Immune cell infiltration and inflammatory landscape in primary brain tumours.

BACKGROUND: Primary malignant brain tumours are more than one-third of all brain tumours and despite the molecular investigation to identify cancer driver mutations, the current therapeutic options available are challenging due to high intratumour heterogeneity. In addition, an immunosuppressive and inflammatory tumour microenvironment strengthens cancer progression. Therefore, we defined an immune and inflammatory profiling of meningioma and glial tumours to elucidate the role of the immune infiltration in these cancer types. METHODS: Using tissue microarrays of 158 brain tumour samples, we assessed CD3, CD4, CD8, CD20, CD138, Granzyme B (GzmB), 5-Lipoxygenase (5-LOX), Programmed Death-Ligand 1 (PD-L1), O-6-Methylguanine-DNA Methyltransferase (MGMT) and Transglutaminase 2 (TG2) expression by immunohistochemistry (IHC). IHC results were correlated using a Spearman correlation matrix. Transcript expression, correlation, and overall survival (OS) analyses were evaluated using public datasets available on GEPIA2 in Glioblastoma (GBM) and Lower Grade Glioma (LGG) cohorts. RESULTS: Seven out of ten markers showed a significantly different IHC expression in at least one of the evaluated cohorts whereas CD3, CD4 and 5-LOX were differentially expressed between GBMs and astrocytomas. Correlation matrix analysis revealed that 5-LOX and GzmB expression were associated in both meningiomas and GBMs, whereas 5-LOX expression was significantly and positively correlated to TG2 in both meningioma and astrocytoma cohorts. These findings were confirmed with the correlation analysis of TCGA-GBM and LGG datasets. Profiling of mRNA levels indicated a significant increase in CD3 (CD3D, CD3E), and CD138 (SDC1) expression in GBM compared to control tissues. CD4 and 5-LOX (ALOX5) mRNA levels were significantly more expressed in tumour samples than in normal tissues in both GBM and LGG. In GBM cohort, GzmB (GZMB), SDC1 and MGMT gene expression predicted a poor overall survival (OS). Moreover, in LGG cohort, an increased expression of CD3 (CD3D, CD3E, CD3G), CD8 (CD8A), GZMB, CD20 (MS4A1), SDC1, PD-L1, ALOX5, and TG2 (TGM2) genes was associated with worse OS. CONCLUSIONS: Our data have revealed that there is a positive and significant correlation between the expression of 5-LOX and GzmB, both at RNA and protein level. Further evaluation is needed to understand the interplay of 5-LOX and immune infiltration in glioma progression.

Estrogen Induces Selective Transcription of Caveolin1 Variants in Human Breast Cancer through Estrogen Responsive Element-Dependent Mechanisms.

The estrogen receptor (ER) signaling regulates numerous physiological processes mainly through activation of gene transcription (genomic pathways). Caveolin1 (CAV1) is a membrane-resident protein that behaves as platform to enable different signaling molecules and receptors for membrane-initiated pathways. CAV1 directly interacts with ERs and allows their localization on membrane with consequent activation of ER-non-genomic pathways. Loss of CAV1 function is a common feature of different types of cancers, including breast cancer. Two protein isoforms, CAV1α and CAV1ß, derived from two alternative translation initiation sites, are commonly described for this gene. However, the exact transcriptional regulation underlying CAV1 expression pattern is poorly elucidated. In this study, we dissect the molecular mechanism involved in selective expression of CAV1ß isoform, induced by estrogens and downregulated in breast cancer. Luciferase assays and Chromatin immunoprecipitation demonstrate that transcriptional activation is triggered by estrogen-responsive elements embedded in CAV1 intragenic regions and DNA-binding of estrogen-ER complexes. This regulatory control is dynamically established by local chromatin changes, as proved by the occurrence of histone H3 methylation/demethylation events and association of modifier proteins as well as modification of H3 acetylation status. Thus, we demonstrate for the first time, an estrogen-ERs-dependent regulatory circuit sustaining selective CAV1ß expression.

The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior.

Overactivation of the c-MET/HGF system is a feature of many cancers. We previously reported that type II testicular germ cell tumor (TGCT) cells express the c-MET receptor, forming non-seminomatous lesions that are more positive compared with seminomatous ones. Notably, we also demonstrated that NT2D1 non-seminomatous cells (derived from an embryonal carcinoma lesion) increase their proliferation, migration, and invasion in response to HGF. Herein, we report that HGF immunoreactivity is more evident in the microenvironment of embryonal carcinoma biopsies with respect to seminomatous ones, indicating a tumor-dependent modulation of the testicular niche. PI3K/AKT is one of the signaling pathways triggered by HGF through the c-MET activation cascade. Herein, we demonstrated that phospho-AKT increases in NT2D1 cells after HGF stimulation. Moreover, we found that this pathway is involved in HGF-dependent NT2D1 cell proliferation, migration, and invasion, since the co-administration of the PI3K inhibitor LY294002 together with HGF abrogates these responses. Notably, the inhibition of endogenous PI3K affects collective cell migration but does not influence proliferation or chemotactic activity. Surprisingly, LY294002 administered without the co-administration of HGF increases cell invasion at levels comparable to the HGF-administered samples. This paradoxical result highlights the role of the testicular microenvironment in the modulation of cellular responses and stimulates the study of the testicular secretome in cancer lesions.

Pentraxin 3 Induces Vascular Endothelial Dysfunction Through a P-selectin/Matrix Metalloproteinase-1 Pathway.

BACKGROUND: Pentraxin 3 (PTX3), the prototype of long pentraxins, has been described to be associated with endothelial dysfunction in different cardiovascular disorders. No study has yet evaluated the possible direct effect of PTX3 on vascular function. METHODS AND RESULTS: Through in vitro experiments of vascular reactivity and ultrastructural analyses, we demonstrate that PTX3 induces dysfunction and morphological changes in the endothelial layer through a P-selectin/matrix metalloproteinase-1 pathway. The latter hampered the detachment of endothelial nitric oxide synthase from caveolin-1, leading to an impairment of nitric oxide signaling. In vivo studies showed that administering PTX3 to wild-type mice induced endothelial dysfunction and increased blood pressure, an effect absent in P-selectin-deficient mice. In isolated human umbilical vein endothelial cells, PTX3 significantly blunted nitric oxide production through the matrix metalloproteinase-1 pathway. Finally, using ELISA, we found that hypertensive patients (n=31) have higher plasma levels of PTX3 and its mediators P-selectin and matrix metalloproteinase-1 than normotensive subjects (n=21). CONCLUSIONS: Our data show for the first time a direct role of PTX3 on vascular function and blood pressure homeostasis, identifying the molecular mechanisms involved. The findings in humans suggest that PTX3, P-selectin, and matrix metalloproteinase-1 may be novel biomarkers that predict the onset of vascular dysfunction in hypertensive patients.

HIV-1 Tat protein vaccination in mice infected with Mycobacterium tuberculosis is safe, immunogenic and reduces bacterial lung pathology.

BACKGROUND: The therapeutic HIV-1 Tat protein vaccine is in advanced clinical development. Tuberculosis, the main AIDS co-infection, is highly endemic in areas where AIDS prevention through vaccination is needed. However, safety and immunogenicity of Tat vaccination in the course of Mycobacterium tuberculosis (Mtb) infection is still unknown and it prevents the possibility to administer the vaccine to Mtb-infected individuals. We addressed the interplay and effects of Tat vaccination on Mtb infection in immunocompetent mice. METHODS: C57BL/6 mice were vaccinated or not with Bacillus Calmette-Guerin (BCG), the current tuberculosis vaccine, and after 5 weeks were infected with Mtb by intravenous route. The Tat protein was injected intradermally at 1, 2 and 4 weeks after Mtb challenge. Eight weeks after Mtb infection, all mice were sacrificed, and both the degree of pathology and immune responses to Mtb and Tat were evaluated. As additional control, some mice were either vaccinated or not with BCG, were not challenged with Mtb, but received the Tat protein. Statistical significances were evaluated by one-way or two-way ANOVA and Tukey's multiple comparisons post-test. RESULTS: In the lungs of Mtb-infected mice, Tat-vaccine did not favour Mtb replication and indeed reduced both area of cellular infiltration and protein levels of Interferon-γ, Chemokine (C-C motif) ligand-4 and Interleukin-1ß, pathological events triggered by Mtb-infection. Moreover, the protection against Mtb infection conferred by BCG remained good after Tat protein treatment. In spleen cells of Mtb-infected mice, Tat vaccination enhanced Mtb-specific Interferon-γ and Interleukin-17 responses, which may have a protective role. Of note, Mtb infection reduced, but did not suppress, the development of anti-Tat antibodies, required for Tat vaccine efficacy and the titer of anti-Tat IgG was potentiated by BCG vaccination in Mtb-free mice. In general, Tat treatment was well tolerated in both Mtb-infected and Mtb-free mice. CONCLUSIONS: Tat protein vaccine, administered in Mtb-infected mice with a protocol resembling that used in the clinical trials, was safe, immunogenic, limited the lung Mtb-associated immunopathology and did not abrogate the protective efficacy of BCG. These data provide preliminary evidence for a safe use of Tat vaccine in people vaccinated with BCG and/or suffering from tuberculosis.

High levels of GPR30 protein in human testicular carcinoma in situ and seminomas correlate with low levels of estrogen receptor-beta and indicate a switch in estrogen responsiveness.

The G protein-coupled estrogen receptor (GPR30) is suggested to be involved in non-nuclear estrogen signalling and is expressed in a variety of hormone dependent cancer entities. It is well established that oestrogens are involved in pathological germ cell proliferation including testicular germ cell tumours. This study was performed to further elucidate the role of this receptor and the possible correlation with the estrogen receptor ß in human testicular carcinoma in situ (CIS), seminomas and in GC1 and TCam-2 germ cell lines; in addition, a Tissue Micro-Array was built using the most representative areas from 25 cases of human testicular seminomas and 20 cases of CIS. The expression of ERß and GPR30 were observed by using Western blot analysis in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that down regulation of ERß associates with GPR30 over-expression both in human testicular CIS and seminomas. In addition, we show that 17ß-oestradiol induces the ERK1/2 activation and increases c-Fos expression through GPR30 associated with ERß down-regulation in TCam-2 cell line. The present results suggest that exposure to oestrogens or oestrogen-mimics, in some as of yet undefined manner, diminishes the ERß-mediated growth restraint in CIS and in human testicular seminoma, probably due to ERß down-regulation associated to GPR30 increased expression indicating that GPR30 could be a potential therapeutic target to design specific inhibitors.

Expression and localization of serine protease Htra1 in neuroblastoma: correlation with cellular differentiation grade.

Neuroblastoma (NB) is a paediatric tumor that arises from neural crest and shows heterogeneous clinical and biological features. The serine-protease high temperature requirement A1 (HtrA1) has a pivotal role in both cell proliferation and differentiation. Here we report the expression and localization of HtrA1 in NB tumor samples to assess HtrA1 role as a possible new biomarker of cellular differentiation in NB patients. HtrA1 protein expression by Western Blot assay was performed in 60 tissue samples of 50 children with NB and 10 children with ganglioneuroblastoma (GNB). HtrA1 was expressed in 56/60 (93.3 %) samples with different expression levels: low levels in 36/56 samples (64.3 %) and high levels in 20/56 (35.7 %). Higher levels were found in 1, 2 and 4s stages (80 %), whereas 3 and 4 stages (20 %) showed a low expression, with a statistically significant difference (p = 0.003). Among not amplified N-MYC group, 28 (60 %) had low/absent expression of HtrA1: seven with recurrent disease and negative outcome and 21 in continuous complete remission (CCR), whereas all samples with high expression of HtrA1 (17/44) were in CCR (p = 0.03). The immunohistochemical analysis showed localization of HtrA1 in differentiated areas higher than in undifferentiated areas where the protein was absent. Moreover, HtrA1 was highly expressed in all GNB samples. In conclusion, the over-expression of HtrA1 is correlated to cellular differentiation grade and stage of NB at diagnosis. Moreover, HtrA1 could represent a new marker of undifferentiation and biological aggressiveness of NB.

Mycobacterium tuberculosis antigen 85B modifies BCG-induced antituberculosis immunity and favors pathogen survival.

Tuberculosis is one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis has developed strategies not only to evade host immunity but also to manipulate it for its survival. We investigated whether Mycobacterium tuberculosis exploited the immunogenicity of Ag85B, one of its major secretory proteins, to redirect host antituberculosis immunity to its advantage. We found that administration of Ag85B protein to mice vaccinated with Bacillus Calmette-Guérin impaired the protection elicited by vaccination, causing a more severe infection when mice were challenged with Mycobacterium tuberculosis. Ag85B administration reduced Bacillus Calmette-Guérin-induced CD4 T-cell activation and IFN-γ, CCL-4, and IL-22 production in response to Mycobacterium tuberculosis-infected cells. On the other hand, it promoted robust Ag85B-responsive IFN-γ-producing CD4 T cells, expansion of a subset of IFN-γ/IL-10-producing CD4+FOXP3+Treg cells, differential activation of IL-17/IL-22 responses, and activation of regulatory and exhaustion pathways, including programmed death ligand 1 expression on macrophages. All this resulted in impaired intracellular Mycobacterium tuberculosis growth control by systemic immunity, both before and after the Mycobacterium tuberculosis challenge. Interestingly, Mycobacterium tuberculosis infection itself generated Ag85B-reactive inflammatory immune cells incapable of clearing Mycobacterium tuberculosis in both unvaccinated and Bacillus Calmette-Guérin-vaccinated mice. Our data suggest that Mycobacterium tuberculosis can exploit the strong immunogenicity of Ag85B to promote its own survival and spread. Since Ag85B is normally secreted by replicating bacteria and is commonly found in the lungs of the Mycobacterium tuberculosis-infected host, our findings may advance the understanding on the mechanisms of Mycobacterium tuberculosis pathogenesis and immune evasion.

Cytosolic phosphorylated EGFR is predictive of recurrence in early stage penile cancer patients: a retropective study.

BACKGROUND: Penile cancer (PC) is a rare tumor, and therapeutic options are limited for this disease, with an overall 5-year overall survival around 65-70%. Adjuvant therapy is not recommended for patients with N0-1 disease, despite up to 60% of these patients will die within 5 years from diagnosis. METHODS: Medical records of all patients who underwent radical surgery at University Federico II of Naples and at National Tumor Institute "Pascale" of Naples for early squamous cell carcinoma of the penis from January, 2000 to December, 2011 were retrieved. Paraffin wax embedded tissue specimens were retrieved from the pathology archives of the participating Institutions for all patients. Expression of p-EGFR, EGFR and positivity to HPV were evaluated along with other histological variables of interest. Demographic data of eligible patients were retrieved along with clinical characteristics such as type of surgical operation, time of follow up, time of recurrence, overall survival. A multivariable model was constructed using a forward stepwise selection procedure. RESULTS: Thirty eligible patients were identified. All patients were positive for EGFR by immunohistochemistry, while 13 and 16 were respectively positive for nuclear and cytosolic p-EGFR. No EGFR amplification was detected by FISH. Eight patients were positive for high-risk HPV by ISH. On univariable analysis, corpora cavernosa infiltration (OR 7.8; 95% CI=0,8 to 75,6; P=0,039) and positivity for cytosolic p-EGFR (OR 7.6; 95% CI =1.49 to 50; P = 0.009) were predictive for recurrence, while only positivity for cytosolic p-EGFR (HR =9.0; 95% CI 1.0-100; P=0,0116) was prognostic for poor survival. CONCLUSION: It is of primary importance to identify patients with N0-1 disease who are at increased risk of recurrence, as they do not normally receive any adjuvant therapy. Expression of p-EGFR was found in this series to be strongly related to increase risk of recurrence and shorter overall survival. This finding is consistent with the role of p-EGFR in other solid malignancies. Integration of p-EGFR with classic prognostic factors and other histology markers should be pursued to establish optimal adjuvant therapy for N0-1 PC patients.

Testicular and testicular adnexa tumors in the elderly.

Neoplasms in the testis and in the testicular adnexa of elderly patients are completely different from those observed in younger patients. Indeed, although conventional seminomas and nonseminomas are mainly observed in the age range of 20-45 years, spermatocytic seminoma, malignant Leydig tumors, and lymphomas in the testis and sarcomas in the paratesticular region are encountered in individuals older than 60 years of age. Here, we discuss the testis and paratesticular region neoplasm more commonly diagnosed in elderly men.

The high-mobility group A1-estrogen receptor ß nuclear interaction is impaired in human testicular seminomas.

It is well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. The effects of estrogen are now known to be mediated by estrogen receptor-α (ERα) and ERß subtypes, but only ERß has been found in human germ cells of normal testis. However, its expression was markedly diminished in human testicular seminomas. The expression and the possible interaction of ERß and HMGA1 were studied in normal germ cells and in human testicular seminomas. GC1 and TCam-2 germ cell lines, were used; in addition, a tissue micro-array (TMA) was built using the most representative areas from 35 cases of human testicular seminomas. The expression and the interaction of ERß and HMGA1 were observed by using immunoprecipitation and Western blot analyses in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that ERß interacts with HMGA1 in normal germ cells, while down regulation of ERß associates with transcriptional co-regulator HMGA1 over-expression and cytoplasmic localization both in human testicular seminomas and in TCam-2 cell line. In addition, we show that 17ß-oestradiol induces an HMGA1 increased cytoplasmic expression associated to an ERß down-regulation in TCam-2 cell line. Taken together, our results suggest that exposure to estrogens or estrogen-mimics, in some as of yet undefined manner, diminishes the ERß-mediated growth restraint in human testicular seminoma, probably due to the HMGA1 cytoplasmic delocalization associated with ERß down-regulation.

HMGA1 induces EZH2 overexpression in human B-cell lymphomas.

EZH2 is an enzymatic subunit of PRC2, an epigenetic regulator that triggers the methylation of the histone H3 lysine 27 silencing the transcription of several genes. EZH2 has a critical role in cancer progression, since its overexpression has been associated with increased cancer cell invasiveness, drug resistance and poor patient survival. However, the mechanisms accounting for EZH2 overexpression in cancer remain still unclear. Intriguingly, also HMGA protein overexpression is a feature of many human malignancies and correlates with the presence of metastases and a poor outcome. The HMGA proteins, including HMGA1 and HMGA2, belong to the architectural transcription factors that play a key role in the organization of chromatin structure. Here, we report a statistically significant correlation between HMGA1 and EZH2 expression in human lymphomas. We demonstrate that HMGA1 is able to bind EZH2 promoter and induce its activity. Consistently, silencing of HMGA1 expression results in the downregulation of the EZH2 levels leading to a decreased proliferation and migration rate of human lymphoma cell lines. Therefore, these data identify HMGA1 as an EZH2 activator, suggesting a novel molecular mechanism contributing to EZH2 overexpression in human malignancies and a synergism of these proteins in cancer progression.

Subcellular Localization of uc.8+ as a Prognostic Biomarker in Bladder Cancer Tissue.

Non-coding RNA transcripts originating from Ultraconserved Regions (UCRs) have tissue-specific expression and play relevant roles in the pathophysiology of multiple cancer types. Among them, we recently identified and characterized the ultra-conserved-transcript-8+ (uc.8+), whose levels correlate with grading and staging of bladder cancer. Here, to validate uc.8+ as a potential biomarker in bladder cancer, we assessed its expression and subcellular localization by using tissue microarray on 73 human bladder cancer specimens. We quantified uc.8+ by in-situ hybridization and correlated its expression levels with clinical characteristics and patient survival. The analysis of subcellular localization indicated the simultaneous presence of uc.8+ in the cytoplasm and nucleus of cells from the Low-Grade group, whereas a prevalent cytoplasmic localization was observed in samples from the High-Grade group, supporting the hypothesis of uc.8+ nuclear-to-cytoplasmic translocation in most malignant tumor forms. Moreover, analysis of uc.8+ expression and subcellular localization in tumor-surrounding stroma revealed a marked down-regulation of uc.8+ levels compared to the paired (adjacent) tumor region. Finally, deep machine-learning approaches identified nucleotide sequences associated with uc.8+ localization in nucleus and/or cytoplasm, allowing to predict possible RNA binding proteins associated with uc.8+, recognizing also sequences involved in mRNA cytoplasm-translocation. Our model suggests uc.8+ subcellular localization as a potential prognostic biomarker for bladder cancer.

Caloric Restriction Promotes Immunometabolic Reprogramming Leading to Protection from Tuberculosis.

There is a strong relationship between metabolic state and susceptibility to Mycobacterium tuberculosis (MTB) infection, with energy metabolism setting the basis for an exaggerated immuno-inflammatory response, which concurs with MTB pathogenesis. Herein, we show that controlled caloric restriction (CR), not leading to malnutrition, protects susceptible DBA/2 mice against pulmonary MTB infection by reducing bacterial load, lung immunopathology, and generation of foam cells, an MTB reservoir in lung granulomas. Mechanistically, CR induced a metabolic shift toward glycolysis, and decreased both fatty acid oxidation and mTOR activity associated with induction of autophagy in immune cells. An integrated multi-omics approach revealed a specific CR-induced metabolomic, transcriptomic, and proteomic signature leading to reduced lung damage and protective remodeling of lung interstitial tightness able to limit MTB spreading. Our data propose CR as a feasible immunometabolic manipulation to control MTB infection, and this approach offers an unexpected strategy to boost immunity against MTB.

Immunohistochemical detection of paralogous 13 HOX genes in phyllodes tumor of the breast as a useful diagnostic tool.

Phyllodes tumor of the breast is a rare fibroepithelial lesion characterized by a propensity for local recurrence and distant metastasis. Its histologic classification, based on morphology, mitotic index and tumor margin, includes malignant, borderline, and benign. These tumors show similar cellular morphology, which may contribute to difficulty in classifying them histologically and in prediction of their clinical behavior. Thus, the identification of new biomarkers detectable also by in situ methods has become indispensable. Paralogous HOX13 genes (HOX A13, HOX B13, HOX C13 and HOX D13) play a relevant role in tumor development and progression in particular in breast cancer. In this study we analyzed the immunohistochemical expression of paralogous HOX13 homeoproteins on a phyllodes tumor case series to validate their usefulness in histologic classification.

The Oncolytic Virus dl922-947 Triggers Immunogenic Cell Death in Mesothelioma and Reduces Xenograft Growth.

Background: Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure that urgently requires effective therapeutic strategies. Current treatments are unable to increase significantly patient survival, which is often limited to <1 year from diagnosis. Virotherapy, based on the use of oncolytic viruses that exert anti-cancer effects by direct cell lysis and through the induction of anti-tumor immune response, represents an alternative therapeutic option for rare tumors with limited life expectancy. In this study, we propose the use of the adenovirus dl922-947, engineered to allow selective replication in cancer cells, to counteract MPM. Methods: We performed a thorough preclinical assessment of dl922-947 effects in a set of MPM cell lines and xenografts. Cytotoxicity of dl922-947 alone and in combination assays was evaluated by sulforhodamine B assay. Cell cycle, calreticulin expression, and high mobility group box protein 1 (HMGB1) secretion were determined by flow cytometry, whereas ATP content was determined by a luminescence-based bioassay. The modulation of angiogenic factors in MPM-infected cells was evaluated through ELISA. Results: We found that dl922-947 infection exhibits cytotoxic effects in MPM cell lines, affecting cell viability, cell cycle progression, and regulating main hallmarks of immunogenic cell death inducing calreticulin surface exposure, HMGB1 and ATP release. Our results also suggest that dl922-947 may affect angiogenic signals by regulation of VEGF-A and IL-8 secretion. Furthermore, dl922-947 shows anti-tumor efficacy in murine xenograft models reducing tumor growth and enhancing survival. Finally, the combination with cisplatin potentiated the cytotoxic effect of dl922-947. Conclusions: Overall our data identify virotherapy, based on the use of dl922-947, as a new possible therapeutic strategy against MPM, which could be used alone, in combination with standard chemotherapy drugs, as shown here, or other approaches also aimed at enhancing the antitumoral immune response elicited by the virus.

Targeting the Formyl Peptide Receptor type 1 to prevent the adhesion of ovarian cancer cells onto mesothelium and subsequent invasion.

BACKGROUND: The biological behavior of epithelial ovarian cancer (EOC) is unique since EOC cells metastasize early to the peritoneum. Thereby, new anti-target agents designed to block trans-coelomic dissemination of EOC cells may be useful as anti-metastatic drugs. The Urokinase Plasminogen Activator Receptor (uPAR) is overexpressed in EOC tissues, and its truncated forms released in sera and/or ascitic fluid are associated with poor prognosis and unfavorable clinical outcome. We documented that uPAR triggers intra-abdominal dissemination of EOC cells through the interaction of its 84-95 sequence with the Formyl Peptide Receptor type 1 (FPR1), even as short linear peptide Ser-Arg-Ser-Arg-Tyr (SRSRY). While the pro-metastatic role of uPAR is well documented, little information regarding the expression and role of FPR1 in EOC is currently available. METHODS: Expression levels of uPAR and FPR1 in EOC cells and tissues were assessed by immunofluorescence, Western blot, or immunohystochemistry. Cell adhesion to extra-cellular matrix proteins and mesothelium as well as mesothelium invasion kinetics by EOC cells were monitored using the xCELLigence technology or assessed by measuring cell-associated fluorescence. Cell internalization of FPR1 was identified on multiple z-series by confocal microscopy. Data from in vitro assays were analysed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons. Tissue microarray data were analyzed with the Pearson's Chi-square (χ2) test. RESULTS: Co-expression of uPAR and FPR1 by SKOV-3 and primary EOC cells confers a marked adhesion to vitronectin. The extent of cell adhesion decreases to basal level by pre-exposure to anti-uPAR84-95 Abs, or to the RI-3 peptide, blocking the uPAR84-95/FPR1 interaction. Furthermore, EOC cells exposed to RI-3 or desensitized with an excess of SRSRY, fail to adhere also to mesothelial cell monolayers, losing the ability to cross them. Finally, primary and metastatic EOC tissues express a high level of FPR1. CONCLUSIONS: Our findings identify for the first time FPR1 as a potential biomarker of aggressive EOC and suggests that inhibitors of the uPAR84-95/FPR1 crosstalk may be useful for the treatment of metastatic EOC.

Large oncosomes overexpressing integrin alpha-V promote prostate cancer adhesion and invasion via AKT activation.

BACKGROUND: Molecular markers for prostate cancer (PCa) are required to improve the early definition of patient outcomes. Atypically large extracellular vesicles (EVs), referred as "Large Oncosomes" (LO), have been identified in highly migratory and invasive PCa cells. We recently developed and characterized the DU145R80 subline, selected from parental DU145 cells as resistant to inhibitors of mevalonate pathway. DU145R80 showed different proteomic profile compared to parental DU145 cells, along with altered cytoskeleton dynamics and a more aggressive phenotype. METHODS: Immunofluorescence staining and western blotting were used to identify blebbing and EVs protein cargo. EVs, purified by gradient ultra-centrifugations, were analyzed by tunable resistive pulse sensing and multi-parametric flow cytometry approach coupled with high-resolution imaging technologies. LO functional effects were tested in vitro by adhesion and invasion assays and in vivo xenograft model in nude mice. Xenograft and patient tumor tissues were analyzed by immunohistochemistry. RESULTS: We found spontaneous blebbing and increased shedding of LO from DU145R80 compared to DU145 cells. LO from DU145R80, compared to those from DU145, carried increased amounts of key-molecules involved in PCa progression including integrin alpha V (αV-integrin). By incubating DU145 cells with DU145R80-derived LO we demonstrated that αV-integrin on LO surface was functionally involved in the increased adhesion and invasion of recipient cells, via AKT. Indeed either the pre-incubation of LO with an αV-integrin blocking antibody, or a specific AKT inhibition in recipient cells are able to revert the LO-induced functional effects. Moreover, DU145R80-derived LO also increased DU145 tumor engraftment in a mice model. Finally, we identified αV-integrin positive LO-like structures in tumor xenografts as well as in PCa patient tissues. Increased αV-integrin tumor expression correlated with high Gleason score and lymph node status. CONCLUSIONS: Overall, this study is the first to demonstrate the critical role of αV-integrin positive LO in PCa aggressive features, adding new insights in biological function of these large EVs and suggesting their potential use as PCa prognostic markers.

LncRNA HOTAIR Polymorphisms Association with Cancer Susceptibility in Different Tumor Types.

Single nucleotide polymorphisms (SNPs) in non-coding RNAs (ncRNA) molecules affect gene and protein expression and generate genetic variability influencing the risk of tumor diseases. HOTAIR locates at the heart of the cell memory gene program and represents a prototype of long non coding RNA (LncRNA) due to its capacity to regulate in-trans a distant chromosome landscape. Aberrant expression of HOTAIR is frequently associated with pathogenesis and mostly with metastatic progression of several human cancers. Different polymorphisms, particularly present in intronic sequences, as well as in promoter regions of HOTAIR, are often associated with its aberrant expression, patient prognosis, and cancer susceptibility in different tumor phenotypes. In this minireview, we have summarized the main SNPs in HOTAIR sequence and their relation with cancer risk in several types of solid tumors.

Correction: c-MET receptor as potential biomarker and target molecule for malignant testicular germ cell tumors.

[This corrects the article DOI: 10.18632/oncotarget.25867.].