The roles of cell motility and angiogenetic processes in metastatic spread and tumor aggressiveness are well established and must be simultaneously targeted to maximize antitumor drug potency. This work evaluated the antitumorigenic capacities of human recombinant RNASET2
ET2 (hrRNASET2), a homologue of the Aspergillus niger T2RNase ACTIBIND, which has been shown to display both antitumorigenic and antiangiogenic activities. hrRNASET2 disrupted intracellular actin filament and actin-rich extracellular extrusion organization in both CT29 colon cancer and A375SM melanoma cells and induced a significant dose-dependent inhibition of A375SM cell migration. hrRNASET2 also induced full arrest of angiogenin-induced tube formation and brought to a three-fold lower relative HT29 colorectal and A375SM melanoma tumor volume, when compared to Avastin-treated animals. In parallel, mean blood vessel counts were 36.9% lower in hrRNASET2-vs. Avastin-treated mice and survival rates of hrRNASET2-treated mice were 50% at 73 days post-treatment, while the median survival time for untreated animals was 22 days. Moreover, a 60-day hrRNASET2 treatment period reduced mean A375SM lung metastasis foci counts by three-fold when compared to untreated animals. Taken together, the combined antiangiogenic and antitumorigenic capacities of hrRNASET2, seemingly arising from its direct interaction with intercellular and extracellular matrices, render it an attractive anticancer therapy candidate.
Sinkevicius KW, etal., Dis Model Mech. 2018 Jun 27;11(6). pii: dmm.032631. doi: 10.1242/dmm.032631.
RNASET2 deficiency in humans is associated with infant cystic leukoencephalopathy, which causes psychomotor impairment, spasticity and epilepsy. A zebrafish mutant model suggests that loss of RNASET2 function leads to neurod
egeneration due to the accumulation of non-degraded RNA in the lysosomes. The goal of this study was to characterize the first rodent model of RNASET2 deficiency. The brains of 3- and 12-month-old RNaseT2 knockout rats were studied using multiple magnetic resonance imaging modalities and behavioral tests. While T1- and T2-weighted images of RNaseT2 knockout rats exhibited no evidence of cystic lesions, the prefrontal cortex and hippocampal complex were enlarged in knockout animals. Diffusion-weighted imaging showed altered anisotropy and putative gray matter changes in the hippocampal complex of the RNaseT2 knockout rats. Immunohistochemistry for glial fibrillary acidic protein (GFAP) showed the presence of hippocampal neuroinflammation. Decreased levels of lysosome-associated membrane protein 2 (LAMP2) and elevated acid phosphatase and ß-N-acetylglucosaminidase (NAG) activities indicated that the RNASET2 knockout rats likely had altered lysosomal function and potential defects in autophagy. Object recognition tests confirmed that RNaseT2 knockout rats exhibited memory deficits. However, the Barnes maze, and balance beam and rotarod tests indicated there were no differences in spatial memory or motor impairments, respectively. Overall, patients with RNASET2 deficiency exhibited a more severe neurodegeneration phenotype than was observed in the RNaseT2 knockout rats. However, the vulnerability of the knockout rat hippocampus as evidenced by neuroinflammation, altered lysosomal function and cognitive defects indicates that this is still a useful in vivo model to study RNASET2 function.
Wang Q, etal., J Dermatol Sci. 2015 Oct;80(1):25-32. doi: 10.1016/j.jdermsci.2015.07.004. Epub 2015 Jul 15.
BACKGROUND: Impaired dendrite outgrowth of melanocytes is one of the reasons triggering vitiligo. RNASET2 was identified as one of the risk genes for vitiligo in a GWAS study conducted in the Chinese Han population. However, the role of Rnaset2
700;'>Rnaset2 in the outgrowth of melanocytes is rarely studied. OBJECTIVE: This study is to investigate the effects of Rnaset2 in regulating the outgrowth of melanocytes and its interacting proteins. METHODS: Stress conditions (UV irradiation, hydrogen peroxide, and lipopolysaccharides) were applied to primary human epidermal melanocytes (HEMs) and epidermal keratinocytes (HEKs). HEKs with Rnaset2 overexpression were co-cultured with HEMs. Rnaset2 expression levels were detected by ELISA. HEMs, HEKs and A375 cells were treated with recombinant Rnaset2 protein and actin network was observed with fluorescence microscope. Cell migration assay was performed using nuclepore filters after incubating A375 cells with recombinant Rnaset2 protein. Human proteome microarray was performed to identify proteins interacting with Rnaset2. Co-immunoprecipitation was conducted to verify the results. RESULTS: Our results showed that after exposing to stress conditions, Rnaset2 expression and secretion by HEKs and HEMs were increased. Co-culture of HEKs and HEMs showed that outgrowth of HEMs was inhibited by Rnaset2 overexpression in HEKs. Additionally, human recombinant Rnaset2 protein treatment altered the actin network of HEMs, HEKs and A375 cells. The migration of A375 cells was also inhibited by human recombinant Rnaset2 protein treatment. Human proteome microarray identified shootin1, an important protein involved in axon outgrowth, as one of the interacting proteins of Rnaset2. Co-immunoprecipitation confirmed that Rnaset2 interacted with shootin1 in vitro. CONCLUSION: Rnaset2 inhibits melanocyte outgrowth through interacting with shootin1 and this effect may be associated with vitiligo pathogenesis. Rnaset2 may be a potential therapeutic target for vitiligo.
Henneke M, etal., Nat Genet. 2009 Jul;41(7):773-5. doi: 10.1038/ng.398. Epub 2009 Jun 14.
Congenital cytomegalovirus brain infection without symptoms at birth can cause a static encephalopathy with characteristic patterns of brain abnormalities. Here we show that loss-of-function mutations in the gene encoding the RNASET2 glycoprotein lead to cystic
leukoencephalopathy, an autosomal recessive disorder with an indistinguishable clinical and neuroradiological phenotype. Congenital cytomegalovirus infection and RNASET2 deficiency may both interfere with brain development and myelination through angiogenesis or RNA metabolism.
Zeng Z, etal., Front Oncol. 2020 May 22;10:836. doi: 10.3389/fonc.2020.00836. eCollection 2020.
Background: In addition to exploiting its ribonuclease capacity, Ribonuclease T2 (RNASET2) has been reported to exert anti-angiogenic and anti-tumorigenic effects in several tumors. However, the role of RNASET2 in gastric ad
enocarcinoma (GAC) remains unclear. The purpose of this study was to explore the expression, location, and clinical implications of RNASET2 in GAC. Methods: Data of RNASET2 mRNA expression in GAC and normal gastric mucosa tissues were extracted from three GSE series and 388 TCGA samples and reanalyzed. Genome-wide CRISPR/Cas9 proliferation screening datasets were used to investigate cell growth changes after RNASET2 knockout in 19 GAC cell lines. The biological processes involved in RNASET2 were studied by the bioinformatics analysis. Furthermore, the corresponding experiments including immunohistochemical staining, clinicopathological features analysis, survival curve, microvessel density detection, cell viability assay, and colony formation assay were performed to validate the expression and function of RNASET2 in GAC. Results: An abundance of RNASET2 was present in the fundus glands and pylorus glands of the normal gastric mucosa. RNASET2 mRNA and protein were down-regulated in GAC compared with adjacent non-cancerous or normal gastric mucosa tissues. The expression of RNASET2 mRNA and protein in early GAC was higher than that in advanced GAC. 79/134 gene sets involved in the early GAC pathway were enriched in the RNASET2 mRNA high expression group. Genome-wide shRNA and CRISPR/Cas9 proliferation screening showed that knockdown or knockout of RNASET2 could not significantly promote GAC cell growth. AlamarBlue cell viability assay and colony formation assay in AGS cells further validated these results. Clinicopathologic features and survival analysis demonstrated that RNASET2 protein was significantly correlated with tumor cell differentiation, Lauren's classification, and TM4SF1 protein expression, but not correlated with lymph nodal metastasis and patient's prognosis. Microvessel density detection indicated that no significant correlation was found between the expression of RNASET2 protein and the angiogenesis of GAC. Conclusions: Down-regulation of RNASET2 in GAC was only the consequence of the GAC, instead of the driver. The expression of RNASET2 could be regarded as a good biomarker for identifying the early stage of GAC.
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
pan> 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.
De Vito A, etal., Cancers (Basel). 2020 Mar 18;12(3). pii: cancers12030717. doi: 10.3390/cancers12030717.
Human RNASET2 acts as a powerful oncosuppressor protein in in vivo xenograft-based murine models of human cancer. Secretion of RNASET2 in the tumor microenvironment seems involved in tumor suppression, following recruitment
of M1-polarized macrophages. Here, we report a murine Rnaset2-based syngeneic in vivo assay. BALB/c mice were injected with parental, empty vector-transfected or murine Rnaset2-overexpressing mouse C51 or TS/A syngeneic cells and tumor growth pattern and immune cells distribution in tumor mass were investigated. Compared to control cells, mouse Rnaset2-expressing C51 cells showed strong delayed tumor growth. CD86+ M1 macrophages were massively recruited in Rnaset2-expressing C51-derived tumors, with concomitant inhibition of MDSCs and CD206+ M2 macrophages recruitment. At later times, a relevant expansion of intra-tumor CD8+ T cells was also observed. After re-challenge with C51 parental cells, most mice previously injected with Rnaset2-expressing C51 cells still rejected C51 tumor cells, suggesting a Rnaset2-mediated T cell adaptive immune memory response. These results point at T2 RNases as evolutionary conserved oncosuppressors endowed with the ability to inhibit cancer growth in vivo through rebalance of intra-tumor M1/M2 macrophage ratio and concomitant recruitment of adaptive anti-tumor CD8+ T cells.
Lualdi M, etal., Oncotarget. 2015 Apr 10;6(10):7851-65.
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 enc
oded 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.
