**1.2. What makes a ribonuclease selectively cytotoxic for cancer cells?**

inducing angiogenesis, activating invasion and metastasis, reprogramming of energy metabolism, and evading immune destruction. Involved in these metaprocesses, there is a deregulation of gene expression. A significant part of the current chemotherapeutic com‐ pounds used to treat cancer patients target different over‐ or under‐expressed genes that take part in the abovementioned processes that drive to malignant cell transformation and/or

Control of gene expression can be carried out at different levels in the flow of genetic infor‐

**Figure 1.** Targets of genotoxic and non‐mutagenic antitumoral drugs along the information pathway. Transmission of biological information in tumor cells occurs from DNA to RNA and proteins that exert their biological function. (A) Classical antitumor therapies like radiotherapy and chemotherapy affect DNA inhibiting cell replication but may also kill normal dividing cells, and since they are genotoxic, they may induce secondary tumors. (B) Alternative damaging RNA therapies inhibit gene expression and its regulation. These therapies exert pleiotropic effects because they affect multiple RNA substrates and are not mutagenic. (C) Therapies affecting a single protein or pathway of the cell are highly specific but sometimes cannot cope with the multifactorial nature of cancer although are also non‐mutagenic.

Drugs that act over DNA have the drawback of being mutagenic and are responsible for the appearance of new cancers, time after the patients have been cured of or controlled their first cancer disease [2]. Instead, drugs that destroy or inactivate RNA are similarly powerful without the associated risk of genotoxicity. In addition, drugs that specifically target a single protein or pathway have the advantage of being highly specific, but they are often insufficient to cope with the multifactorial complexity of the cancer phenotype. Several approaches are used to target RNAs, the use of antisense oligonucleotides, small interfering RNA (siRNAs), and the use of ribozymes or proteins with ribonucleolytic activity [3]. In the present chapter, we will focus on ribonucleases (RNases) as antitumor agents and how the knowledge gained so far about their mechanism of action has inspired researchers in the design of more powerful and selective RNases that can overcome tumor resistance as well as minimize the toxic effects

RNases are enzymes present in all life kingdoms that degrade RNA and in cells are responsible for RNA turnover [4]. Their interest as antitumor agents started early in the fifties of the last

to normal cells, properties strongly desired for any antitumor drug.

metastasis.

**1.1. Why target RNA to treat cancer diseases?**

mation from DNA to proteins (**Figure 1**).

136 Anti-cancer Drugs - Nature, Synthesis and Cell

Although some RNases have reached clinical trials for treatment of different types of cancer [19–25], their mechanism of action is not well understood. Nevertheless, RNases share some steps of cell intoxication with most cytotoxins.

**Figure 2.** Multifactorial causes of RNase cytotoxicity. Some RNases are able to reach the cytosol but are not cytotoxic because they are unable to evade the action of the RI (A). Other RNases are not cytotoxic because they cannot reach the cytosol, either because they are degraded during its internalization (B) or because they follow an intracellular pathway that does not allow them to reach this compartment (C). Some RNases are cytotoxic because they reach the cytosol and are not inhibited by the RI (D). Other RNases are cytotoxic although they do not evade the RI either because they reach the cytosol with high efficiency allowing to saturate the RI present in the cytosol (E) or because they can reach the nu‐ cleus where the RI cannot inhibit them (F).

To be cytotoxic an RNase has to reach the tumor cells. This implies two basic steps: to attain target cells from the administration point (RNases are mainly administered i.v.) and to be able to enter these cells. The first step means that the RNase has to be stable enough in blood to reach their target cells and not to be cleared rapidly from circulation through glomerular filtration. The second step [26–28] implies an interaction with a specific or a nonspecific component of the target cell surface in order to be endocytosed. Then, during its journey, at some point of the endocytic pathway (**Figure 2B**), the RNase has to translocate to the cytosol to avoid lysosomal degradation and, obviously, follow a productive endocytic pathway. Once in the cytosol, it has to be stable and resistant to proteases, and at the same time, it has to evade the ribonuclease inhibitor (RI) to preserve its ribonucleolytic activity and therefore be able to degrade RNAs and induce cell death by apoptosis. The RI is a protein present in the cytosol of mammalian cells that binds to some RNases with high affinity [29]. It is hypothesized that the RI acts as a safeguard for the potential entry of any external RNase [30]. Alternatively to the evasion strategy, an RNase can also have the ability to enter the cell very efficiently to saturate the RI and to leave free RNase molecules able to degrade RNAs. Finally, an RNase can also be driven to any organelle devoid of RI where it can degrade RNAs, for instance, the cell nucleus [31] (**Figure 2F**).

The paradigmatic native cytotoxic RNase that evades the RI is onconase (ONC), a member of the vertebrate‐secreted RNase family of amphibian origin (isolated from oocytes and early embryos of *Rana pipiens*). ONC reached phases II/IIIb for treatment of malignant pleural mesothelioma [21] although it presents renal toxicity that is reversed when the treatment is discontinued [32]. It exhibits selective cytostatic and cytotoxic activities against many tumor models both *in vitro* and *in vivo* [22, 33] and presents synergy, proved also *in vivo* and *in vitro*, with a significant number of compounds [34]. ONC induces apoptosis or in some cases autophagy previously to apoptosis [35, 36]. These processes present characteristics different from those of indiscriminate protein synthesis arrest and are due to the degradation of different target RNAs, rRNAs [37], mRNAs [38], tRNAs [39], and miRNAs or their precursors [40–42]. It has been described that ONC up‐ or downregulates genes that code for proteins involved in cell cycle control or transcription factors that are also responsible for its cytotoxicity [43]. Although from the literature the apoptotic effects seem to be cell‐type dependent [34], recently it has been found that the activating transcription factor 3 (ATF3) controls ONC‐induced apoptosis in a cell‐type independent manner (Vert et al., submitted). Other tumoricidal amphibian RNases are Amphinase (Amph), also isolated from oocytes of *R. pipiens* [44] and the sialic acid‐binding lectins (leczymes) found in *Rana catesbeiana* (RC‐RNase) and *Rana japonica* (RJ‐RNase) oocytes [45, 46]. Unlike ONC and Amph, these latter ones agglutinate cancer cells [47–49] binding to cell membrane glycoproteins with a high content of sialic acids [47, 49]. It is also proposed, like for ONC and Amph, that these leczymes require an internal‐ ization process to trigger apoptosis [50]; however compared to them, clinical trials and studies on animal models are needed to unveil their mechanism of antitumor activity and clinical potential.

The critical process of ONC internalization is still an open question. This is not a minor issue because it is strongly related to the RNase cytotoxic selectivity for cancer cells. For ONC it has been described both the existence of a specific receptor [37] and an entry through a non‐ saturable process [51] as well as an entry through the clathrin/AP‐2‐mediated endocytic pathway [52] and a non‐dynamin‐dependent pathway [51]. These discrepancies may be explained by the model cell lines used in the different works. In addition, electrostatics are described as necessary for the cellular uptake of ONC, while for other RNases, an specific interaction with cell surface structures seems to contribute more decisively to their internali‐ zation [53]. Essentially, RNases are cationic proteins, and since the surface of most cancer cells is more electronegative [54] than that of normal cells, the electrostatic interactions that they establish may dictate their selectivity. Very recently, both RNase A and its human counterpart, the human pancreatic ribonuclease (HP‐RNase), have been described to interact with a neutral hexasaccharide glycosphingolipid, Globo H [55], a component of a glycolipid or a glycoprotein located on the outer membrane of epithelial cells and detected in high levels in the outer membrane of several tumor cells [56]. The authors suggest that this interaction is not only substantial for the internalization of these RNases but for their release from the lumen of endosomes allowing for the access to the cytosol [55], although if they are not engineered to avoid the RI, they are not cytotoxic (see below). In addition, for RNase A, it has been described a multipathway of internalization that involves both clathrin‐coated vesicles and macropino‐ somes [57]. Finally, through an *in silico* study by sliding‐window hydrophobicity analysis, it has been hypothesized that some cytotoxic RNases have a hydrophobic segment sterically available for a hydrophobic interaction with both tumor cell and endosomal membranes that would facilitate their internalization [58]. The more it is known about the membrane structures that are recognized by RNases or the productive pathway, by which they enter the cell, the better they can be engineered to increase their selectivity and potency.

some point of the endocytic pathway (**Figure 2B**), the RNase has to translocate to the cytosol to avoid lysosomal degradation and, obviously, follow a productive endocytic pathway. Once in the cytosol, it has to be stable and resistant to proteases, and at the same time, it has to evade the ribonuclease inhibitor (RI) to preserve its ribonucleolytic activity and therefore be able to degrade RNAs and induce cell death by apoptosis. The RI is a protein present in the cytosol of mammalian cells that binds to some RNases with high affinity [29]. It is hypothesized that the RI acts as a safeguard for the potential entry of any external RNase [30]. Alternatively to the evasion strategy, an RNase can also have the ability to enter the cell very efficiently to saturate the RI and to leave free RNase molecules able to degrade RNAs. Finally, an RNase can also be driven to any organelle devoid of RI where it can degrade RNAs, for instance, the cell

The paradigmatic native cytotoxic RNase that evades the RI is onconase (ONC), a member of the vertebrate‐secreted RNase family of amphibian origin (isolated from oocytes and early embryos of *Rana pipiens*). ONC reached phases II/IIIb for treatment of malignant pleural mesothelioma [21] although it presents renal toxicity that is reversed when the treatment is discontinued [32]. It exhibits selective cytostatic and cytotoxic activities against many tumor models both *in vitro* and *in vivo* [22, 33] and presents synergy, proved also *in vivo* and *in vitro*, with a significant number of compounds [34]. ONC induces apoptosis or in some cases autophagy previously to apoptosis [35, 36]. These processes present characteristics different from those of indiscriminate protein synthesis arrest and are due to the degradation of different target RNAs, rRNAs [37], mRNAs [38], tRNAs [39], and miRNAs or their precursors [40–42]. It has been described that ONC up‐ or downregulates genes that code for proteins involved in cell cycle control or transcription factors that are also responsible for its cytotoxicity [43]. Although from the literature the apoptotic effects seem to be cell‐type dependent [34], recently it has been found that the activating transcription factor 3 (ATF3) controls ONC‐induced apoptosis in a cell‐type independent manner (Vert et al., submitted). Other tumoricidal amphibian RNases are Amphinase (Amph), also isolated from oocytes of *R. pipiens* [44] and the sialic acid‐binding lectins (leczymes) found in *Rana catesbeiana* (RC‐RNase) and *Rana japonica* (RJ‐RNase) oocytes [45, 46]. Unlike ONC and Amph, these latter ones agglutinate cancer cells [47–49] binding to cell membrane glycoproteins with a high content of sialic acids [47, 49]. It is also proposed, like for ONC and Amph, that these leczymes require an internal‐ ization process to trigger apoptosis [50]; however compared to them, clinical trials and studies on animal models are needed to unveil their mechanism of antitumor activity and clinical

The critical process of ONC internalization is still an open question. This is not a minor issue because it is strongly related to the RNase cytotoxic selectivity for cancer cells. For ONC it has been described both the existence of a specific receptor [37] and an entry through a non‐ saturable process [51] as well as an entry through the clathrin/AP‐2‐mediated endocytic pathway [52] and a non‐dynamin‐dependent pathway [51]. These discrepancies may be explained by the model cell lines used in the different works. In addition, electrostatics are described as necessary for the cellular uptake of ONC, while for other RNases, an specific interaction with cell surface structures seems to contribute more decisively to their internali‐

nucleus [31] (**Figure 2F**).

138 Anti-cancer Drugs - Nature, Synthesis and Cell

potential.

Another RNase that naturally shows antitumor activity by RI evasion is bovine seminal ribonuclease (BS‐RNase), present in the bull seminal plasma. In this case the quaternary structure attained by this enzyme is responsible for its low RI affinity due to steric hindrance, while the monomeric form is strongly inhibited by the RI [59]. BS‐RNase exists as a mixture of two dimeric forms, M=M and MxM, each monomer being a structural homolog of RNase A [60]. The MxM dimer exchanges the N‐terminal α‐helices forming a 3D‐swapped structure and is the form that even in the reducing conditions of the cell cytosol is cytotoxic [61] (for a comparative review on the RNase structures, see [62, 63]). BS‐RNase binds to the extracellular matrix, and this interaction seems to be important for its cytotoxic effect [64, 65] even though it does not bind to cell membranes, suggesting an adsorption cell entry mechanism [66]. BS‐ RNase has been localized in endosomes and its cytotoxicity is blocked by inhibition of this energy‐dependent entry mechanism [65]. It has also been localized in the trans‐Golgi network of treated malignant cells, which may be indicative that this organelle is an effective site for translocation providing an explanation for its selectivity [65, 67]. Although, it has been described that BS‐RNase can destabilize artificial membranes [68, 69], it is not exactly known how BS‐RNase permeates the trans‐Golgi membranes. Like for ONC, rRNA is a target of BS‐ RNase and its cleavage induces apoptosis [64], but the enzyme has also been found in the nucleolus of cancer cells [65], and although it is not known how it reaches the cell nucleus, a correlation between cytotoxicity and a decrease of telomerase activity and its associated RNA has been found [70]. Recently, it has been described that BS‐RNase triggers Beclin1‐mediated autophagy in treated cancer cells being ineffective in normal cells, suggesting that autophagy more than apoptosis can be the mechanism of cancer cell death induced by BS‐RNase [71]. Comparable to ONC, this selective autophagy for cancer cells seems to be related to the basic charge distribution in the surface of these RNases [36, 71].

Apart from RNases of animal origin, it is worth mentioning that there are a vast array of RNases from fungal, bacterial, and plant origin that natively present remarkable cytotoxic properties. Among them we can mention mushroom RNases [72, 73]; microbial RNases such as α‐sarcin from *Aspergillus*; the two well‐known T1 ribonuclease members from *Bacillus*, binase (*B. intermedius*), and barnase (*B. amyloliquefaciens*); and RNase Sa (*S. aureofaciens*) [28, 74] as well as plant RNases from ginseng, wheat leaf, mung bean, black pine pollen, seeds of bitter gourd, tomato, and hop [12, 15, 75–79]. Although adverse effects due to immunogenicity and nonspecific binding [12] have been described for some of them, others are described to have a lower immunogenicity than ONC [28]. In addition, they have a remarkable resistance to RI, and in some cases, the cytotoxic effect is comparable to that of ONC. However, in terms of knowledge of their cytotoxic mechanism and clinical applications, they are still lagging behind when compared to the animal counterparts. In the last years, especially for binase, a significant advance has been attained in the understanding of its mechanism of cell intoxication. The cytotoxic effect of binase is effected via induction of both intrinsic and extrinsic apoptotic pathways [80], and evidence is provided that targets KIT and AML1‐ETO oncogenes in human leukemia Kasumi‐1 cells [81]. It has also shown a positive effect on the liver of tumor‐bearing mice, articulated as a tumor reduction in the volume of destructive changes in the live parenchyma as well as of being effective in tumor growth suppression [82].
