The dual role of ribonucleases in tumor-host relationship
Summary. Ribonucleases are enzymes that destroy RNA, play an important role in protein synthesis, epigenetic regulation of genetic activity, cell proliferation and apoptosis. Ribonucleases are important antimicrobial, antiviral and immune defense factors. Despite the same biochemical properties, they exhibit unequal, sometimes opposite biological effects. While mostly ribonucleases inhibit cell proliferation, induce apoptosis and inhibit the growth of tumors, some ribonucleases stimulate vascular growth, proliferation and tumor development. RNase inhibitors have an opposite effect. The correct use of these features of RNases can provide additional opportunities for the development of a strategy of targeted influence on tumor growth.
Submitted: July 5, 2019.
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Abbreviations used: ANG — angiogenin; mRNA — messenger RNA; ONC — onconase; PARN — poly(A)-specific ribonuclease; RNases — ribonucleases; RNAs — ribonucleic acids; TLRs — toll-like receptors.
Ribonucleases (RNases) are a group of enzymes that cleave ribonucleic acids (RNAs) at phosphodiester bonds resulting in remarkably diverse biological consequences. Endoribonucleases cleave RNA molecule endoribonucleolytically (in 5’-3’ direction) while exoribonucleases degrade RNA molecule in 3’-5’ direction. This review focuses on mammalian RNases that are capable of, or potentially capable of, cleaving messenger RNA (mRNA) as well as other RNAs in cells and play a role in the development or inhibition of human cancer. The aims of this review are to provide an overview of the role of currently known mammalian RNases, and the evidence on their involvement in regulation of tumor development. RNase-controlled RNA degradation is a determining step in gene regulation, maturation and metabolic turnover which is further associated with progression of cancers or infectious diseases. The roles of these RNases as oncoproteins and/or tumor suppressors influencing cell growth, apoptosis, angiogenesis, and other cellular hallmarks of cancer is presented and discussed. The RNases under discussion include RNases from the conventional mRNA decay pathways, RNases that are activated under cellular stress, RNases from the miRNA pathway, and RNases with multifunctional activity. Some of them, by contrast, have a stimulating effect on the proliferation and growth of tumors. This must be taken into account when assessing the role of RNases in oncogenesis.
To date, more than 120 different mammalian RNases have been described and characterized . In ruminants and other mammals with ruminant-like digestion the major function of pancreatic RNases is to degrade dietary RNA. However, many members of the vertebrate RNase superfamily evolved to perform important nondigestive functions including neurotoxicity, angiogenesis, immunosuppressivity, and antibacterial as well as antiviral actions . All human RNase genes are localized on chromosome 14 (Figure).
Figure. Chromosomal locations of human RNase genes. The transcription of the genes coding for both canonical (RNases 1–8; red) and noncanonical (RNases 9–13; blue). RNASE2-ps presents pseudogene. Arrows indicate direction of transcription (compiled based on the data presented in )
The loss of the function of individual RNases leads to severe disorders of development and even death of the organism [3, 4].
Biochemically, the function of RNase is to cleave different types of RNA. At the same time, it has been found that the physiological consequences of RNases activities are very diverse. In addition to regulation of the RNA metabolism RNases demonstrate wide range of therapeutic effects [5, 6]. RNase activity was dramatically reduced in blood serum of cancer patients . To understand the physiological role of RNAses, it is important to use the modern methods for the isolation of individual enzymes with the retention of their enzymatic activity.
Up-to-date, many RNases have been discovered, which destroy the various types of RNA in cells, and induce apoptosis [5, 6, 8].
Degradation of RNA into smaller nucleotides is performed by a wide variety of cellular RNases, which are also responsible for regulating the functional expression of several fundamental genes in living systems. This property of RNases has attracted the attention of researchers as a possible means for the treatment of malignant tumors [9–12]. The implementation of complex relationships between the tumor and the host, in addition to the mechanisms established earlier, also occurs through the mutual influence of enzyme systems and their inhibitors. In this case, enzymes that perform the same biochemical function (splitting of RNA), sometimes show completely opposite physiological effect, which manifests itself in the stimulation or suppression of cell proliferation.
The multiplicity of the physiological functions of RNases suggests the existence of many molecular forms of the enzymes. The existence of a plurality of RNase isoforms in insects, animals and plants has already been shown [10, 13–15]. The study of the multiplicity of molecular forms of RNases would allow to study in detail the properties and physiological functions of individual RNases and their participation in the numerous signal pathways.
Currently, the best methods for the separation of enzymes while maintaining enzymatic activity are chromatographic methods, various methods of polyacrylamide gel electrophoresis and the methods of isoelectric focusing. However, the elucidation of the specific physiological functions of each of the detected isoforms of RNases presently remains an unsolved problem. Due to the ability to destroy various types of RNA, RNAses can block protein synthesis, stop cell proliferation and induce cell apoptosis . In vitro and in vivo experiments showed a direct correlation between radiation- and cytostatics-induced inhibition of cell proliferation and an increase of RNase activity . Several RNases isolated from various sources are shown to exhibit antitumor activity and their number continues to grow.
The bovine seminal RNase (BS-RNase, EC 18.104.22.168) is expressed in the seminal vesicles and testes of Bos taurus . It is a secretory ribonuclease. Its native form exists as a dimer in which both subunits are held together by two disulfide linkages between Cys-31 and Cys-32. Secretory homodimeric enzyme binds to and destabilizes the membrane bilayer and thus reaches the cytosol and degrades the cellular RNA; it exerts antitumor activity, in particular, against thyroid cancer . This enzyme can be hardly differentiated from other members of RNase superfamily because it shares with them many common features. The studies of evolutionary acquirement of new functions of proteins revealed an uncommon consequence for a usual biological event called gene conversion in the case of the RNase protein family . The most well-known member of this family, RNase A (also called pancreatic RNase), is expressed in the bovine pancreas. It digests RNA in intestine, and evolved from bacteria in the bovine stomach [18–20]. Anaplastic thyroid carcinoma is an aggressive solid tumor that fails to adequately respond to any known chemotherapy but turned out to be sensitive to the action of the BS-RNase .
Another enzyme exhibiting pronounced antitumor activity is RNase from embryonic tissues of the frog Rana pipiens.
Onconase (Ranpirnase) was isolated from oocytes and early embryos of Rana pipiens. Enzyme exhibits thermal and guanidine stability, degrades tRNA, inhibits protein synthesis that leads to cell apoptosis. Onconase (ONC) is a member of the RNase A superfamily that is toxic to cancer cells in vitro and in vivo. ONC is now in phase IIIb clinical trials for the treatment of malignant mesothelioma. Internalization of ONC to the cytosol of cancer cells is essential for its cytotoxic activity, despite the apparent absence of a cell-surface receptor protein. Endocytosis and cytotoxicity do, however, appear to correlate with the net positive charge of RNases [21–23]. ONC demonstrated significant cytotoxic effects against cancer cell lines HL-60, HT-29, 9L rat glioma, K-562, Colo-320, JCA-1, U937, A549 and ASPC-1. Cytotoxicity of ranpirnase (ONC) in combination with components of R-CHOP regimen against diffuse large B cell lymphoma cell line was significantly higher than that of each of the drugs used separately . ONC conjugated with chlorotoxin has been found to be active against glioma cells . ONC and its derivatives exhibited potent antitumor effects against cervical, breast, colon, pancreatic, ovarian and prostate cancers. It catalyzes the formation of interfering RNAs (RNAi), degrades tRNAs and inhibits protein synthesis, which results in cell apoptosis . The antitumor activity of the enzyme can be significantly increased in conditions of hyperthermia . ONC modulates cytokine-receptor interactions, MAPK, Jak-STAT, Bcl-2, Bax and various other signaling pathways in cancer models . Currently, ONC is tested in clinical trials for treatment of malignant mesothelioma, human lung carcinoma and human pancreatic adenocarcinoma . It has been shown that ONC alters the expression profile of siRNA in several cell lines of pleural mesothelioma, destroying the precursors of these molecules and thus reducing the amount of substrate for RNase Dicer . Using recombinant ONC, Qiao et al. found that miR-155 and miR-21, two well-known oncogenic miRNAs with high endogenous levels in mesothelioma cell line Msto-211h, were dose- and time-dependently downregulated by ONC in these cells . Resulting damaged molecular patterns of RNA fragments stimulate immune sensors such as toll-like receptors (TLRs), and activated TLRs provoke immunokines which further induce production of cytokines, growth factors and angiogenic modulators influencing tumor progression . It was shown that ranpirnase eradicates human papillomavirus in cultured cells and heals anogenital warts in a phase I study .
Binase, an enzyme from Bacillus intermedius, display antitumor activities against K562, A549, ovary cancer cells and Kasumi-1 cells . It renders antiviral effect against rabies virus, plant virus, influenza virus strains  and does not interact with mammalian RNase inhibitor evading its attack. ONC downregulates microRNA expression through targeting microRNA precursors .
Several attempts are made to isolate cytotoxic RNases from plants , molds , and mushrooms [23, 33] with varying success in the treatment of experimental tumors.
In addition to resistance to natural inhibitors for the implementation of the antitumor action of RNases, it’s important to achieve a net positive charge of the enzyme molecule [21, 22]. The cytotoxic properties of naturally occurring or engineered RNases are correlated with their efficiency of cellular internalization and digestion level of cellular RNA. Artificially сationized RNases are considered to adsorb to the anionic cellular surface by Coulombic interactions, and then become efficiently internalized into cells by an endocytosis-like pathway. The design of cytotoxic RNases by chemical modification of surface carboxylic residues is one of the powerful strategies for enhancing cellular internalization and is accompanied with a decreased sensitivity for the cytoplasmic RNase inhibitor. Although chemically modified cationized RNases showed decreased ribonucleolytic activity, improved endocytosis and decreased affinity to the endogenous RNase inhibitor conclusively contribute to their ability to digest cellular RNA. Furthermore, the cytotoxicity of cationized RNases can be drastically enhanced by co-endocytosis with an endosome destabilizing peptide . Since efficient cellular internalization of proteins into living cells is important in biotechnology, the studies of cytotoxic RNases provided general rules for protein-based drug design. It was shown that targeted delivery of immuno-RNase may noticeably improve cancer therapy . Besides, chemical compounds of non-biological origin imitating the action of RNases are of interest as well .
It has also been shown that the antitumor activity of RNases can be significantly enhanced by suppressing the activity of its natural inhibitor , chemical modification of its molecule, in particular, dimerization , or imparting an increased net positive charge to the enzyme molecule [21, 22]. It is also possible to increase the effectiveness of antitumor therapy by the combined effect of RNases and conventional cytostatics [24, 25].
It turned out, however, that RNases from various sources have a different impact on the сеll proliferation and tumor growth. In the separate cases of selective destruction of mRNA that encodes the synthesis of a tumor suppressor protein, the result will be inverse, i.e. acceleration of tumor growth.
Of interest in this respect is angiogenin (ANG), a ribonuclease with stimulating effect on tumor growth. ANG initiates vascularization of tumors and consequently tumor growth.
As a member of the vertebrate-specific secreted RNase, ANG was firstly isolated from tumor cells and identified solely by its ability to induce the formation of new blood vessels, and now it has been recognized to play an important role in various physiological and pathological processes through regulating cell proliferation, survival, migration, invasion, and/or differentiation . ANG exhibits ribonucleolytic activity that is critical for its biological functions exerted through activating different signaling transduction pathways in different target cells. A series of recent studies have indicated that ANG contributes to cellular nucleic acid metabolism . Moreover, current problems and future research directions of ANG are actively discussed. ANG promotes cell growth and survival. Under certain cell growth conditions, ANG undergoes nuclear translocation and accumulates in the nucleolus where it stimulates rRNA transcription. When cells are under stress, ANG mediates the production of tRNA-derived stress-induced small RNA (tiRNA), which reprograms protein translation into a survival mechanism. The ribonucleolytic activity of ANG is essential for both processes, but the way of regulation of such activity remains unknown . It was found that ribonuclease/ANG inhibitor 1 (RNH1) controls both the localization and activity of ANG. Under growth conditions, ANG is located in the nucleus and is not associated with RNH1 so that the ribonucleolytic activity is retained to ensure rRNA transcription. Cytoplasmic ANG is associated with and inhibited by RNH1 so that random cleavage of cellular RNA is prevented . Under stress conditions, ANG is localized to the cytoplasm and is concentrated in stress granules where it is not associated with RNH1 and thus remains enzymatically active for tiRNA production . By contrast, nuclear ANG is associated with RNH1 in stressed cells to ensure that the enzymatic activity is inhibited and no unnecessary rRNA is produced to save anabolic energy. Knockdown of RNH1 abolished stress-induced relocalization of ANG and decreased cell growth and survival . The understanding of the function of ANG will help to better delineate its role in diseases, especially in cancer. Blockade of nuclear translocation of ANG by the aminoglycoside antibiotic neomycin inhibited PC-3 cell tumor growth in athymic mice and was accompanied by a decrease in both cancer cell proliferation and angiogenesis. RNase inhibitor expresses anti-angiogenic properties and leads to reduced tumor growth in mice [36, 43–45].
These results suggest that ANG has a dual effect on angiogenesis and cancer cell proliferation, and may serve as a molecular target for drug development. Blocking nuclear translocation of ANG could be beneficial in combined cancer therapy [39, 42, 43].
The ambivalent role of RNase in relation to tumor growth is also manifested at other levels of regulation. Thus, RNases that synthesize numerous microRNAs can affect the activation of both oncogenes and tumor suppressor genes with corresponding consequences.
Regulation of mRNA decay plays a crucial role in the post-transcriptional control of cell growth, survival, differentiation, death and senescence. Deadenylation is a rate-limiting step in the silencing and degradation of the bulk of highly regulated mRNAs. However, the physiological functions of various deadenylases have not been fully deciphered. It was found that poly(A)-specific ribonuclease (PARN) was upregulated in gastric tumor tissues and gastric cancer cell lines MKN28 and AGS . The cellular function of PARN was investigated by stable knockdown of the endogenous PARN in the MKN28 and AGS cells. It was showed that PARN-depletion significantly inhibited the proliferation of the two types of gastric cancer cells and promoted cell death, but did not significantly affect cell motility and invasion. The depletion of PARN arrested the gastric cancer cells at the G0/G1 phase by upregulating the expression levels of p53 and p21 but not p27. The mRNA stability of p53 was unaffected by PARN-knockdown in both types of cells. A significant stabilizing effect of PARN-depletion on p21 mRNA was observed in the AGS cells but not in the MKN28 cells. It was further shown that the p21 3´-UTR triggered the action of PARN in AGS cells. The dissimilar observations on the MKN28 and AGS cells as well as various stress conditions suggested that the action of PARN strongly relied on protein expression profiles of the cells, which led to heterogeneity in the stability of PARN-targeted mRNAs [40, 46, 47]. Therefore, PARN has been proposed to be considered as a potential target for cancer treatment .
These data show that a variety of RNases have different effects on metabolic processes, in particular, on the processes of proliferation, malignant transformation and tumor growth. Thus, RNases exert powerful natural defense against viruses, microorganisms [48–51], as well as against cancer. In some cases, it is necessary to increase the activity of the enzymes that hydrolyze certain types of RNA, while in others it is necessary to use specific inhibitors, both naturally occurring and artificially created, to achieve the desired antitumor effect.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
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