liver cancer. TERT in association with RNA polymerase III (pol III) subunit RPC32 resulting in increased RNA pol III occupancy, and tRNA expression in cancers lead to cancer cell proliferation [23]. Noteworthy is that the tRNA pool changes among several cancer types. Although it is determined that a significant increase in the absolute levels of tRNA genes occur in cancerous cells in comparison with healthy cells, the relative composition of tRNA isoacceptors in healthy, cancerous, or transformed cells remains almost the same. That means, maintaining the relative tRNA levels in cancerous cells is advantageous as it stabilizes the effectiveness of translation [24]. Thus, tRNAs are active modulators of gene expression and the tRNA codon landscape can causally and explicitly impact cancer progression. tRNAs linked to cancers are in Table 9.1.
tRNA FUNCTIONS BEYOND TRANSLATION IN CANCER At the level of cyt c-mediated apoptosome formation, tRNAs can regulate apoptosis of cancer cells. Cyt c is an essential part in the intrinsic apoptosis pathway crucial to shielding healthy tissues from cancer. It was recently reported that tRNAs inhibit the proapoptotic activity of cyt c by preventing the interaction of cyt c with the caspase activator Apaf-1. tRNAs act as an effective scavenger of cyt c, directly bind to it and inhibit cyt c-initiated apoptosome formation and caspase activation. Elevated amounts of tRNA in tumor cells contribute to apoptosis resistance and tumorigenesis, and targeting tRNAs are important in tumor therapy. Microinjection of tRNAs can inhibit cyt c-induced apoptosis [6,7,25,26]. tRNAs interact with MEK2 (mitogen-activated protein kinase kinase 2, MAP2K2) and its mutants in pancreatic cancer cells altering the catalytic activity of the MEK2 protein, promoting cancer progression. The MEK-specific inhibitor U0126 can reduce the tRNA–MEK2 interaction in cells. MEK2 is a dual-specificity protein kinase of the STE7 kinase family. The MEK pathway functions downstream of Kras and is mutated in >95% of human pancreatic ductal adenocarcinoma (PDAC) tumors, contributing to pancreatic cancer development and progression [27].
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Table 9.1 Alteration of Expression Levels of tRNA Genes Linked to Different Cancers tRNA Name
Type of Cancer
Alteration Type
Breast cancer Cervical cancer Myeloma cancer Ovarian cancer Breast cancer Myeloma cancer Breast cancer Cervical cancer Myeloma cancer Breast cancer Breast cancer Colorectal cancer Gastric cancer Melanoma Breast cancer Breast cancer Breast cancer Ovarian cancer Breast cancer
Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Downregulated
Liver cancer Cervical cancer Lung cancer Colorectal cancer Head and neck cancer Cervical cancer Lymphoma Gastric cancer Head and neck cancer Lung cancer Liver cancer Lung cancer Liver cancer
Polymorphism Upregulated Polymorphism Polymorphism Polymorphism Upregulated Point mutation Polymorphism Polymorphism Polymorphism Polymorphism Polymorphism Polymorphism
Cytoplasmic tRNA tRNA-Arg
tRNA-Glu tRNA-Ile tRNA-Leu
tRNA-Lys tRNA-iMet
tRNA-Ser tRNA-Thr tRNA-Tyr tRNA-Val Mitochondrial tRNA tRNA-Ala tRNA-Asp tRNA-His tRNA-Leu tRNA-Lys tRNA-Met tRNA-Phe tRNA-Pro tRNA-Ser tRNA-Trp tRNA-Val
Mutations and polymorphisms of tRNA genes lead to cancer. The major allele, A, at 12,308 np on mt-tRNALeu-CUN, increased the risk of oral cancer but not that of leukoplakia [28]. It was also established that polymorphisms of tRNA genes in breast cancer may affect the cells and cause dysregulation [9]. The A12308G alteration in mt-tRNALeu-CUN was also observed in colorectal cancer [10]. In case of lung cancer, association of A12172G mutation in tRNAHis gene was reported [29].
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ROLE OF tRNA-DERIVED FRAGMENTS IN CANCER PREDISPOSITION According to recent reports, among the regulators in cancer pathways, tRNA and tRNA-derived small RNAs have significant impact [30]. tRNA-derived small RNAs are of two major classes, defined by the position of cleavage induced by various conditions. Stress-induced cleavage of mature tRNA by angiogenin (ANG) on anticodon loop produces 5′- and 3′-tRNA halves called tiRNAs. Mature or precursor tRNAs are cleaved constitutively by Dicer, or RNase enzymes, in D-loop, D-arm, anticodon stem, T-loop, and 3′-trailer sequence to produce tRNA fragments tRF-5 (a, b, and c), tRF-3 (a and b), and tRF-1 [31]. tRNA and tRNA halves/fragments affect cell proliferation in several ways. They can regulate a target either by binding directly to the target using a complementary sequence and inhibiting/ inducing its function (tRFs) or by competitively binding to RNA-binding proteins and displacing the targets in the cell (tiRNAs) [32]. In breast cancer cells and serum, the pattern of 5′-tRNA halves’ expressions revealed significant variations. Thus, breast cancer may, directly or indirectly, exert diverse effects on levels of circulating 5′-tRNA halves. While these molecules may be used as biomarkers, they might also provide evidence of the pathophysiology of systemic effects during cancer progression [33]. Previous study revealed that tiRNAs consistently increase in number when cells are exposed to low oxygen levels and other forms of cellular stress [34]. Recently, it was discovered that breast cancer cells generate tiRNAs when exposed to low levels of oxygen and cancer cells that carry more of these genetic fragments are less likely to metastasize. Remarkably, adding tiRNAs to cells repressed the growth and progression of cancer, and blocking tiRNAs led to the opposite effect. Among different drivers of metastasis, hypoxia, a major stress encountered by cancer cells, mediates pathways of cancer metastasis selectively enhancing the expression level of genes. Cancer cells adapt to hypoxic conditions by increasing levels of hypoxia-inducible factors (HIFs) that induce the expression of multiple genes involved in angiogenesis, glucose utilization, resistance to oxidative stress, cell proliferation, resistance to apoptosis, invasion, and metastasis [35]. Cancer metastasis is a cancer cell’s escape from hypoxic death in primary site. HIF-induced ANG is known to cleave mature tRNAs to produce tiRNAs that curb metastasis via transcript displacement-based mechanism. Specific tRNAs (glutamic acid, aspartic acid, glycine, and tyrosine) mainly give rise to tiRNAs. They bind to the oncogene, YBX1, in turn displacing prooncogenic transcripts bound to YBX1 whose expression is increased by YBX1. This displacement action of tiRNA reduces cancer cells’ ability to grow and metastasize. This function of tiRNAs makes them tumor suppressors [36]. tRFs, derived from mature or precursor tRNAs, are involved in cell proliferation in cancer initiation, progression, and metastasis, however, their modes of functions are currently being investigated [37,38,39]. It has been proposed that modifications of tRNAs are important in tRF biogenesis and functions. tRFs can associate with Argonaute (Ago) proteins, particularly Ago 1, 3, and 4, and perform RNA silencing. While acting as RNA silencers, overexpressed tRFs mostly need to compete with miRNAs for same gene [13,40]. Different tRFs associated with cancers are cataloged in tRF2Cancer database. tRF2Cancer is the first web server for identifying tRFs and their expression in cancers from small RNA deep-sequencing data [41]. A tRF was isolated from human urinary bladder carcinoma and functionally studied. It specifically inhibited growth of endothelial cells, but not smooth muscle cells, bovine kidney cells, 3T3 fibroblasts, and several cancer cell lines [42]. tRFs exhibit some surprising roles, such as for cell proliferation in
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PCa. tRF-1001 derived from tRNASer-TGA, expressed in a wide range of cancer cell lines, is strongly linked to cell proliferation. Cell proliferation was reduced when tRF-1001 was knocked down using small interfering RNA , leading to accumulation of cells in G2. tRF-1001 is produced in the cytoplasm by tRNA 3′-endonuclease ELAC2, a PCa susceptibility gene [13]. Following this study, Olvedy found recently that 5′-tRFs are mostly upregulated, and 3′-tRFs are mostly downregulated in PCa. The expression ratio tRF-544 generated from tRNAPhe-GAA and tRF-315 produced from tRNALys-CTT effectively categorize high- from low-grade prostate tumors cured from recurrent disease. This establishes tRFs as novel candidate biomarkers for the early detection of recurrent aggressive PCa [43]. The recently discovered i-tRFs are derived from the interior of the mature tRNAs overlapping the anticodon. Mitochondrial tRFs have also been detected. Using short-RNA sequencing profiles of lymphoblastoid cell lines (LCLs) and breast cancer samples from the BRCA repository of The Cancer Genome Atlas (TCGA), it was revealed that different cell types have different tRF abundance profiles [44]. tRF-5 have been demonstrated to bind to the human Piwi protein Hiwi2 in a breast adenocarcinoma cell line, MDAMB231 [45]. Aside from the tRNA halves described above, Honda et al. recently reported of new SHOT-RNAs, for sex hormone–dependent tRNA-derived RNAs. SHOT-RNAs are specifically expressed in large quantities in sex hormone–dependent cancers, i.e., estrogen receptor (ER)–positive breast cancer and androgen receptor (AR)–positive Pca that are driven by estrogen and testosterone. SHOT-RNAs are produced by ANG, whose activity is promoted in sex hormone signaling pathways, from anticodon loop of tRNA, similar to tiRNA, resulting in the accumulation of SHOT-RNAs. Depending on the biogenesis, there are two types of SHOT-RNAs: 5′-SHOT-RNAs containing a phosphate at the 5′-end and a 2′, 3′-cyclic phosphate (cP) at the 3′-end, and of 3′-SHOT-RNAs containing a 5′-hydroxyl group at the 5′-end and an amino acid at the 3′-end. Elevated levels of SHOT-RNAs were observed in patients with ER-positive luminal-type cancers, but not those that were negative for ER expression, implying their potential use as novel biomarkers. Expression of SHOT-RNA was linked to cell proliferation. Using cP-RNA-seq on breast cancer cells, eight cytoplasmic tRNAs were identified as primary sources of 50-SHOT-RNAs, of which SHOT-RNAs from tRNALys-CUU and tRNAHis-GUG were abundant, covering approximately 60% and 28% of the total reads, respectively. Interestingly, knockdowns of three distinct 5′-SHOT-RNAs resulted in severe impairment in cell proliferation [46,47,48]. The 22-nucleotide miRNA, CU1276, derived from 3′-end of at least five annotated human tRNAGly-GCC is abundant in normal germinal center (GC) B cells, but strongly downregulated in GC-derived lymphomas. Like for miRNAs, biogenesis of CU1276 is dependent on DICER1 and can bind to all four Ago proteins. CU1276 represses endogenous RPA1 (replication protein A1), an essential gene for DNA dynamics, including genome replication. It has been demonstrated that CU1276 expression in a Burkitt lymphoma–derived cell line results in an RPA1-dependent suppression of their proliferation rate. CU1276 is proposed to be an important noncoding RNA (ncRNA) involved in the regulation of DNA damage response pathways in the GC, and loss of its expression in lymphomas may decrease their sensitivity to ongoing DNA damage, thereby helping them to tolerate the accumulation of mutations and genomic aberrations during tumor evolution [38]. Moreover, miRNA-like tRNA fragments are found to be abundant in breast cancer–derived extracellular vesicles (EVs) MCF7 and not in MCF10A. This high levels of tRNA-derived miRNA-like fragments can discriminate tumor-derived EVs in circulation. The cellular levels of these tRNA-derived miRNA-like fragments are significantly increased in MCF7 EVs [49]. tRNA-derived miRNAs, miR-3676, and miR-4521, also known as ts-3676 and ts-4521, are downregulated and mutated in chronic lymphocytic leukemia and lung cancer.
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ts-3676 and ts-4521 are also found in complexes containing Piwi-like protein 2 (PIWIL2). miR-3676, derived from tRNAThr is a powerful regulator of T-cell leukemia/lymphoma 1 (TCL1) expression, an important oncogene involved in the development of the aggressive CLLs [50]. A snapshot of production and functionality of different tRNA fragments is depicted in Fig. 9.1.
FIGURE 9.1 tRNA fragment generation and function. The types of tRNA fragments generated from mature and pre-tRNAs and their functionalities. ANG, angiogenin, CDS, coding sequence, RBP, RNA-binding proteins, UTR, untranslated region.
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tRNA ACTS AS AN ANTICANCER DRUG TARGET AND THERAPEUTIC AGENT The traditional paradigm in cancer drug development is to target aberrant proteins or stretches of DNAs. Recently, scientists are looking at entirely new set of potential targets, namely, ncRNAs, including tRNAs. Following the detection of the interaction of the potent anticancer agent cis-(Pt(NH3)2Cl2) to yeast tRNAPhe by X-ray crystallography, many other anticancer agents have been found to target tRNAs [14]. The breast anticancer drug tamoxifen and its metabolites (4-hydroxitamoxifen and endoxifen), have been detected to bind to three different sites of tRNAs without altering the A-family structural conformation. Tamoxifen–tRNA interactions involve both hydrophilic and hydrophobic contacts. Tamoxifen belongs to a class of drugs known as selective estrogen receptor modulators (SERMs). It blocks the action of estradiol, the female sex hormone that is implicated in the origin and growth of breast cancer [51]. Using spectroscopic approach, the interactions of tRNAs with natural anticancer compounds, the two vinca alkaloids, vincristine (VCR) and vinblastine (VBS), were established. VCR and VBS interacted with tRNAs with major reaction occurring between the aromatic molecules and the nitrogenous bases (guanine, cytosine, and uracil) of tRNA [52]. The vinca alkaloids are a subset of drugs derived from the Madagascar periwinkle plant. VBS inhibits angiogenesis and VCR stops microtubule formation. VCR is an FDA-approved drug to treat acute leukemia, rhabdomyosarcoma, neuroblastoma, Wilms tumor, Hodgkin disease, and other lymphomas; VBS is used to treat breast cancer and osteosarcoma [53]. tRNA also binds to antitumor drug doxorubicin (DOX) and its analogue, N-(trifluoroacetyl) doxorubicin (FDOX). FDOX forms more stable complexes than DOX. DOX is a chemotherapeutic agent to treat breast cancer, bladder cancer, Kaposi sarcoma, lymphoma, and acute lymphocytic leukemia [54,55]. Another drug, mitoxantrone, binds to uracil (C]O) and adenine (C]N) sites of tRNA. Mitoxantrone (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione) is a synthetically designed antineoplastic agent and structurally similar to classical anthracyclines. It is used to treat certain types of cancer, mostly metastatic breast cancer, acute myeloid leukemia, and non–Hodgkin lymphoma. Mitoxantrone–tRNA association is moderate to strong [56]. Thus, tRNA-targeting chemotherapeutic agents are in focus. tRNASer-AAU, an engineered human tRNASer, strongly affect breast cancer translation, cell viability and tumor formation by forcefully inhibiting translation. Translation inhibition is brought about by generating mutant proteins owing to this chimeric tRNA, corrupting the cellular proteome. As for example, tRNASer-AAU leads to the substitution of isoleucine with serine within the proteome and is particularly proapoptotic. Thus, this engineered human tRNASer has anticancer effects and is potentially an antitumor agent. Since, tRNASer-AAU destroys the protein synthesis mechanism, it would presumably be difficult for tumor cells to evolve resistance [57]. Hereditary diffuse gastric cancer (HDGC) is an aggressive disease, difficult to diagnose and with a high risk for GC development in CDH1 germline mutation carriers. Like other cancer syndromes, therapeutic options beyond surgery and conventional chemo- and radio-therapy are unavailable. In recent times, suppressor-tRNAs are proposed as a possible therapeutic tool for inherited cancer syndromes, where about 10%–20% of the mutations described in are nonsense mutations. SuppressortRNAs are mutant tRNAs that insert a cognate amino acid at a mutant site in protein-encoding genes, promoting their read-through to produce full-length proteins. It has been demonstrated in HDGC, which is associated with mutations in CDH1 gene encoding the adhesion molecule E-cadherin, that a full length and functional E-cadherin can be efficiently recovered from a nonsense-mutated allele using a suppressor-tRNA in GC cells. This therapeutic treatment delays cancer onset. This approach may be functional for other genetic diseases, being especially important for inherited cancer syndromes [58].
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FUTURE PERSPECTIVE At present, the interest in tRNAs and their involvement in cancer is rapidly growing and has provided fresh perspectives for the investigation and expansion of new biomarkers and novel therapeutic strategies for the detection, monitoring, and treatment of cancer [59]. Explication of the molecular mechanisms by which tRFs and tiRNAs function in controlling gene expression can generate novel molecular tools for the modulation of functional restoration of other oncogenes or tumor-suppressor genes in the malignant cell [60]. In conclusion, the further development of next-generation sequencing technologies, computational algorithms for the detection and analysis of tRNA, and the expanding data on the (aberrant) expression of known and newly discovered tRF and tiRNAs will deepen our knowledge of their functions in the malignant cells.
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[41] Zheng LL, Xu WL, Liu S, Sun WJ, Li JH, Wu J, et al. tRF2Cancer: a web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers. Nucleic Acids Res 2016;44(W1): W185–93. [42] Zhao H, Bojanowski K, Ingber DE, Panigrahy D, Pepper MS, Montesano R, et al. New role for tRNA and its fragment purified from human urinary bladder carcinoma conditioned medium: inhibition of endothelial cell growth. J Cell Biochem 1999;76(1):109–17. [43] Olvedy M, Scaravilli M, Hoogstrate Y, Visakorpi T, Jenster G, Martens-Uzunova ES. A comprehensive repertoire of tRNA-derived fragments in prostate cancer. Oncotarget 2016;7(17):24766–77. [44] Telonis AG, Loher P, Honda S, Jing Y, Palazzo J, Kirino Y, et al. Dissecting tRNA-derived fragment complexities using personalized transcriptomes reveals novel fragment classes and unexpected dependencies. Oncotarget 2015;6(28):24797–822. [45] Keam SP, Young PE, McCorkindale AL, Dang TH, Clancy JL, Humphreys DT, et al. The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells. Nucleic Acids Res 2014;42(14): 8984–95. [46] Honda S, Loher P, Shigematsu M, Palazzo JP, Suzuki R, Imoto I, et al. Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proc Natl Acad Sci 2015;112(29):E3816–25. [47] Honda S, Kirino Y. SHOT-RNAs: a novel class of tRNA-derived functional RNAs expressed in hormone dependent cancers. Mol Cell Oncol 2015;3(2):e1079672. [48] Honda S, Morichika K, Kirino Y. Selective amplification and sequencing of cyclic phosphate–containing RNAs by the cP-RNA-seq method. Nat Protoc 2016;11(3):476–89. [49] Guzman N, Agarwal K, Asthagiri D, Yu L, Saji M, Ringel MD, et al. Breast cancer–specific miR signature unique to extracellular vesicles includes “microRNA-like” tRNA fragments. Mol Cancer Res 2015;13(5):891–901. [50] Pekarsky Y, Balatti V, Palamarchuk A, Rizzotto L, Veneziano D, Nigita G, et al. Dysregulation of a family of short noncoding RNAs, tsRNAs, in human cancer. Proc Natl Acad Sci 2016;113(18):5071–6. [51] Bourassa P, Thomas TJ, Bariyanga J, Tajmir-Riahi HA. Breast anticancer drug tamoxifen and its metabolites bind tRNA at multiple sites. Int J Biol Macromol 2015;72:692–8. [52] Tyagi G, Agarwal S, Mehrotra R. tRNA binding with anti-cancer alkaloids–nature of interaction and comparison with DNA–alkaloids adducts. J Photochem Photobiol B Biol 2015;142:250–6. [53] Himes RH, Kersey RN, Heller-Bettinger I, Samson FE. Action of the vinca alkaloids vincristine, vinblastine, and desacetyl vinblastine amide on microtubules in vitro. Cancer Res 1976;36(10):3798–802. [54] Agudelo D, Bourassa P, Beauregard M, Bérubé G, Tajmir-Riahi HA. tRNA binding to antitumor drug doxorubicin and its analogue. Cancer Res 1976;36(10):3798–802. [55] Agudelo D, Bourassa P, Bérubé G, Tajmir-Riahi HA. Review on the binding of anticancer drug doxorubicin with DNA and tRNA: structural models and antitumor activity. J Photochem Photobiol B Biol 2016;158:274–9. [56] Ray B, Agarwal S, Kadian H, Gambhir K, Sharma P, Mehrotra R. Deciphering molecular aspects of interaction between anticancer drug mitoxantrone and tRNA. J Biomol Struct Dyn 2016;9:1–13. [57] Zhou DH, Lee J, Frankenberger C, Geslain R, Rosner M, Pan T. Anti-tumor effects of an engineered ‘‘killer’’ transfer RNA. Biochem Biophys Res Commun 2012;427(1):148–53. [58] Bordeira-Carriço R, Ferreira D, Mateus DD, Pinheiro H, Pêgo AP, Santos MA, et al. Rescue of wildtype E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA. Eur J Hum Genet 2014;22(9):1085–92. [59] Mleczko AM, Celichowski P, Bąkowska-Żywicka K. Ex-translational function of tRNAs and their fragments in cancer. Acta Biochim Pol 2014;61(2):211–6. [60] Geslain R, Eriani G. Regulation of translation dynamic and neoplastic conversion by tRNA and their pieces. Translation (Austin) 2014;2(1):e28586.
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Sanga Mitra1,2, Jayprokas Chakrabarti1,3,* 1Indian Association
for the Cultivation of Science, Kolkata, India; 2National Institute of Health, Bethesda, MD, United States; 3Gyanxet, Kolkata, India
CHAPTER OUTLINE Introduction������������������������������������������������������������������������������������������������������������������������������������������ 151 tRNA Mediates Translation of Proliferating Cancer Cells������������������������������������������������������������������������� 152 tRNA Functions Beyond Translation in Cancer����������������������������������������������������������������������������������������� 153 Role of tRNA-Derived Fragments in Cancer Predisposition����������������������������������������������������������������������� 155 tRNA Acts as an Anticancer Drug Target and Therapeutic Agent��������������������������������������������������������������� 158 Future Perspective�������������������������������������������������������������������������������������������������������������������������������� 159 References������������������������������������������������������������������������������������������������������������������������������������������� 159
INTRODUCTION Functions of transfer RNA (tRNA) in cellular pathways other than in translation are of recent interest. Alterations in the expression levels of tRNAs are pervasive in cancer. Studies indicated the roles of tRNA in the efficiency of translation in cancer and tRNA breakdown products were detected in the urine of cancer patients. In 1976, Mukerjee and Goldfeder determined that tRNAs play a role in cell growth, differentiation, and carcinogenesis in mouse [1]. Following that, in 1977, it was reported that some tRNAs have regulatory roles in mammalian cells and show high turnover rates in tumor tissues and produce tRNA-derived nucleotides in urine [2]. In continuation, in 1979, Speer et al. reported that the breakdown nucleotides, by-products of very high turnover of tRNAs in tumor tissues, might be used as reliable biomarkers of cancer [3]. After a gap, in 2009, Zhou et al. demonstrated that tRNA levels were drastically elevated in multiple myeloma (MM) cell lines compared with normal bone marrow cells. The differences in abundances in MM cell lines highlighted that the tRNA pools varied between cell lines [4]. The same year, Eternod et al., in cancer-derived versus non–cancer-derived breast cancer cell lines, reported that nuclear tRNAs showed a threefold and mitochondrial tRNAs (mt-tRNAs) a fivefold overexpression. In tumor versus *Senior Author. Cancer and Noncoding RNAs. http://dx.doi.org/10.1016/B978-0-12-811022-5.00009-7 Copyright © 2018 Elsevier Inc. All rights reserved.
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normal breast tissues, both the tRNAs showed a 10-fold overexpression. Expression levels of tRNA isoacceptors specific to housekeeping genes remained the same in normal and cancer tissue, but tRNA isoacceptors required to boost the translation of oncogenes showed a hike in their expression [5]. Thus, tRNAs augmented translation efficiency in proliferating cancer cells. tRNAs, particularly mt-tRNAs, act as scavengers of proapoptotic protein cytochrome c (cyt c), both inside and outside of the mitochondria, and help to inhibit cyt c-mediated apoptosis. This new finding of tRNA–protein interaction gives us a sign that tRNA can be used as potential target for anticancer drugs, making tumor cells more disposed to apoptosis induced by degrading tRNA or inhibiting tRNA synthesis [6]. As both cyt c and tRNA are ancient molecules, cyt c–tRNA interaction might be evolutionarily ancient [7]. Mutations and polymorphisms of tRNA genes in cancer cell lines/tumors are of growing interest. A novel point mutation, a G-to-A transition at position 4450 of mt-tRNAiMet, was involved in lymphoproliferative process [8]. In 2007, Datta et al., reported that the major allele, A, at 12,308 nucleotide pairs on mt-tRNALeu-CUN, increased the risk of oral cancer. In 2012, it was reported that mutations in mt-tRNA genes in breast cancer could affect the cell and cause its dysfunction [9]. Following that in 2015, a polymorphic mutation in V-loop of mt-tRNALeu-CUN, A12308G, was detected in colorectal tumors [10]. Polymorphism may alter the efficiency of mt-tRNALeu-CUN causing disruption in the mitochondrial protein synthesis leading to a decrease in oxidative phosphorylation and an increase in reactive oxygen species (ROS) production. tRNAs are heavily modified posttranscriptionally during maturation. Aberrant expression of tRNA modification enzymes are linked to cancer [11]. In 2007, using deep sequencing, Lui et al. determined the small RNA profiles for six human cervical carcinoma cell lines; out of more than 7000 small RNA clones, 8% were tRNA fragments [12]. Following intensive studies, it transpired that these fragments were the remnants of mature tRNAs and had unique roles to play. Lee and coworkers reported that tRF-1001, derived from the 3′-end of a precursor of tRNASer-TGA, was required for cell proliferation and was highly expressed in prostate cancer (PCa) cell lines [13]. Interestingly, anticancer drugs bind to multiple sites on tRNAs without changing tRNA conformation from A-family structure, rather inducing tRNA aggregation. tRNAs are one of the critical entities driving tumorigenesis, hence the rationale for targeting tRNA by anticancer drugs to treat cancer. The interaction of the potent anticancer agent cis-(Pt(NH3)2Cl2) to yeast tRNAPhe has been investigated using X-ray crystallography [14].
tRNA MEDIATES TRANSLATION OF PROLIFERATING CANCER CELLS A wide variety of biological processes, such as cell proliferation [15], differentiation [15,16], and apoptosis [7] have varied tRNA levels. Change of tRNA levels can greatly transform the cell state by various mechanisms. For example, codon usage is different between the genes serving cell-autonomous functions and the genes involved in multicellularity. tRNAs induced by proliferation and differentiation often carry anticodons that correspond to the codons enriched for these genes, suggesting coordination between tRNA production and messenger RNA (mRNA) translation [15,17,18]. Gingold et al. demonstrated that the tRNA pools are different between cancer and differentiated noncancer cells. tRNAs that are elevated in differentiated/arrested cells are repressed in proliferating cells. On the other hand, tRNAs whose levels are increased in proliferating cells become minimal in differentiated/arrested cells. Cancer cells regulate their tRNA repertoire to selectively boost translation
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of the mRNAs that are required for tumor progression [15,19]. Comparing tRNA expression in tumor versus normal breast tissues via tRNA-customized array, Pavon-Eternod et al. found that nuclear and mt-tRNAs exhibit distinct expression patterns, indicating the potential of using tRNAs as biomarkers for breast cancer [5]. Overexpression of tRNAiMet significantly fine-tunes the global tRNA expression profile and increases the cell metabolic activity and cell proliferation, whereas tRNAiMet remains repressed in differentiated and arrested cells [16]. Increased levels of the translation initiator tRNAiMet can destabilize the balance leading to transformation [20]. Elevated tRNAiMet in melanoma coerces migratory, invasive behavior and metastatic potential without affecting cell proliferation and primary tumor growth [21]. Further studies showed tRNAGlu-UUC and tRNAArg-CCG promote breast cancer metastasis by directly enhancing EXOSC2 (exosome complex component RRP4) and GRIPAP1 (GRIP1-associated protein 1) expression [22]. Using tRNA-specific microarrays, Zhou et al. revealed that tRNA levels are considerably high in MM cell lines compared with normal bone marrow cells. It was also established that the proteasome inhibitor, bortezomib (Velcade, PS-341), could decrease charging levels of tRNAs, specifically those coding for hydrophobic amino acids. These results suggested that amount of tRNA fluctuated in MM in accordance with the type of protein production, and that proteasome inhibition directly affected protein synthesis in MM through effects on tRNA charging [4]. Expression levels of telomerase reverse transcriptase (TERT) and tRNA are correlated in breast and liver cancer. TERT in association with RNA polymerase III (pol III) subunit RPC32 resulting in increased RNA pol III occupancy, and tRNA expression in cancers lead to cancer cell proliferation [23]. Noteworthy is that the tRNA pool changes among several cancer types. Although it is determined that a significant increase in the absolute levels of tRNA genes occur in cancerous cells in comparison with healthy cells, the relative composition of tRNA isoacceptors in healthy, cancerous, or transformed cells remains almost the same. That means, maintaining the relative tRNA levels in cancerous cells is advantageous as it stabilizes the effectiveness of translation [24]. Thus, tRNAs are active modulators of gene expression and the tRNA codon landscape can causally and explicitly impact cancer progression. tRNAs linked to cancers are in Table 9.1.
tRNA FUNCTIONS BEYOND TRANSLATION IN CANCER At the level of cyt c-mediated apoptosome formation, tRNAs can regulate apoptosis of cancer cells. Cyt c is an essential part in the intrinsic apoptosis pathway crucial to shielding healthy tissues from cancer. It was recently reported that tRNAs inhibit the proapoptotic activity of cyt c by preventing the interaction of cyt c with the caspase activator Apaf-1. tRNAs act as an effective scavenger of cyt c, directly bind to it and inhibit cyt c-initiated apoptosome formation and caspase activation. Elevated amounts of tRNA in tumor cells contribute to apoptosis resistance and tumorigenesis, and targeting tRNAs are important in tumor therapy. Microinjection of tRNAs can inhibit cyt c-induced apoptosis [6,7,25,26]. tRNAs interact with MEK2 (mitogen-activated protein kinase kinase 2, MAP2K2) and its mutants in pancreatic cancer cells altering the catalytic activity of the MEK2 protein, promoting cancer progression. The MEK-specific inhibitor U0126 can reduce the tRNA–MEK2 interaction in cells. MEK2 is a dual-specificity protein kinase of the STE7 kinase family. The MEK pathway functions downstream of Kras and is mutated in >95% of human pancreatic ductal adenocarcinoma (PDAC) tumors, contributing to pancreatic cancer development and progression [27].
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Table 9.1 Alteration of Expression Levels of tRNA Genes Linked to Different Cancers tRNA Name
Type of Cancer
Alteration Type
Breast cancer Cervical cancer Myeloma cancer Ovarian cancer Breast cancer Myeloma cancer Breast cancer Cervical cancer Myeloma cancer Breast cancer Breast cancer Colorectal cancer Gastric cancer Melanoma Breast cancer Breast cancer Breast cancer Ovarian cancer Breast cancer
Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated Downregulated
Liver cancer Cervical cancer Lung cancer Colorectal cancer Head and neck cancer Cervical cancer Lymphoma Gastric cancer Head and neck cancer Lung cancer Liver cancer Lung cancer Liver cancer
Polymorphism Upregulated Polymorphism Polymorphism Polymorphism Upregulated Point mutation Polymorphism Polymorphism Polymorphism Polymorphism Polymorphism Polymorphism
Cytoplasmic tRNA tRNA-Arg
tRNA-Glu tRNA-Ile tRNA-Leu
tRNA-Lys tRNA-iMet
tRNA-Ser tRNA-Thr tRNA-Tyr tRNA-Val Mitochondrial tRNA tRNA-Ala tRNA-Asp tRNA-His tRNA-Leu tRNA-Lys tRNA-Met tRNA-Phe tRNA-Pro tRNA-Ser tRNA-Trp tRNA-Val
Mutations and polymorphisms of tRNA genes lead to cancer. The major allele, A, at 12,308 np on mt-tRNALeu-CUN, increased the risk of oral cancer but not that of leukoplakia [28]. It was also established that polymorphisms of tRNA genes in breast cancer may affect the cells and cause dysregulation [9]. The A12308G alteration in mt-tRNALeu-CUN was also observed in colorectal cancer [10]. In case of lung cancer, association of A12172G mutation in tRNAHis gene was reported [29].
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ROLE OF tRNA-DERIVED FRAGMENTS IN CANCER PREDISPOSITION According to recent reports, among the regulators in cancer pathways, tRNA and tRNA-derived small RNAs have significant impact [30]. tRNA-derived small RNAs are of two major classes, defined by the position of cleavage induced by various conditions. Stress-induced cleavage of mature tRNA by angiogenin (ANG) on anticodon loop produces 5′- and 3′-tRNA halves called tiRNAs. Mature or precursor tRNAs are cleaved constitutively by Dicer, or RNase enzymes, in D-loop, D-arm, anticodon stem, T-loop, and 3′-trailer sequence to produce tRNA fragments tRF-5 (a, b, and c), tRF-3 (a and b), and tRF-1 [31]. tRNA and tRNA halves/fragments affect cell proliferation in several ways. They can regulate a target either by binding directly to the target using a complementary sequence and inhibiting/ inducing its function (tRFs) or by competitively binding to RNA-binding proteins and displacing the targets in the cell (tiRNAs) [32]. In breast cancer cells and serum, the pattern of 5′-tRNA halves’ expressions revealed significant variations. Thus, breast cancer may, directly or indirectly, exert diverse effects on levels of circulating 5′-tRNA halves. While these molecules may be used as biomarkers, they might also provide evidence of the pathophysiology of systemic effects during cancer progression [33]. Previous study revealed that tiRNAs consistently increase in number when cells are exposed to low oxygen levels and other forms of cellular stress [34]. Recently, it was discovered that breast cancer cells generate tiRNAs when exposed to low levels of oxygen and cancer cells that carry more of these genetic fragments are less likely to metastasize. Remarkably, adding tiRNAs to cells repressed the growth and progression of cancer, and blocking tiRNAs led to the opposite effect. Among different drivers of metastasis, hypoxia, a major stress encountered by cancer cells, mediates pathways of cancer metastasis selectively enhancing the expression level of genes. Cancer cells adapt to hypoxic conditions by increasing levels of hypoxia-inducible factors (HIFs) that induce the expression of multiple genes involved in angiogenesis, glucose utilization, resistance to oxidative stress, cell proliferation, resistance to apoptosis, invasion, and metastasis [35]. Cancer metastasis is a cancer cell’s escape from hypoxic death in primary site. HIF-induced ANG is known to cleave mature tRNAs to produce tiRNAs that curb metastasis via transcript displacement-based mechanism. Specific tRNAs (glutamic acid, aspartic acid, glycine, and tyrosine) mainly give rise to tiRNAs. They bind to the oncogene, YBX1, in turn displacing prooncogenic transcripts bound to YBX1 whose expression is increased by YBX1. This displacement action of tiRNA reduces cancer cells’ ability to grow and metastasize. This function of tiRNAs makes them tumor suppressors [36]. tRFs, derived from mature or precursor tRNAs, are involved in cell proliferation in cancer initiation, progression, and metastasis, however, their modes of functions are currently being investigated [37,38,39]. It has been proposed that modifications of tRNAs are important in tRF biogenesis and functions. tRFs can associate with Argonaute (Ago) proteins, particularly Ago 1, 3, and 4, and perform RNA silencing. While acting as RNA silencers, overexpressed tRFs mostly need to compete with miRNAs for same gene [13,40]. Different tRFs associated with cancers are cataloged in tRF2Cancer database. tRF2Cancer is the first web server for identifying tRFs and their expression in cancers from small RNA deep-sequencing data [41]. A tRF was isolated from human urinary bladder carcinoma and functionally studied. It specifically inhibited growth of endothelial cells, but not smooth muscle cells, bovine kidney cells, 3T3 fibroblasts, and several cancer cell lines [42]. tRFs exhibit some surprising roles, such as for cell proliferation in
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PCa. tRF-1001 derived from tRNASer-TGA, expressed in a wide range of cancer cell lines, is strongly linked to cell proliferation. Cell proliferation was reduced when tRF-1001 was knocked down using small interfering RNA , leading to accumulation of cells in G2. tRF-1001 is produced in the cytoplasm by tRNA 3′-endonuclease ELAC2, a PCa susceptibility gene [13]. Following this study, Olvedy found recently that 5′-tRFs are mostly upregulated, and 3′-tRFs are mostly downregulated in PCa. The expression ratio tRF-544 generated from tRNAPhe-GAA and tRF-315 produced from tRNALys-CTT effectively categorize high- from low-grade prostate tumors cured from recurrent disease. This establishes tRFs as novel candidate biomarkers for the early detection of recurrent aggressive PCa [43]. The recently discovered i-tRFs are derived from the interior of the mature tRNAs overlapping the anticodon. Mitochondrial tRFs have also been detected. Using short-RNA sequencing profiles of lymphoblastoid cell lines (LCLs) and breast cancer samples from the BRCA repository of The Cancer Genome Atlas (TCGA), it was revealed that different cell types have different tRF abundance profiles [44]. tRF-5 have been demonstrated to bind to the human Piwi protein Hiwi2 in a breast adenocarcinoma cell line, MDAMB231 [45]. Aside from the tRNA halves described above, Honda et al. recently reported of new SHOT-RNAs, for sex hormone–dependent tRNA-derived RNAs. SHOT-RNAs are specifically expressed in large quantities in sex hormone–dependent cancers, i.e., estrogen receptor (ER)–positive breast cancer and androgen receptor (AR)–positive Pca that are driven by estrogen and testosterone. SHOT-RNAs are produced by ANG, whose activity is promoted in sex hormone signaling pathways, from anticodon loop of tRNA, similar to tiRNA, resulting in the accumulation of SHOT-RNAs. Depending on the biogenesis, there are two types of SHOT-RNAs: 5′-SHOT-RNAs containing a phosphate at the 5′-end and a 2′, 3′-cyclic phosphate (cP) at the 3′-end, and of 3′-SHOT-RNAs containing a 5′-hydroxyl group at the 5′-end and an amino acid at the 3′-end. Elevated levels of SHOT-RNAs were observed in patients with ER-positive luminal-type cancers, but not those that were negative for ER expression, implying their potential use as novel biomarkers. Expression of SHOT-RNA was linked to cell proliferation. Using cP-RNA-seq on breast cancer cells, eight cytoplasmic tRNAs were identified as primary sources of 50-SHOT-RNAs, of which SHOT-RNAs from tRNALys-CUU and tRNAHis-GUG were abundant, covering approximately 60% and 28% of the total reads, respectively. Interestingly, knockdowns of three distinct 5′-SHOT-RNAs resulted in severe impairment in cell proliferation [46,47,48]. The 22-nucleotide miRNA, CU1276, derived from 3′-end of at least five annotated human tRNAGly-GCC is abundant in normal germinal center (GC) B cells, but strongly downregulated in GC-derived lymphomas. Like for miRNAs, biogenesis of CU1276 is dependent on DICER1 and can bind to all four Ago proteins. CU1276 represses endogenous RPA1 (replication protein A1), an essential gene for DNA dynamics, including genome replication. It has been demonstrated that CU1276 expression in a Burkitt lymphoma–derived cell line results in an RPA1-dependent suppression of their proliferation rate. CU1276 is proposed to be an important noncoding RNA (ncRNA) involved in the regulation of DNA damage response pathways in the GC, and loss of its expression in lymphomas may decrease their sensitivity to ongoing DNA damage, thereby helping them to tolerate the accumulation of mutations and genomic aberrations during tumor evolution [38]. Moreover, miRNA-like tRNA fragments are found to be abundant in breast cancer–derived extracellular vesicles (EVs) MCF7 and not in MCF10A. This high levels of tRNA-derived miRNA-like fragments can discriminate tumor-derived EVs in circulation. The cellular levels of these tRNA-derived miRNA-like fragments are significantly increased in MCF7 EVs [49]. tRNA-derived miRNAs, miR-3676, and miR-4521, also known as ts-3676 and ts-4521, are downregulated and mutated in chronic lymphocytic leukemia and lung cancer.
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ts-3676 and ts-4521 are also found in complexes containing Piwi-like protein 2 (PIWIL2). miR-3676, derived from tRNAThr is a powerful regulator of T-cell leukemia/lymphoma 1 (TCL1) expression, an important oncogene involved in the development of the aggressive CLLs [50]. A snapshot of production and functionality of different tRNA fragments is depicted in Fig. 9.1.
FIGURE 9.1 tRNA fragment generation and function. The types of tRNA fragments generated from mature and pre-tRNAs and their functionalities. ANG, angiogenin, CDS, coding sequence, RBP, RNA-binding proteins, UTR, untranslated region.
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tRNA ACTS AS AN ANTICANCER DRUG TARGET AND THERAPEUTIC AGENT The traditional paradigm in cancer drug development is to target aberrant proteins or stretches of DNAs. Recently, scientists are looking at entirely new set of potential targets, namely, ncRNAs, including tRNAs. Following the detection of the interaction of the potent anticancer agent cis-(Pt(NH3)2Cl2) to yeast tRNAPhe by X-ray crystallography, many other anticancer agents have been found to target tRNAs [14]. The breast anticancer drug tamoxifen and its metabolites (4-hydroxitamoxifen and endoxifen), have been detected to bind to three different sites of tRNAs without altering the A-family structural conformation. Tamoxifen–tRNA interactions involve both hydrophilic and hydrophobic contacts. Tamoxifen belongs to a class of drugs known as selective estrogen receptor modulators (SERMs). It blocks the action of estradiol, the female sex hormone that is implicated in the origin and growth of breast cancer [51]. Using spectroscopic approach, the interactions of tRNAs with natural anticancer compounds, the two vinca alkaloids, vincristine (VCR) and vinblastine (VBS), were established. VCR and VBS interacted with tRNAs with major reaction occurring between the aromatic molecules and the nitrogenous bases (guanine, cytosine, and uracil) of tRNA [52]. The vinca alkaloids are a subset of drugs derived from the Madagascar periwinkle plant. VBS inhibits angiogenesis and VCR stops microtubule formation. VCR is an FDA-approved drug to treat acute leukemia, rhabdomyosarcoma, neuroblastoma, Wilms tumor, Hodgkin disease, and other lymphomas; VBS is used to treat breast cancer and osteosarcoma [53]. tRNA also binds to antitumor drug doxorubicin (DOX) and its analogue, N-(trifluoroacetyl) doxorubicin (FDOX). FDOX forms more stable complexes than DOX. DOX is a chemotherapeutic agent to treat breast cancer, bladder cancer, Kaposi sarcoma, lymphoma, and acute lymphocytic leukemia [54,55]. Another drug, mitoxantrone, binds to uracil (C]O) and adenine (C]N) sites of tRNA. Mitoxantrone (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione) is a synthetically designed antineoplastic agent and structurally similar to classical anthracyclines. It is used to treat certain types of cancer, mostly metastatic breast cancer, acute myeloid leukemia, and non–Hodgkin lymphoma. Mitoxantrone–tRNA association is moderate to strong [56]. Thus, tRNA-targeting chemotherapeutic agents are in focus. tRNASer-AAU, an engineered human tRNASer, strongly affect breast cancer translation, cell viability and tumor formation by forcefully inhibiting translation. Translation inhibition is brought about by generating mutant proteins owing to this chimeric tRNA, corrupting the cellular proteome. As for example, tRNASer-AAU leads to the substitution of isoleucine with serine within the proteome and is particularly proapoptotic. Thus, this engineered human tRNASer has anticancer effects and is potentially an antitumor agent. Since, tRNASer-AAU destroys the protein synthesis mechanism, it would presumably be difficult for tumor cells to evolve resistance [57]. Hereditary diffuse gastric cancer (HDGC) is an aggressive disease, difficult to diagnose and with a high risk for GC development in CDH1 germline mutation carriers. Like other cancer syndromes, therapeutic options beyond surgery and conventional chemo- and radio-therapy are unavailable. In recent times, suppressor-tRNAs are proposed as a possible therapeutic tool for inherited cancer syndromes, where about 10%–20% of the mutations described in are nonsense mutations. SuppressortRNAs are mutant tRNAs that insert a cognate amino acid at a mutant site in protein-encoding genes, promoting their read-through to produce full-length proteins. It has been demonstrated in HDGC, which is associated with mutations in CDH1 gene encoding the adhesion molecule E-cadherin, that a full length and functional E-cadherin can be efficiently recovered from a nonsense-mutated allele using a suppressor-tRNA in GC cells. This therapeutic treatment delays cancer onset. This approach may be functional for other genetic diseases, being especially important for inherited cancer syndromes [58].
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FUTURE PERSPECTIVE At present, the interest in tRNAs and their involvement in cancer is rapidly growing and has provided fresh perspectives for the investigation and expansion of new biomarkers and novel therapeutic strategies for the detection, monitoring, and treatment of cancer [59]. Explication of the molecular mechanisms by which tRFs and tiRNAs function in controlling gene expression can generate novel molecular tools for the modulation of functional restoration of other oncogenes or tumor-suppressor genes in the malignant cell [60]. In conclusion, the further development of next-generation sequencing technologies, computational algorithms for the detection and analysis of tRNA, and the expanding data on the (aberrant) expression of known and newly discovered tRF and tiRNAs will deepen our knowledge of their functions in the malignant cells.
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