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Ovarian cancer represents one of the most malignant gynecological cancers worldwide, with an overall 5-year survival rate, being locked in the 25-30% range in the last decade. Cancer immunotherapy is currently one of the most intensively investigated and promising therapeutic strategy and as such, is expected to provide in the incoming years significant benefits for ovarian cancer treatment as well. Here, we provide a detailed survey on the highly pleiotropic oncosuppressive roles played by the human RNASET2 gene, whose protein product has been consistently reported to establish a functional crosstalk between ovarian cancer cells and key cellular effectors of the innate immune system (the monocyte/macrophages lineage), which is in turn able to promote the recruitment to the cancer tissue of M1-polarized, antitumoral macrophages. This feature, coupled with the ability of T2 ribonucleases to negatively affect several cancer-related parameters in a cell-autonomous manner on a wide range of ovarian cancer experimental models, makes human RNASET2 a very promising candidate to develop a "multitasking" therapeutic approach for innovative future applications for ovarian cancer treatment.

Hypoxia is a key component of the tumor microenvironment (TME) and promotes not only tumor growth and metastasis, but also negatively affects infiltrating immune cells by impairing host immunity. Dendritic cells (DCs) are the most potent antigen-presenting cells and their biology is weakened in the TME in many ways, including the modulation of their viability. RNASET2 belongs to the T2 family of extracellular ribonucleases and, besides its nuclease activity, it exerts many additional functions. Indeed, RNASET2 is involved in several human pathologies, including cancer, and it is functionally relevant in the TME. RNASET2 functions are not restricted to cancer cells and its expression could be relevant also in other cell types which are important players in the TME, including DCs. Therefore, this study aimed to unravel the effect of hypoxia (2% O 2 ) on the expression of RNASET2 in DCs. Here, we showed that hypoxia enhanced the expression and secretion of RNASET2 in human monocyte-derived DCs. This paralleled the HIF-1α accumulation and HIF-dependent and -independent signaling, which are associated with DCs' survival/autophagy/apoptosis. RNASET2 expression, under hypoxia, was regulated by the PI3K/AKT pathway and was almost completely abolished by TLR4 ligand, LPS. Taken together, these results highlight how hypoxia- dependent and -independent pathways shape RNASET2 expression in DCs, with new perspectives on its implication for TME and, therefore, in anti-tumor immunity.

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme Hv RNASET2 modulates the leech's innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, Hv RNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the Hv RNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli . For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate Hv RNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that Hv RNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health.

Members of the T2 extracellular ribonucleases family have long been reported as stress response proteins, often involved in host defence, in many different taxonomic groups. In particular, the human RNASET2 protein (hRNASET2) has been reported as an extracellular tumor suppressor protein, endowed with the ability to act as an "alarmin" signalling molecule following its expression and secretion in the tumor microenvironment by cancer cells and the subsequent recruitment and activation of cells belonging to the host innate immune system. Many in vitro and in vivo assays have been recently reported in support of the oncosuppressive role of hRNASET2: most of them relied on genetically engineered cell lines and the use of recombinant proteins from non-mammalian sources. In order to ensure a human-like glycosylation pattern, here we report for the first time the expression of recombinant hRNASET2 in the CHO-S cell line. We established a simple one-step chromatographic purification procedure that resulted in the production of 5 mg of endotoxin-free hRNASET2 per liter of culture, with a >95% purity degree. hRNASET2 expressed in CHO-S cells displayed a high degree of glycosylation homogeneity and a secondary structure content in agreement with that determined from the crystal structure. Indeed, recombinant hRNASET2 was active at both enzymatic and functional level, as stated by a biological activity assay. The availability of a pure, homogeneous recombinant human RNASET2 would provide a key tool to better investigate its non cell-autonomous roles in the context of cancer development and growth., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The RNASET2 ribonuclease, belonging to the highly conserved RH/T2/s RNase gene family, has been recently shown to modulate inflammatory processes in both vertebrates and invertebrates. Indeed, the RNASET2 protein acts as a chemoattractor for macrophages in both in vitro and in vivo experimental settings and its expression significantly increases following bacterial infections. Moreover, we recently observed that injection of human recombinant RNASET2 protein in the body wall of the medicinal leech (a consolidated invertebrate model for both immune response and tissue regeneration) not only induced immune cell recruitment but also apparently triggered massive connective tissue remodelling as well. Based on these data, we evaluate here a possible role of leech recombinant RNASET2 protein (rHvRNASET2) in connective tissue remodelling by characterizing the cell types involved in this process through histochemical, morphological and immunofluorescent assays. Moreover, a time-course expression analysis of newly synthesized pro-collagen1α1 (COL1α1) and basic FGF receptor (bFGFR, a known fibroblast marker) following rHvRNASET2 injection in the leech body wall further supported the occurrence of rHvRNASET2-mediated matrix remodelling. Human MRC-5 fibroblast cells were also investigated in order to evaluate their pattern of collagen neosynthesis driven by rHvRNASET2 injection.Taken together, the data reported in this work provide compelling evidence in support of a pleiotropic role for RNASET2 in orchestrating an evolutionarily conserved crosstalk between inflammatory response and regenerative process, based on macrophage recruitment and fibroblast activation, coupled to a massive extracellular reorganization.

The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate Hirudo verbana (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein ( Hv RNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, Hv RNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of Hv RNASET2 was also evaluated on Staphylococcus aureus living cells by means of light, transmission, and scanning electron microscopy analysis. Hv RNASET2 injection triggered the formation of S. aureus clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released Hv RNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area., (Copyright © 2020 Baranzini, De Vito, Orlandi, Reguzzoni, Monti, de Eguileor, Rosini, Pollegioni, Tettamanti, Acquati and Grimaldi.)

The link between cancer development or progression and immune system dysregulation has long been established. Virtually every cell type belonging to both the innate and adaptive immune system has been reported to be involved in a complex interplay that might culminate into either a pro- or anti-tumorigenic response. Among the cellular components of the innate immune system, cells belonging to the monocyte/macrophage lineage have been consistently shown to play a key role in the tumorigenic process. The most advanced human tumors are reported to be strongly infiltrated with Tumor-Associated Macrophages (TAMs) endowed with the ability to contribute to tumor growth and dissemination. However, given their widely acknowledged functional plasticity, macrophages can display anti-tumor properties as well. Based on these premises, experimental approaches to promote the in vivo macrophage shift from pro-tumor to anti-tumor phenotype represent one of the most promising research field aimed at developing immune system-mediated tumor suppressive therapies. In this context, the human RNASET2 oncosuppressor gene has emerged as a potential tool for macrophage-mediated tumor suppression. A growing body of experimental evidence has been reported to suggest a role for this gene in the regulation of macrophage activity in both in vitro and in vivo experimental models. Moreover, several recent reports suggest a role for this gene in a broad range of cell types involved in immune response, pointing at RNASET2 as a putative regulator of several functional features within the immune system., (Copyright © 2019 Acquati, Mortara, De Vito, Baci, Albini, Cippitelli, Taramelli and Noonan.)

Macrophages represent key inflammatory cellular effectors of the innate immune response. Despite being widely acknowledged as professional phagocytes, the functional roles played by these cells have been progressively widened over the years to encompass regulation of the adaptive immune system, stimulation or suppression of cancer cell growth and tissue remodeling. These diverse functional features have led to the concept of "macrophage plasticity", i.e. the ability of these cells to express a wide range of phenotypes endowed with different functional roles. Several activation programs have been described for mammalian macrophages, based mainly on their differential transcriptional profiles. Based on established in vitro experimental conditions, many researchers currently refer to the M1 (or M1-like) and M2 (or M2-like) terms to describe the two extremes of a rather broad spectrum of polarization states that macrophages can experience in vivo. In light of the widely recognized opposite roles of M1-like and M2-like macrophages on cancer growth, and our largely incomplete knowledge of the cellular and molecular mechanisms underlying the establishment of the M1-like versus M2-like balance within a tumor mass, we report here results from in vitro assays pointing at the human RNASET2 gene as a potential regulator of the balance between M1-like/M2-like macrophage polarization. Not only do our results confirm previous in vivo data, thus further supporting a role for this pleiotropic protein in the innate immune system, but they also define RNASET2 as a new molecular target with potential applications for in vivo reprogramming of macrophage polarization, an increasingly appraised anticancer strategy., (Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Ribonuclease T2 (RNASET2) is a pleiotropic and polyfunctional protein, which exerts several different activities in neoplastic cells since the early steps of tumor development. Besides having an antitumorigenic activity, RNASET2 inhibits both bFGF-induced and VEGF-induced angiogenesis and has a role as a stress-response, alarmin-like, protein. In this study, we investigated RNASET2 expression in well-differentiated and poorly differentiated neuroendocrine neoplasms of the lung (Lu-NENs), which are known to show clear-cut differences in morphology, biology and clinical behavior. In addition, we explored possible relationships between RNASET2 expression and a series of immunohistochemical markers related to hypoxic stress, apoptosis, proliferation and angiogenesis. Our results showed a significantly higher expression of RNASET2, HIF-1α, and its target CA IX in poorly differentiated than in well-differentiated Lu-NENs, the former also showing higher proliferation and apoptotic rates, as well as a lower microvessel density (MVD) than the latter. Moreover, we were able to demonstrate in vitro an overexpression of RNASET2 in consequence of the activation of HIF-1α. In conclusion, we suggest that in poorly differentiated Lu-NENs, RNASET2 expression may be induced by HIF-1α, behaving as an alarmin-like molecule. In this aggressive group of cancers, which have highly deregulated proliferation pathways, RNASET2 fails to exert the growth-inhibiting effects described in other types of neoplasms. Its increased expression, however, may contribute to the typical phenotypic alterations seen in poorly differentiated Lu-NENs, such as the high apoptotic rate and the extensive necrosis, and may also enhance the low MVD observed in these neoplasms., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68 + and HmAIF-1 + macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

Ribonucleases (RNases) are hydrolytic enzymes endowed with the ability to either process or degrade ribonucleic acids. Among the many biological functions assigned to RNases, a growing attention has been recently devoted to the control of cancer growth, in the attempt to bring novel therapeutic approaches to clinical oncology. Indeed, several enzymes belonging to different ribonuclease families have been reported in the last decade to display a marked oncosuppressive activity in a wide range of experimental models. The human RNASET2 gene, the only member of the highly conserved T2/Rh/S family of endoribonucleolytic enzymes described in our species, has been shown to display oncosuppressive roles in both in vitro and in vivo models representing several human malignancies. In the present study, we extend previous findings obtained in ovarian cancer models to shed further light on the cell-autonomous roles played by this gene in the context of its oncosuppresive role and to show that RNASET2 silencing can significantly affect the transcriptional output in one of the most thoroughly investigated human ovarian cancer cell lines. Moreover, we report for the first time that RNASET2-mediated changes in the cell transcriptome are in part mediated by its apparent ability to affect the cell's microRNA expression pattern.

Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosuppressors called "tumor antagonizing genes," whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary, and availability of high-quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression.

As widely recognized, tumor growth entails a close and complex cross-talk among cancer cells and the surrounding tumor microenvironment. We recently described the human RNASET2 gene as one key player of such microenvironmental cross-talk. Indeed, the protein encoded by this gene is an extracellular RNase which is able to control cancer growth in a non-cell autonomous mode by inducing a sustained recruitment of immune-competent cells belonging to the monocyte/macrophage lineage within a growing tumor mass. Here, we asked whether this oncosuppressor gene is sensitive to stress challenges and whether it can trigger cell-intrinsic processes as well. Indeed, RNASET2 expression levels were consistently found to increase following stress induction. Moreover, changes in RNASET2 expression levels turned out to affect several cancer-related parameters in vitro in an ovarian cancer cell line model. Of note, a remarkable rearrangement of the actin cytoskeleton organization, together with changes in cell adhesion and motility, emerged as putative mechanisms by which such cell-autonomous role could occur. Altogether, these biological features allow to put forward the hypothesis that the RNASET2 protein can act as a molecular barrier for limiting the damages and tissue remodeling events occurring during the earlier step of cell transformation.

Characterising tumour-associated antigens (TAAs) not only represents an important approach to the identification of new diagnostic/prognostic markers, but can also provide information on disease processes and additional potential therapeutic targets. Preliminary screening of a protein macroarray, containing more than 12,000 different proteins, with sera from anaplastic lymphoma kinase (ALK)-negative and ALK-positive anaplastic large cell lymphoma (ALCL) patients identified ribonuclease and tumour suppressor protein Ribonuclease T2 (RNASET2), phosphatase lipid phosphate phosphatase-related protein type 3 (LPPR3) and apoptotic adaptor molecule Fas-associating protein (FADD) as ALK-negative ALCL-associated TAAs. Further validation of these observations was confirmed using the ALCL sera in reverse ELISAs. The circulating anti-RNASET2 autoantibodies present in ALCL patients' sera also recognised eukaryotically expressed RNASET2 protein. RNASET2 expression was then investigated in normal tissues and in lymphomas to explore its clinical potential. RNASET2 protein and mRNA levels showed highest expression in the spleen, leucocytes and pancreas. RNASET2 protein expression was not restricted to ALK-negative ALCL (81%), being expressed in ALK-positive ALCL (65%) as well as in a number of other lymphomas. The immunological recognition of RNASET2, its expression in ALCL and other lymphomas together with its known tumourigenic properties suggest that further studies on this autoantigen are warranted., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Background Information: The ribonucleases (RNases) constitute a heterogeneous group of enzymes, which exert diverse and specific biological functions. Several RNases have been shown to control gene expression and cell differentiation. RNASET2, a novel member of the Rh/T2/S family of RNases, exerts micro-environmental control of malignancy in different experimental models with a general onco-suppressor activity, since it prevents cancer proliferation. Indeed, RNASET2 was found to be downregulated at the transcript level in several primary ovarian tumours or cell lines and in melanoma cell lines. Although recent works shed light on the biological role of RNASET2 in delaying tumour growth, its trafficking within the cell is still poorly understood. RNASET2 seems to play diverse biological roles including turnover of tRNA in yeast as well as rRNA degradation in zebrafish., Results: Here, we have studied the intracellular trafficking of RNASET2 in mammalian cells. RNASET2 co-localizes with markers for the trans-Golgi network (TGN), which is the central sorting and processing station of the secretory pathway. Moreover, using the temperature-sensitive vesicular stomatitis glycoprotein, we demonstrate that RNASET2 undergoes delivery to the plasma membrane. In contrast to other RNA-interacting proteins, RNASET2 does not accumulate in stress granules upon metabolic stress in mammalian cells. Surprisingly, RNASET2 shows co-localization with processing bodies (P-bodies), which increases upon metabolic stress. Finally, cells lacking RNASET2 show a reduced numbers of P-bodies., Conclusions: In this study, we have identified two distinct cellular pools of RNASET2-containing granules. One pool undergoes membrane delivery using the TGN, and it is released to the extracellular environment. The second pool is recruited into P-bodies, suggesting a possible involvement of RNASET2 in P-body formation in mammalian cells., (Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.)

Using the Hey3Met2 human ovarian cancer cell line, we previously found the RNASET2 gene to possess a remarkable in vivo tumor suppressor activity, although no in vitro features such as inhibition of cell proliferation, clonogenic potential, impaired growth in soft agar and increase in apoptotic rate could be detected. This is reminiscent of the behavior of genes belonging to the class of tumor antagonizing genes (TAG) which act mainly within the context of the microenvironment. Here we present transcriptional profiles analysis which indicates that investigations of the mechanisms of TAG biological functions require a comparison between the in vitro and in vivo expression patterns. Indeed several genes displaying a biological function potentially related to tumor suppression could not be validated by subsequent in vivo expression analysis. On the other hand the fact that we could find congruency for three genes both in vivo and in vitro adds a warning to a too much stringent categorization of this class of genes which relies on the sensitivity of the methodological approaches.

A recent body of evidence indicates an active role for stromal (mis)-regulation in the progression of neoplasias. Within this conceptual framework, genes belonging to the growing but still poorly characterized class of tumor antagonizing/malignancy suppressor genes (TAG/MSG) seem to play a crucial role in the regulation of the cross-talk between stromal and epithelial cells by controlling malignant growth in vivo without affecting any cancer-related phenotype in vitro. Here, we have functionally characterized the human RNASET2 gene, which encodes the first human member of the widespread Rh/T2/S family of extracellular RNases and was recently found to be down-regulated at the transcript level in several primary ovarian tumors or cell lines and in melanoma cell lines. Although we could not detect any activity for RNASET2 in several functional in vitro assays, a remarkable control of ovarian tumorigenesis could be detected in vivo. Moreover, the control of ovarian tumorigenesis mediated by this unique tumor suppressor gene occurs through modification of the cellular microenvironment and the induction of immunocompetent cells of the monocyte/macrophage lineage. Taken together, the data presented in this work strongly indicate RNASET2 as a previously unexplored member of the growing family of tumor-antagonizing genes.

We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family., (Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.)

The RNASET2 gene, mapped in 6q27, was previously found to exert control of tumorigenesis in an ovarian cancer system. We present here results indicating a similar control in a melanoma cancer model. Thus, this gene is most likely involved in a common general pathway of tumorigenesis. Moreover, its antitumorigenic activity is manifested in vivo but not in vitro, suggesting that this gene belongs to the growing category of tumor antagonizing/malignancy suppressor genes. A possible role of RNASET2 in the activation of a senescence program, whose responsible locus was mapped in the same chromosomal 6q27 region, seems to be inconsistent with our data.

Ribonucleases are ubiquitous enzymes involved in RNA metabolism and are classified in several families on the basis of their structural, catalytic, and biological properties. Here, we describe characterization of the only human member of the Rh/T2/S family of acid hydrolases so far described, named RNASET2. This protein was previously reported to have an interesting biological function in the control of tumourigenesis and metastatization. We show that RNASET2 is present in multiple forms in human cell lines and mouse tissues, one of which represents the full length, glycosylated and secreted form, while the others are proteolytic products. RNASET2 is endowed with catalytic activity as demonstrated with purified recombinant protein expressed in the Baculovirus Expression Vector System and in a human cell line ectopically expressing various types of constructs. Furthermore, we document for this protein a lysosomal localization as described for other members of the Rh/T2/S family of ribonucleases. The results presented herein represent a further advancement toward the molecular understanding of the tumour suppressive properties of the human RNASET2 protein.

The region 6q27 from human chromosome 6 has been reported to contain one or more tumor suppressor genes on the basis of cytogenetic, molecular and functional studies. We have recently carried out a detailed analysis of a candidate gene from 6q27 to evaluate its putative role as a tumor suppressor gene involved in ovarian cancer pathogenesis. The RNASET2 gene was shown to behave as a class II tumor suppressor and abolish the tumorigenic potential of an ovarian cancer-derived cell line. In this study, we have started the cellular and biochemical characterization of RNASET2 and showed that it is a secreted glycoprotein. Moreover, we have extended our previous studies by evaluating the effect of RNASET2 on the metastatic behavior of the highly-invasive ovarian cancer cell line HEY3MET2. From such analysis, RNASET2 was found to significantly decrease the metastatic potential of this cell line in vivo. Moreover, RNASET2-mediated suppression of tumorigenesis and metastasis was not affected by a double point mutation targeted to the putative ribonuclease catalytic sites, suggesting that tumor suppression by RNASET2 is not mediated by its ribonuclease activity. The potential biological implications of this unexpected finding are discussed in relation to the current evolutionary models.

In recent years, there has been a growing interest in developing innovative anticancer therapies targeting the tumor microenvironment (TME). The TME is a complex and dynamic milieu surrounding the tumor mass, consisting of various cellular and molecular components, including those from the host organism, endowed with the ability to significantly influence cancer development and progression. Processes such as angiogenesis, immune evasion, and metastasis are crucial targets in the search for novel anticancer drugs. Thus, identifying molecules with "multi-tasking" properties that can counteract cancer cell growth at multiple levels represents a relevant but still unmet clinical need. Extensive research over the past two decades has revealed a consistent anticancer activity for several members of the T2 ribonuclease family, found in evolutionarily distant species. Initially, it was believed that T2 ribonucleases mainly acted as anticancer agents in a cell-autonomous manner. However, further investigation uncovered a complex and independent mechanism of action that operates at a non-cell-autonomous level, affecting crucial processes in TME-induced tumor growth, such as angiogenesis, evasion of immune surveillance, and immune cell polarization. Here, we review and discuss the remarkable properties of ribonucleases from the T2 family in the context of "multilevel" oncosuppression acting on the TME. [ABSTRACT FROM AUTHOR]

Ribonucleases (RNases) are key players of the host immunity and contribute to maintaining tissue homeostasis and body fluid sterility. Strikingly, RNases belonging to the T2 protein family have also recently been implicated in tumor suppression ([8]). Of note, tumor suppression turned out to be an evolutionarily conserved feature of T2 RNases ([12]) and to be carried out by means of both cell-autonomous and non-autonomous mechanisms, among which the establishment of cancer cell-tumor microenvironment cross-talk was established to play a key role ([13]). Within the frame of this Research Topic, the review by Acquati et al. summarizes the role played by members of the very ancient ribonuclease T2 gene family in innate immunity-mediated tumor suppression. [Extracted from the article]

In recent years, the role played by the stromal microenvironment has been given growing attention in order to achieve a full understanding of cancer initiation and progression. Because cancer is a tissue-based disease, the integrity of tissue architecture is a major constraint toward cancer growth. Indeed, a large contribution of the natural resistance to cancer stems from stromal microenvironment components, the dysregulation of which can facilitate cancer occurrence. For instance, recent experimental evidence has highlighted the involvement of stromal cells in ovarian carcinogenesis, as epitomized by ovarian xenografts obtained by a double KO of the murine Dicer and Pten genes. Likewise, we reported the role of an ancient extracellular RNase, called Ribonuclease T2 (RNASET2), within the ovarian stromal microenvironment. Indeed, hyperexpression of RNASET2 is able to control tumorigenesis by recruiting macrophages (mostly of the anticancer M1 subtype) at the tumor sites. We present biological data obtained by RNASET2 silencing in the poorly tumorigenetic and highly RNASET2-expressing human OVCAR3 cell line. RNASET2 knockdown was shown to stimulate in vivo tumor growth early after microinjection of OVCAR3 cells in nude mice. Moreover, we have investigated by molecular profiling the in vivo expression signature of human and mouse cell xenografts and disclosed the activation of pathways related to activation of the innate immune response and modulation of ECM components. Finally, we provide evidence for a role of RNASET2 in triggering an in vitro chemotactic response in macrophages. These results further highlight the critical role played by the microenvironment in RNASET2-mediated ovarian tumor suppression, which could eventually contribute to better clarify the pathogenesis of this disease. [ABSTRACT FROM AUTHOR]