Plasma microRNA Signature as Companion Diagnostic for Abiraterone Acetate Treatment in Metastatic Castration-Resistant Prostate Cancer: A Pilot Study.

Abiraterone acetate (AA) serves as a medication for managing persistent testosterone production in patients with metastatic castration-resistant prostate cancer (mCRPC). However, its efficacy varies among individuals; thus, the identification of biomarkers to predict and follow treatment response is required. In this pilot study, we explored the potential of circulating microRNAs (c-miRNAs) to stratify patients based on their responsiveness to AA. We conducted an analysis of plasma samples obtained from a cohort of 33 mCRPC patients before and after three, six, and nine months of AA treatment. Using miRNA RT-qPCR panels for candidate discovery and TaqMan RT-qPCR for validation, we identified promising miRNA signatures. Our investigation indicated that a signature based on miR-103a-3p and miR-378a-5p effectively discriminates between non-responder and responder patients, while also following the drug's efficacy over time. Additionally, through in silico analysis, we identified target genes and transcription factors of the two miRNAs, including PTEN and HOXB13, which are known to play roles in AA resistance in mCRPC. In summary, our study highlights two c-miRNAs as potential companion diagnostics of AA in mCRPC patients, offering novel insights for informed decision-making in the treatment of mCRPC.

Proline Dehydrogenase (PRODH) Is Expressed in Lung Adenocarcinoma and Modulates Cell Survival and 3D Growth by Inducing Cellular Senescence.

The identification of markers for early diagnosis, prognosis, and improvement of therapeutic options represents an unmet clinical need to increase survival in Non-Small Cell Lung Cancer (NSCLC), a neoplasm still characterized by very high incidence and mortality. Here, we investigated whether proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key step in proline degradation, played a role in NSCLC tumorigenesis. PRODH expression was investigated by immunohistochemistry; digital PCR, quantitative PCR, immunoblotting, measurement of reactive oxygen species (ROS), and functional cellular assays were carried out. PRODH expression was found in the majority of lung adenocarcinomas (ADCs). Patients with PRODH-positive tumors had better cancer-free specific and overall survival compared to those with negative tumors. Ectopic modulation of PRODH expression in NCI-H1299 and the other tested lung ADC cell lines decreased cell survival. Moreover, cell proliferation curves showed delayed growth in NCI-H1299, Calu-6 and A549 cell lines when PRODH-expressing clones were compared to control clones. The 3D growth in soft agar was also impaired in the presence of PRODH. PRODH increased reactive oxygen species production and induced cellular senescence in the NCI-H1299 cell line. This study supports a role of PRODH in decreasing survival and growth of lung ADC cells by inducing cellular senescence.

The Potential Role of the T2 Ribonucleases in TME-Based Cancer Therapy.

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.

Signals of pseudo-starvation unveil the amino acid transporter SLC7A11 as key determinant in the control of Treg cell proliferative potential.

Human CD4+CD25hiFOXP3+ regulatory T (Treg) cells are key players in the control of immunological self-tolerance and homeostasis. Here, we report that signals of pseudo-starvation reversed human Treg cell in vitro anergy through an integrated transcriptional response, pertaining to proliferation, metabolism, and transmembrane solute carrier transport. At the molecular level, the Treg cell proliferative response was dependent on the induction of the cystine/glutamate antiporter solute carrier (SLC)7A11, whose expression was controlled by the nuclear factor erythroid 2-related factor 2 (NRF2). SLC7A11 induction in Treg cells was impaired in subjects with relapsing-remitting multiple sclerosis (RRMS), an autoimmune disorder associated with reduced Treg cell proliferative capacity. Treatment of RRMS subjects with dimethyl fumarate (DMF) rescued SLC7A11 induction and fully recovered Treg cell expansion. These results suggest a previously unrecognized mechanism that may account for the progressive loss of Treg cells in autoimmunity and unveil SLC7A11 as major target for the rescue of Treg cell proliferation.

Human Primary Dermal Fibroblasts Interacting with 3-Dimensional Matrices for Surgical Application Show Specific Growth and Gene Expression Programs.

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications.

Measurements Methods for the Development of MicroRNA-Based Tests for Cancer Diagnosis.

Studies investigating microRNAs as potential biomarkers for cancer, immune-related diseases, or cardiac pathogenic diseases, among others, have exponentially increased in the last years. In particular, altered expression of specific miRNAs correlates with the occurrence of several diseases, making these molecules potential molecular tools for non-invasive diagnosis, prognosis, and response to therapy. Nonetheless, microRNAs are not in clinical use yet, due to inconsistencies in the literature regarding the specific miRNAs identified as biomarkers for a specific disease, which in turn can be attributed to several reasons, including lack of assay standardization and reproducibility. Technological limitations in circulating microRNAs measurement have been, to date, the biggest challenge for using these molecules in clinical settings. In this review we will discuss pre-analytical, analytical, and post-analytical challenges to address the potential technical biases and patient-related parameters that can have an influence and should be improved to translate miRNA biomarkers to the clinical stage. Moreover, we will describe the currently available methods for circulating miRNA expression profiling and measurement, underlining their advantages and potential pitfalls.

TRPV4 and TRPM8 as putative targets for chronic low back pain alleviation.

The purpose of this study is to investigate the presence of nervous fibers and expression of TRP channels in samples harvested during decompressive/fusion spine surgeries from patients affected by chronic low back pain (CLBP). The aim was to understand if members of this family of receptors played a role in detection and processing of painful stimuli, to eventually define them as potential targets for CLBP alleviation. Expression of transient receptor potential (TRP) channels (A1, V1, V2, V4, and M8) was evaluated in samples from different periarticular sites of 6 patients affected by CLBP, at both protein and transcript levels. The capsular connective pathological tissue appeared infiltrated by sensitive unmyelinated nervous fibers. An increase in TRP channel mRNAs and proteins was observed in the pathological capsule compared with tissues collected from the non-symptomatic area in five of the six analyzed patients, independently by the location and number of affected sites. In particular, TRPV4 and TRPM8 were consistently upregulated in pathological tissues. Interestingly, the only patient showing a different pattern of expression also had a different clinical history. TRPV4 and TRPM8 channels may play a role in CLBP and warrant further investigations as possible therapeutic targets.

Beyond Conventional: The New Horizon of Anti-Angiogenic microRNAs in Non-Small Cell Lung Cancer Therapy.

GLOBOCAN 2018 identified lung cancer as the leading oncological pathology in terms of incidence and mortality rates. Angiogenesis is a key adaptive mechanism of numerous malignancies that promotes metastatic spread in view of the dependency of cancer cells on nutrients and oxygen, favoring invasion. Limitation of the angiogenic process could significantly hamper the disease advancement through starvation of the primary tumor and impairment of metastatic spread. This review explores the basic molecular mechanisms of non-small cell lung cancer (NSCLC) angiogenesis, and discusses the influences of the key proangiogenic factors-the vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF2), several matrix metalloproteinases (MMPs-MMP-2, MMP-7, MMP-9) and hypoxia-and the therapeutic implications of microRNAs (miRNAs, miRs) throughout the entire process, while also providing critical reviews of a number of microRNAs, with a focus on miR-126, miR-182, miR-155, miR-21 and let-7b. Finally, current conventional NSCLC anti-angiogenics-bevacizumab, ramucirumab and nintedanib-are briefly summarized through the lens of evidence-based medicine.

Serum miR-223: A Validated Biomarker for Detection of Early-Stage Non-Small Cell Lung Cancer.

BACKGROUND: The published circulating miRNA signatures proposed for early-stage non-small cell lung cancer (NSCLC) detection are inconsistent and difficult to replicate. Reproducibility and validation of an miRNA simple signature of NSCLC are prerequisites for translation to clinical application. METHODS: The serum level of miR-223 and miR-29c, emerging from published studies, respectively, as a highly sensitive and a highly specific biomarker of early-stage NSCLC, was measured with droplet digital PCR (ddPCR) technique in an Italian cohort of 75 patients with stage I-II NSCLC and 111 tumor-free controls. By ROC curve analysis we evaluated the miR-223 and miR-29c performance in discerning NSCLC cases from healthy controls. RESULTS: Reproducibility and robust measurability of the two miRNAs using ddPCR were documented. In a training set (40 stage I-II NSCLCs and 56 controls), miR-223 and miR-29c, respectively, showed an AUC of 0.753 [95% confidence interval (CI), 0.655-0.836] and 0.632 (95% CI, 0.527-0.729) in identifying NSCLC. Combination of miR-223 with miR-29c yielded an AUC of 0.750, not improved over that of miR-223 alone. Furthermore, in an independent blind set (35 stage I-II NSCLCs and 55 controls), we validated serum miR-223 as an effective biomarker of stage I-II NSCLC (AUC = 0.808; 95% CI, 0.712-0.884), confirming the miR-223 diagnostic performance reported by others in Chinese cohorts. CONCLUSIONS: Using ddPCR technology, miR-223 was externally validated as a reproducible, effective serum biomarker of early-stage NSCLC in ethnically different subjects. Combination with miR-29c did not improve the miR-223 diagnostic performance. IMPACT: Serum miR-223 determination may be proposed as a tool for refining NSCLC risk stratification, independent of smoking habit and age.

Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line.

L-Proline is a multifunctional amino acid that plays an essential role in primary metabolism and physiological functions. Proline is oxidized to glutamate in the mitochondria and the FAD-containing enzyme proline oxidase (PO) catalyzes the first step in L-proline degradation pathway. Alterations in proline metabolism have been described in various human diseases, such as hyperprolinemia type I, velo-cardio-facial syndrome/Di George syndrome, schizophrenia and cancer. In particular, the mutation giving rise to the substitution Leu441Pro was identified in patients suffering of schizophrenia and hyperprolinemia type I. Here, we report on the expression of wild-type and L441P variants of human PO in a U87 glioblastoma human cell line in an attempt to assess their effect on glutamate metabolism. The subcellular localization of the flavoenzyme is not altered in the L441P variant, for which specific activity is halved compared to the wild-type PO. While this decrease in activity is significantly less than that previously proposed, an effect of the substitution on the enzyme stability is also apparent in our studies. At 24 hours of growth from transient transfection, the intracellular level of proline, glutamate, and glutamine is decreased in cells expressing the PO variants as compared to control U87 cells, reaching a similar figure at 72 h. On the other hand, the extracellular levels of the three selected amino acids show a similar time course for all clones. Furthermore, PO overexpression does not modify to a significant extent the expression of GLAST and GLT-1 glutamate transporters. Altogether, these results demonstrate that the proline pathway links cellular proline levels with those of glutamate and glutamine. On this side, PO might play a regulatory role in glutamatergic neurotransmission by affecting the cellular concentration of glutamate.

Systematic review and critique of circulating miRNAs as biomarkers of stage I-II non-small cell lung cancer.

Selected circulating microRNAs (miRNAs) have been suggested for non-invasive screening of non-small cell lung cancer (NSCLC), however the numerous proposed miRNA signatures are inconsistent. Aiming to identify miRNAs suitable specifically for stage I-II NSCLC screening in serum/plasma samples, we searched the databases "Pubmed", "Medline", "Scopus", "Embase" and "WOS" and systematically reviewed the publications reporting quantitative data on the efficacy [sensitivity, specificity and/or area under the curve (AUC)] of circulating miRNAs as biomarkers of NSCLC stage I and/or II. The 20 studies fulfilling the search criteria included 1110 NSCLC patients and 1009 controls, and were of medium quality according to Quality Assessment of Diagnostic Accuracy Studies checklist. In these studies, the patient cohorts as well as the control groups were heterogeneous for demographics and clinicopathological characteristics; moreover, numerous pre-analytical and analytical variables likely influenced miRNA determinations, and potential bias of hemolysis was often underestimated. We identified four circulating miRNAs scarcely influenced by hemolysis, each featuring high sensitivity (> 80%) and AUC (> 0.80) as biomarkers of stage I-II NSCLC: miR-223, miR-20a, miR-448 and miR-145; four other miRNAs showed high specificity (> 90%): miR-628-3p, miR-29c, miR-210 and miR-1244. In a model of two-step screening for stage I-II NSCLC using first the above panel of serum miRNAs with high sensitivity and high AUC, and subsequently the panel with high specificity, the estimated overall sensitivity is 91.6% and overall specificity is 93.4%. These and other circulating miRNAs suggested for stage I-II NSCLC screening require validation in multiple independent studies before they can be proposed for clinical application.

Effect of Inulin on Proteome Changes Induced by Pathogenic Lipopolysaccharide in Human Colon.

In the present study, the protective role of inulin against lipopolysaccharide (LPS)-induced oxidative stress was evaluated on human colonic mucosa using a proteomic approach. Human colonic mucosa and submucosa were sealed between two chambers, with the luminal side facing upwards and overlaid with Krebs (control), LPS or LPS+ inulin IQ solution. The solutions on the submucosal side (undernatants) were collected following 30 min of mucosal exposure. iTRAQ based analysis was used to analyze the total soluble proteomes from human colonic mucosa and submucosa treated with different undernatants. Human colonic muscle strips were exposed to the undernatants to evaluate the response to acetylcholine. Inulin exposure was able to counteract, in human colonic mucosa, the LPS-dependent alteration of some proteins involved in the intestinal contraction (myosin light chain kinase (MLCK), myosin regulatory subunit (MYL)), to reduce the up-regulation of two proteins involved in the radical-mediated oxidative stress (the DNA-apurinic or apyrimidinic site) lyase) APEX1 and the T-complex protein 1 subunit eta (CCT7) and to entail a higher level of some detoxification enzymes (the metallothionein-2 MT2A, the glutathione-S-transferase K GSTk, and two UDP- glucuronosyltransferases UGT2B4, UGT2B17). Inulin exposure was also able to prevent the LPS-dependent intestinal muscle strips contraction impairment and the mucosa glutathione level alterations. Exposure of colonic mucosa to inulin seems to prevent LPS-induced alteration in expression of some key proteins, which promote intestinal motility and inflammation, reducing the radical-mediated oxidative stress.

TAS2R38 taste receptor gene and chronic rhinosinusitis: new data from an Italian population.

BACKGROUND: Chronic rhinosinusitis (CRS) is a frequent disease with high social impact and multifactorial pathogenesis. Recently, single nucleotide polymorphisms within the TAS2R38 gene have been implicated as possible contributors to the complex gene-environment interactions in CRS. The purpose of this study was to confirm the proposed correlation between TAS2R38 genotype, CRS and related comorbidities. METHODS: Fifty-three CRS patients and 39 healthy individuals were genotyped at the TAS2R38 locus. CRS patients were treated by endoscopic sinus surgery and medical therapies and subdivided in CRS with nasal polyps (CRSwNPs) and CRS without nasal polyps (CRSsNPs). The effect of genotype on CRS and CRS-related comorbidities was assessed. RESULTS: The distribution of the different genotypes at the TAS2R38 locus was not significantly different between CRS patients, either with or without nasal polyps, and controls. Besides, no association was found between the different genotypes at the TAS2R38 locus and CRS-related comorbidities. CONCLUSIONS: No association was found between TAS2R38 alleles or genotypes and CRS, thus questioning its role in the pathogenesis of CRS.

A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer.

BACKGROUND: Selected microRNAs (miRNAs) that are abnormally expressed in the serum of patients with lung cancer have recently been proposed as biomarkers of this disease. The measurement of circulating miRNAs, however, requires a highly reliable quantification method. Quantitative real-time PCR (qPCR) is the most commonly used method, but it lacks reliable endogenous reference miRNAs for normalization of results in biofluids. When used in absolute quantification, it must rely on the use of external calibrators. Droplet digital PCR (ddPCR) is a recently introduced technology that overcomes the normalization issue and may facilitate miRNA measurement. Here we compared the performance of absolute qPCR and ddPCR techniques for quantifying selected miRNAs in the serum. RESULTS: In the first experiment, three miRNAs, proposed in the literature as lung cancer biomarkers (miR-21, miR-126 and let-7a), were analyzed in a set of 15 human serum samples. Four independent qPCR and four independent ddPCR amplifications were done on the same samples and used to estimate the precision and correlation of miRNA measurements obtained with the two techniques. The precision of the two methods was evaluated by calculating the Coefficient of Variation (CV) of the four independent measurements obtained with each technique. The CV was similar or smaller in ddPCR than in qPCR for all miRNAs tested, and was significantly smaller for let-7a (p = 0.028). Linear regression analysis of the miRNA values obtained with qPCR and ddPCR showed strong correlation (p < 0.001). To validate the correlation obtained with the two techniques in the first experiment, in a second experiment the same miRNAs were measured in a larger cohort (70 human serum samples) by both qPCR and ddPCR. The correlation of miRNA analyses with the two methods was significant for all three miRNAs. Moreover, in our experiments the ddPCR technique had higher throughput than qPCR, at a similar cost-per-sample. CONCLUSIONS: Analyses of serum miRNAs performed with qPCR and ddPCR were largely concordant. Both qPCR and ddPCR can reliably be used to quantify circulating miRNAs, however, ddPCR revealed similar or greater precision and higher throughput of analysis.

Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

∆N-P63α and TA-P63α exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites.

TP63 is a member of the TP53 gene family that encodes for up to ten different TA and ∆N isoforms through alternative promoter usage and alternative splicing. Besides being a master regulator of gene expression for squamous epithelial proliferation, differentiation and maintenance, P63, through differential expression of its isoforms, plays important roles in tumorigenesis. All P63 isoforms share an immunoglobulin-like folded DNA binding domain responsible for binding to sequence-specific response elements (REs), whose overall consensus sequence is similar to that of the canonical p53 RE. Using a defined assay in yeast, where P63 isoforms and RE sequences are the only variables, and gene expression assays in human cell lines, we demonstrated that human TA- and ∆N-P63α proteins exhibited differences in transactivation specificity not observed with the corresponding P73 or P53 protein isoforms. These differences 1) were dependent on specific features of the RE sequence, 2) could be related to intrinsic differences in their oligomeric state and cooperative DNA binding, and 3) appeared to be conserved in evolution. Sicen genotoxic stress can change relative ratio of TA- and ∆N-P63α protein levels, the different transactivation specificity of each P63 isoform could potentially influence cellular responses to specific stresses.

The degradation (by distinct pathways) of human D-amino acid oxidase and its interacting partner pLG72--two key proteins in D-serine catabolism in the brain.

Human D-amino acid oxidase (EC 1.4.3.3; hDAAO) is a peroxisomal flavoenzyme significantly enriched in the mammalian brain. hDAAO has been proposed to play (with serine racemase; EC 5.1.1.18) an essential role in the catabolism of D-serine, an 'atypical' key signalling molecule that acts as allosteric activator of the N-methyl-D-aspartate-type glutamate receptor (NMDAr). hDAAO and its interacting partner pLG72 have been related to schizophrenia, a highly disabling psychiatric disorder in which a dysfunction of NMDA-mediated neurotransmission is widely assumed to occur. We previously demonstrated that the D-serine cellular concentration depends on hDAAO and pLG72 expression levels and that newly-synthesized hDAAO interacts with its modulator in the cytosol, being progressively destabilized and inactivated. To obtain insight into the mechanisms regulating cellular D-serine levels, we investigated the degradation pathways of hDAAO and pLG72 in U87 glioblastoma cells stably expressing enhanced yellow fluorescent protein-hDAAO (peroxisomal), hDAAO-enhanced yellow fluorescent protein (cytosolic) or pLG72-enhanced cyan fluorescent protein (mitochondrial) proteins. hDAAO is a long-lived protein: the peroxisomal fraction of this flavoprotein is degraded via the lysosomal/endosomal pathway (and blocking this pathway increases the cellular hDAAO activity and decreases D-serine levels), whereas the cytosolic portion is ubiquitinated and targeted to the proteasome. By contrast, pLG72 shows a rapid turnover (t(1/2) ≈ 25-40 min) and is degraded via the proteasome system, albeit not ubiquitinated. Overexpression of pLG72 increases the turnover of hDAAO, in turn playing a protective role against excessive D-serine depletion.

P53 family members modulate the expression of PRODH, but not PRODH2, via intronic p53 response elements.

The tumor suppressor p53 was previously shown to markedly up-regulate the expression of the PRODH gene, encoding the proline dehydrogenase (PRODH) enzyme, which catalyzes the first step in proline degradation. Also PRODH2, which degrades 4-hydroxy-L-proline, a product of protein (e.g. collagen) catabolism, was recently described as a p53 target. Here, we confirmed p53-dependent induction of endogenous PRODH in response to genotoxic damage in cell lines of different histological origin. We established that over-expression of TAp73ß or TAp63ß is sufficient to induce PRODH expression in p53-null cells and that PRODH expression parallels the modulation of endogenous p73 by genotoxic drugs in several cell lines. The p53, p63, and p73-dependent transcriptional activation was linked to specific intronic response elements (REs), among those predicted by bioinformatics tools and experimentally validated by a yeast-based transactivation assay. p53 occupancy measurements were validated in HCT116 and MCF7 human cell lines. Conversely, PRODH2 was not responsive to p63 nor p73 and, at best, could be considered a weak p53 target. In fact, minimal levels of PRODH2 transcript induction by genotoxic stress was observed exclusively in one of four p53 wild-type cell lines tested. Consistently, all predicted p53 REs in PRODH2 were poor matches to the p53 RE consensus and showed very weak responsiveness, only to p53, in the functional assay. Taken together, our results highlight that PRODH, but not PRODH2, expression is under the control of p53 family members, specifically p53 and p73. This supports a deeper link between proteins of the p53-family and metabolic pathways, as PRODH modulates the balance of proline and glutamate levels and those of their derivative alpha-keto-glutarate (α-KG) under normal and pathological (tumor) conditions.

Comparison of the baculovirus-insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2.

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.

Microenvironmental control of malignancy exerted by RNASET2, a widely conserved extracellular RNase.

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.