Invasive Non-functioning Pituitary Adenomas Research Articles

CD87-targeted BiTE and CAR-T cells potently inhibit invasive nonfunctional pituitary adenomas.

Recently, bispecific T-cell engagers (BiTEs) and chimeric antigen receptor-modified T cells (CAR-Ts) have been shown to have high therapeutic efficacy in hematological tumors. CD87 is highly expressed in solid tumors with an oncogenic function. To assess their cytotoxic effects on invasive nonfunctioning pituitary adenomas (iNFPAs), we first examined CD87 expression and its effects on the metabolism of iNFPA cells. We generated CD87-specific BiTE and CAR/IL-12 T cells, and their cytotoxic effects on iNFPAs cells and in mouse models were determined. CD87 had high expression in iNFPA tissue and cell samples but was undetected in noncancerous brain samples. CD87×CD3 BiTE and CD87 CAR/IL-12 T-cells showed antigenic specificity and exerted satisfactory cytotoxic effects, decreasing tumor cell proliferation in vitro and reducing existing tumors in experimental mice. Overall, the above findings suggest that CD87 is a promising target for the immunotherapeutic management of iNFPAs using anti-CD87 BiTE and CD87-specific CAR/IL-12 T cells.

Construction of circRNA-miRNA-mRNA Network Involved in Pituitary Adenoma using High-Throughput Sequencing and Bioinformatics Analysis

We conducted a study to identify circular RNAs associated with invasive non-functioning pituitary adenomas and chemoresistance, as well as to construct a competitive endogenous RNA network. We performed circular RNA expression profiling of 10 specimens using RNA sequencing and identified 37 differentially expressed circular RNAs, which were validated using real-time quantitative polymerase chain reaction. We also detected 26 differentially expressed microRNAs and 388 differentially expressed messenger RNAs using microarray expression profiles downloaded from GSE191113. We predicted the interaction among differentially expressed circular RNAs, differentially expressed microRNAs and differentially expressed messenger RNAs and constructed a circular RNA-microRNA-messenger RNA regulatory network. Our comprehensive bioinformatic analysis revealed the regulatory pathways associated with PA invasion and chemoresistance. Enrichment analysis of differentially expressed circular RNAs revealed their association with non-functioning pituitary adenomas invasion and drug resistance. We established a protein-protein interaction network and identified four hub genes (suppressor of cytokine signaling 3, leptin receptor, interleukin 6 receptor and protein tyrosine phosphatase receptor-type C). Overall, our study provides valuable insights into the invasive and drug-resistance nature of non-functioning pituitary adenomas and could be useful in identifying potential biomarkers or therapeutic targets for invasive non-functioning pituitary adenomas

Expression and clinical significance of Cathepsin K and MMPs in invasive non-functioning pituitary adenomas.

BackgroundCathepsin K (CTSK) is a protease that degrades type I collagen and extracellular matrix, thereby contributing to bone resorption and tumor invasion. Some pituitary adenomas (PAs) could invade the sphenoid sinus (SS) and cavernous sinus (CS).PurposeThis retrospective cohort study aimed to study the expression of tumoral biomarkers (CTSK, MMP9, MMP2, TIMP2, and PTTG1) and evaluate their clinical significance in non-functioning pituitary adenomas (NFPAs) with different invasion patterns.MethodsWe assessed the expression levels of candidate invasion-specific protein biomarkers CTSK, MMP9, MMP2, TIMP2, and PTTG1 by immunohistochemical staining in paraffin-embedded NFPA tumor tissues. Variations in staining intensity were analyzed in cases with SS and CS invasion and non-invasive NFPAs.ResultsWe found that the levels of CTSK were higher in PA cases with SS invasion than that in PA cases with CS invasion (95.57 ± 31.57 vs. 65.29 ± 29.64, P < 0.001), and the expression of MMP9 and MMP2 was higher in CS-invasive cases than that in SS-invasive cases (145.02 ± 49.25 vs. 111.80 ± 51.37, P = 0.002, and 138.67 ± 52.06 vs. 108.30 ± 41.70, P = 0.002). Multiple Cox regression demonstrated that higher CTSK expression (P=0.011), subtotal resection (P<0.001), invasion (P=0.037), and larger tumor diameter (P=0.001) were independent risk factors for recurrence. A positive correlation was observed between CTSK expression and tumor size (r=0.671, p<0.001). There was no significant difference in TIMP2 and PTTG1 levels between CS-and SS-invasive cases (97.42± 39.80 vs. 102.10± 43.22, P = 0.58 and 13.89 ± 4.59 vs. 12.56 ± 3.96, P = 0.14).ConclusionOur data indicated that CTSK has the potential as a marker for SS invasion of PAs, whereas MMP9 and MMP2 may be markers for CS invasion. And CTSK may play an important role in tumor relapse.

A systematic review of molecular alterations in invasive non-functioning pituitary adenoma.

Invasive non-functional pituitary adenomas (NFPAs) constitute 35% of NFPAs. Despite a relatively large body of molecular investigations on the invasiveness of NFPA, the underlying molecular mechanisms of invasiveness are yet to be determined. Herein, we aimed to provide an overview of gene/microRNA(miRNAs) expression alterations in invasive NFPA. This article describes a systematic literature review of articles published up to March 23, 2021, on the transcriptional alterations of invasive NFPA. Five digital libraries were searched, and 42 articles in total fulfilled the eligibility criteria. Pathway enrichment was conducted, and protein interactions among the identified deregulated genes were inferred. In total 133 gene/protein transcriptional alterations, comprising 87 increased and 46 decreased expressions, were detected in a collective number of 1001 invasive compared with 1007 non-invasive patients with NFPA. Deregulation of CDH1, PTTG1, CCNB1, SNAI1, SLUG, EZR, and PRKACB, which are associated with epidermal-mesenchymal transition (EMT), was identified. Moreover, six members of the angiogenesis pathway, i.e., VEGFA, FLT1, CCND1, CTNNB1, MYC(c-MYC), and PTTG1, were detected. SLC2A1, FLT1, and VEGFA were also recognized in the hypoxia pathway. Physical interactions of CTNNB1 with FLT1, CCND1, and EZR as well as its indirect interactions with VEGFA, MYC, CCNB1, and PCNA indicate the tight interplay between EMT, angiogenesis, and hypoxia pathways in invasive NFPAs. In addition, Hippo, JAK-STAT, MAPK, Wnt, PI3K-Akt, Ras, TGF-b, VEGF, and ErbB were identified as interwoven signaling pathways. In conclusion, invasive NFPA shares very common deregulated signaling pathways with invasive cancers. A large amount of heterogeneity in the reported deregulations in different studies necessitates the validation of the expressional changes of the suggested biomarkers in a large number of patients with invasive NFPA.

Diagnosis of invasive non-functional pituitary adenomas using exosomal biomarkers

Background and aimsIncomplete surgical resection of invasive non-functional pituitary adenomas (NFPAs) produces a risk of the subsequent development of complications which will require treatment with powerful drugs and adjuvant radiotherapy. Materials and methodsThe degree of invasiveness of NFPA can be established using biomarkers to help clinicians choose appropriate treatment for these patients. ResultsThis research explored transcriptomic and proteomic variations of non-invasive and invasive NFPAs, other forms of pituitary adenomas and evaluated exosomal genetic markers associated with these diseases. Increased expression of matrix metalloproteinase-1 (MMP1) and its formation in exosomes (exo-MMP1) were correlated with the characteristic invasiveness of NFPAs. Changes in the expression of MMP1 in the exosome was synchronized with transduction of NFPA cells. Enrichment of MMP1 stimulated migration, growth and angiogenesis in tumors through the protease-activated receptor-1 signaling pathway in cells. ConclusionThe results revealed that MMP1 activity has obligatory actions in promoting tumor invasion and angiogenesis, and that the exosome-mediated regulatory pathway for MMP1 may be a novel therapeutic target.

Association of PTTG1 expression with invasiveness of non-functioning pituitary adenomas

BackgroundPituitary tumor transforming gene 1 (PTTG1), paired-like homeodomain 2 (PITX2), and galectin-3 have been widely studied as predictive biomarkers for various tumors and are involved in tumorigenesis and tumor progression. We evaluated the usefulness of PTTG1, PITX2, and galectin-3 as predictive biomarkers for invasive non-functioning pituitary adenomas (NFPAs) by determining the relationship between the expressions of these three proteins and the invasiveness of the NFPAs. We also investigated whether PTTG1, E-cadherin, and Ki-67, which are known to be related to each other, show a correlation with NFPA features.MethodsA retrospective study was conducted on 87 patients with NPFAs who underwent surgical removal. The NFPAs were classified into three groups based on magnetic resonance imaging findings of suprasellar extension and cavernous sinus invasion. Immunohistochemical staining for PTTG1, PITX2, galectin-3, E-cadherin, and Ki-67 was performed on tissue microarrays.ResultsPTTG1 expression showed a statistically significant correlation with the invasiveness of NFPAs, whereas PITX2 and galectin-3 did not have a relationship with the invasiveness of NFPAs. Moreover, there was no association among PTTG1, E-cadherin, and Ki-67 expression.ConclusionsPTTG1 has the potential to serve as a predictive biomarker for invasive NFPA. Furthermore, this study may serve as a reference for the development of PTTG1-targeted therapeutic agents.

Alterations in CD8+ Tregs, CD56+ Natural Killer Cells and IL-10 Are Associated With Invasiveness of Nonfunctioning Pituitary Adenomas (NFPAs).

Invasive nonfunctioning pituitary adenomas (NFPAs) grow rapidly and the mechanisms are unclear. Among many complex mechanisms, the role of immunity in the development of NFPAs has not been fully explored. Here, we analyzed the clinical features 146 NFPA patients who underwent trans-sphenoidal surgery or craniotomy and examined the effects of immune tolerance in invasiveness of NFPA patients using fluorescence-activated cell sorting and immunohistochemical methods. We found patients with invasive NFPAs had more visual deficits and defective fields, higher tumor size, and lower white blood cell count compared with patients with noninvasive NFPAs. Additionally, compared with patients with noninvasive NFPAs, patients with invasive NFPAs had conspicuously lower CD3−CD56+ natural killer (NK) cells and significantly higher levels of CD3+CD8+CD28-T cells (CD8+ Tregs) and interleukin-10 (IL-10) in peripheral blood. Moreover, patients with invasive NFPAs had lower infiltrated CD56+ cells, less infiltrated CD28+ cells, and significantly greater IL-10 expression. These results demonstrated that low CD56+ cells infiltration and CD28+ cells infiltration, as well as high IL-10 expression in pituitary tumor tissues, were related with increased invasiveness of NFPAs. Levels of CD3−CD56+ NK cells, CD8+ Tregs and IL-10 in the peripheral blood could be feasible diagnostic markers for invasive NFPAs.

Integration of quantitative phosphoproteomics and transcriptomics revealed phosphorylation-mediated molecular events as useful tools for a potential patient stratification and personalized treatment of human nonfunctional pituitary adenomas.

Invasiveness is a very challenging clinical problem in nonfunctional pituitary adenomas (NFPAs), and currently, there are no effective invasiveness-related molecular biomarkers. The post-neurosurgery treatment is much different as for invasive and noninvasive NFPAs. The aim of this study was to integrate phosphoproteomics and transcriptomics data to reveal phosphorylation-mediated molecular events for invasive characteristics of NFPAs to achieve a potential tool for patient stratification, and prognostic/predictive assessment to discriminate invasive from noninvasive NFPAs for personalized attitude. The 6-plex tandem mass tag (TMT) labeling reagents coupled with TiO2 enrichment of phosphopeptides and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify and quantify each phosphoprotein and phosphosite in NFPAs and controls. Differentially expressed genes (DEGs) between invasive NFPA and control tissues were obtained from the Gene Expression Omnibus (GEO) database. The overlapping analysis was performed between phosphoprotiens and invasive DEGs. Gene Ontology (GO) enrichment, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein-protein interaction (PPI) analyses were used to analyze these overlapped molecules. In total, 1035 phosphoproteins with 2982 phosphorylation sites were identified in NFPAs vs. controls, and 2751 DEGs were identified in invasive NFPAs vs. controls. Overlapping analysis of these phosphoproteins and DEGs exposed 130 overlapped molecules (phosphoproteins; invasive DEGs). GO enrichment and KEGG pathway analyses of 130 overlapped molecules revealed multiple biological processes and signaling pathway network alterations, including cell-cell adhesion, platelet activation, GTPase signaling pathway, protein kinase signaling, calcium signaling pathway, estrogen signaling pathway, glucagon signaling pathway, cGMP-PKG signaling pathway, GnRH signaling pathway, inflammatory mediator regulation of TRP channels, vascular smooth muscle contraction, and Fc gamma R-mediated phagocytosis, which were obviously associated with tumor invasive characteristics. For 130 overlapped molecules, PPI network-based molecular complex detection (MCODE) identified 10 hub molecules, namely SLC2A4, TSC2, AKT1, SCG3, ALB, APOL1, ACACA, SPARCL1, CHGB, and IGFBP5. These hub molecules are involved in multiple signaling pathways and represent potential predictive/prognostic markers in NFPA patients as well as they represent potential therapeutic targets. This study provided the first large-scale phosphoprotein profiling and phosphorylation-related signaling pathway network alterations in human NFPA tissues. Further, overlapping analysis of phosphoproteins and invasive DEGs revealed the phosphorylation-mediated signaling pathway network changes in invasive NFPAs. These findings are the precious resource for in-depth insight into the molecular mechanisms of NFPAs, as well as for the discovery of effective phosphoprotein biomarkers and therapeutic targets for invasive NFPAs.

Multiomics-Based Signaling Pathway Network Alterations in Human Non-functional Pituitary Adenomas.

Non-functional pituitary adenoma (NFPA) seriously affects hypothanamus-pituitary-target organ axis system, with a series of molecule alterations in the multiple levels of genome, transcriptome, proteome, and post-translational modifications, and those molecules mutually interact in a molecular-network system. Meta analysis coupled with IPA pathway-network program was used to comprehensively analyze nine sets of documented NFPA omics data, including NFPA quantitative transcriptomics data [280 differentially expressed genes (DEGs)], NFPA quantitative proteomics data [50 differentially expressed proteins (DEPs)], NFPA mapping protein data (218 proteins), NFPA mapping protein nitration data (9 nitroproteins and 3 non-nitrated proteins), invasive NFPA quantitative transriptomics data (346 DEGs), invasive NFPA quantitative proteomics data (57 DEPs), control mapping protein data (1469 proteins), control mapping protein nitration data (8 nitroproteins), and control mapping phosphorylation data (28 phosphoproteins). A total of 62 molecular-networks with 861 hub-molecules and 519 canonical-pathways including 54 cancer-related canonical pathways were revealed. A total of 42 hub-molecule panels and 9 canonical-pathway panels were identified to significantly associate with tumorigenesis. Four important molecular-network systems, including PI3K/AKT, mTOR, Wnt, and ERK/MAPK pathway-systems, were confirmed in NFPAs by PTMScan experiments with altered expression-patterns and phosphorylations. Nineteen high-frequency hub-molecules were also validated in NFPAs with PTMScan experiment with at least 2.5-fold changes in expression or phosphorylation, including ERK, ERK1/2, Jnk, MAPK, Mek, p38 MAPK, AKT, PI3K complex, p85, PKC, FAK, Rac, Shc, HSP90, NFκB Complex, histone H3, AP1, calmodulin, and PLC. Furthermore, mTOR and Wnt pathway-systems were confirmed in NFPAs by immunoaffinity Western blot analysis, with significantly decreased expression of PRAS40 and increased phosphorylation levels of p-PRAS40 (Thr246) in mTOR pathway in NFPAs compared to controls, and with the decreased protein expressions of GSK-3β and GSK-3β, significantly increased phosphorylation levels of p-GSK3α (Ser21) and p-GSK3β (Ser9), and increased expression level of β-catenin in Wnt pathway in NFPAs compared to controls. Those findings provided a comphrensive and large-scale pathway network data for NFPAs, and offer the scientific evidence for insights into the accurate molecular mechanisms of NFPA and discovery of the effective biomarkers for diagnosis, prognosis, and determination of therapeutic targets.

Correlation between the Expression of Interleukin-6, STAT3, E-Cadherin and N-Cadherin Protein and Invasiveness in Nonfunctional Pituitary Adenomas

Objective This study aimed to investigate the expression of interleukin (IL)-6, signal transducer and activator of transcription 3 (STAT3), epithelial-cadherin (E- cadherin) and neural-cadherin (N-cadherin) proteins in nonfunctional pituitary adenomas, and their correlation with invasiveness. Methods Thirty cases of nonfunctional pituitary adenoma pathological wax specimens were selected from our hospital, including 20 cases of invasive nonfunctional pituitary adenoma (INFPA) and 10 noninvasive nonfunctional pituitary adenomas (NNFPAs). Envision was used to detect IL-6, STAT3, E-cadherin , and N-cadherin in specimens. Statistical methods were used to analyze the correlation between the four proteins and the Knosp classification of nonfunctional pituitary adenomas. Result IL-6 and STAT3 were highly expressed in INFPAs but poorly expressed in NNFPAs. E-cadherin expression in INFPAs was lower than that in NNFPAs. N-cadherin was positive or strongly positive in both groups. Spearman's correlation analysis showed that the expression of IL-6 and STAT3 was positively correlated with Knosp's classification, whereas the expression of E-cadherin was negatively correlated with Knosp classification. Meanwhile, the expression of N-cadherin was not correlated with Knosp's classification. Conclusion The expression of the IL-6, STAT3, E-cadherin proteins were associated nonfunctional pituitary adenomas. However, the expression of N-cadherin was not correlated with nonfunctional pituitary adenomas.

Multimaterial and multicolor 3D-printed model in training of transnasal endoscopic surgery for pituitary adenoma.

The aim of the present study was to investigate the practical value of a multimaterial and multicolor 3D-printed model in anatomical teaching, surgical training, and preoperative planning of transnasal endoscopic surgery for pituitary adenoma. Multimodality neuroimaging data were obtained in a 42-year-old healthy male volunteer and a 40-year-old female patient with an invasive nonfunctional pituitary adenoma. Three 3D-printed models were produced: a monomaterial and monocolor model, a monomaterial and multicolor model, and a multimaterial and multicolor model. The effects on anatomical teaching and surgical training for exposing the vidian nerve were assessed by 12 residents, and the training effect was validated on cadavers. The practical values for preoperative planning were evaluated by 6 experienced neurosurgeons. All evaluations were based on 5-point Likert questionnaires. The multimaterial and multicolor model was superior to the monomaterial models in surgical training for exposing the vidian nerve (Fisher test; p < 0.05). In addition, the multimaterial and multicolor model was superior to the monomaterial models in anatomical teaching and preoperative planning (Friedman test; p < 0.05). Multimaterial and multicolor 3D printing technology makes it convenient and efficient to produce a practical model for simulating individualized and complex anatomical structures in the sellar region. Furthermore, the multimaterial model can provide a more realistic manipulative experience for surgical training and facilitate the preoperative planning.

Diagnosis of Invasive Nonfunctional Pituitary Adenomas by Serum Extracellular Vesicles.

The invasiveness evaluation of nonfunctional pituitary adenoma (NFPAs) is crucial for the prediction of the malignant potential and for making surgical plans of NFPAs. Current invasiveness evaluation of NFPAs is based on neuroimaging, which can hardly predict the invasive potential and dynamically monitor disease progress. Here we used microbead-assisted flow cytometry to detect and analyze the serum extracellular vesicles (EVs) from 30 NFPAs patients (15 invasive and 15 noninvasive). Lower expressions of folate receptor 1 (FOLR1) and epithelial cell adhesion molecule (EpCAM) were found in serum EVs from the invasive NFPAs patients compared to the noninvasive ones [area under the curve (AUC) of 0.94 for FOLR1 and 0.88 for EpCAM]. Meanwhile, increased mRNA expression of vimentin and N-cadherin, two mesenchymal markers, was found in serum EVs from the invasive NFPAs patients compared to the noninvasive ones. Consistent results were observed in the tumor tissue that invasive NFPAs have lower expression of the epithelial markers while higher expression of the mesenchymal markers. These results suggested the possible role of epithelial-mesenchymal transition (EMT) in the invasiveness of NFPAs. Pituitary tumor transforming gene 1 (PTTG1) mRNA in serum EVs was also found to be an indicator for invasive NFPAs and is related with EMT. These results provide a method for the blood-based diagnosis and invasiveness evaluation of NFPAs and would be beneficial to the diagnosis, prognosis prediction, and surgical risk evaluation of NFPAs.

Identification of Important Invasion-Related Genes in Non-functional Pituitary Adenomas.

Non-functioning pituitary adenomas (NFPAs) are locally invasive with high morbidity. The objective of this study was to diagnose important genes and pathways related to the invasiveness of NFPAs and gain more insights into the underlying molecular mechanisms of NFPAs. The gene expression profiles of GSE51618 were downloaded from the Gene Expression Omnibus database with 4 non-invasive NFPA samples, 3 invasive NFPA samples, and 3 normal pituitary gland samples. Differentially expressed genes (DEGs) are screened between invasive NFPA samples and normal pituitary gland samples, followed by pathway and ontology (GO) enrichment analyses. Subsequently, a protein-protein interaction (PPI) network was constructed and analyzed for these DEGs, and module analysis was performed. In addition, a target gene-miRNA network and target gene-TF (transcription factor) network were analyzed for these DEGs. A total of 879 DEGs were obtained. Among them, 439 genes were upregulated and 440 genes were downregulated. Pathway enrichment analysis indicated that the upregulated genes were significantly enriched in cysteine biosynthesis/homocysteine degradation (trans-sulfuration) and PI3K-Akt signaling pathway, while the downregulated genes were mainly associated with docosahexaenoate biosynthesis III (mammals) and chemokine signaling pathway. GO enrichment analysis indicated that the upregulated genes were significantly enriched in animal organ morphogenesis, extracellular matrix, and hormone activity, while the downregulated genes were mainly associated with leukocyte chemotaxis, dendrites, and RAGE receptor binding. Subsequently, ESR1, SOX2, TTN, GFAP, WIF1, TTR, XIST, SPAG5, PPBP, AR, IL1R2, and HIST1H1C were diagnosed as the top hub genes in the upregulated and downregulated PPI networks and modules. In addition, HS3ST1, GPC4, CCND2, and SCD were diagnosed as the top hub genes in the upregulated and downregulated target gene-miRNA networks, while CISH, ISLR, UBE2E3, and CCNG2 were diagnosed as the top hub genes in the upregulated and downregulated target gene-TF networks. The new important DEGs and pathways diagnosed in this study may serve key roles in the invasiveness of NFPAs and indicate more molecular targets for the treatment of NFPAs.

Transcriptome Analysis Identifies an Attenuated Local Immune Response in Invasive Nonfunctioning Pituitary Adenomas.

BackgroundInvasive nonfunctioning pituitary adenomas (NFPAs) remain challenging due to their high complication rate and poor prognosis. We aimed to identify the distinctive molecular signatures of invasive NFPAs, compared with noninvasive NFPAs, using gene expression profiling by RNA sequencing.MethodsWe obtained frozen fresh tissue samples from 14 patients with NFPAs who underwent primary transsphenoidal surgery. Three non-invasive and 11 invasive NFPAs were used for RNA sequencing. The bioinformatics analysis included differential gene expression, gene ontology analysis, and pathway analysis.ResultsA total of 700 genes were differentially expressed (59 up-regulated and 641 down-regulated genes) between invasive and non-invasive NFPAs (false discovery rate <0.1, and |fold change| ≥2). Using the down-regulated genes in invasive NFPAs, gene ontology enrichment analyses and pathway analyses demonstrated that the local immune response was attenuated and that transforming growth factor-β (TGF-β) RII-initiated TGF-β signaling was down-regulated in invasive NFPAs. The overexpression of claudin-9 (CLDN9) and the down-regulation of insulin-like growth factor-binding protein 5 (IGFBP5), death-associated protein kinase 1 (DAPK1), and tissue inhibitor of metalloproteinase-3 (TIMP3) may be related with invasiveness in NFPAs.ConclusionInvasive NFPAs harbor different gene expression profiles relative to noninvasive NFPAs. In particular, local suppression of the immune response and TGF-β signaling can make PAs prone to invasiveness.

PO-250 Phospho S82 Hsp27: a marker of invasive and recurrent non-functioning pituitary adenoma

IntroductionIntroduction:Non-functioning pituitary adenomas (NFPA) usually present as macroadenomas and 42% shows invasion of the adjacent structures, such as cavernous sinus, sphenoid bone or nasopharynx and some recur locally. The incomplete surgery section of invasive non-functional pituitary adenomas (NFPAs) carries the increased risks of recurrence and requires adjuvant radiotherapy and surgeries. It is necessary to clarify the molecular mechanisms and markers of invasiveness to guide the management of NFPA patients. Here we describe a comprehensive phosphoproteomic evaluation of 20 NFPAs followed by validation in a larger set of samples.Material and methodsPeptides from 20 tumours were enriched with TiO2 beads, fractionated using bRPLC and subjected to high-throughput LC-MS/MS-Orbitrap Fusion Tribrid Mass Spectrometer. Upto 5 precursor ions were chosen for MS/MS analysis. Following MASCOT and SEQUEST analysis our bioinformatics pipeline (PhosphositePlus, Gene Ontology, DAVID, and KEGG) identified 1345 phosphoprotein groups and 2233 unique phosphopeptides. Eight candidate phosphoproteins involved in cell proliferation and growth were selected for validation using immunohistochemistry(tissue microarray containing 200 NFPA samples) and immunoblotting (n=18).Results and discussionsHsp27 phospho-Ser82 was found 2.1 fold upregulated in invasive and 7.8 fold hyperphosphorylated in recurrent group while Hsp phospho-Ser15 was found 2.1 fold upregulated in invasive and 7.8 fold hyperphosphorylated in recurrent group in our mass spectrometry experiment. Immunohistochemistry for Ser82 revealed 2.1-fold upregulation (p<0.0001) in the recurrent and 1.6 (p<0.01) fold upregulation in InvasiveNFPA. There was no significant upregulation in Ser15 was observed in invasive and recurrent groups compared to non-invasive and non-recurrent. In agreement with the mass spectrometry and IHC data, immunoblotting also relvealed significantly upregulated Hsp27 phospho-Ser 82 in invasive and recurrent group. HSPB1 serine 82 phosphorylation by VEGF activation of PKC-mediated PKD induces endothelial migration and tubulogenesis, indicating the potential importance of HSPB1 in VEGF-dependent angiogenesis and hence pituitary tumorigenesis.ConclusionHsp27 phosphorylated at Ser82 is significantly associated with NFPA invasion and recurrence. It Provides a roadmap for patient stratification, and prognostication for recurrence and trials for targeted therapy.

Aberrant expression of the sFRP and WIF1 genes in invasive non-functioning pituitary adenomas

Non-functioning pituitary adenomas (NFPAs) are the most common pituitary tumors and mainly invade the sphenoid, cavernous sinus or dura mate. Aberrant regulation of the Wnt signaling pathway plays an important role in tumorigenesis. This study was designed to investigate the relationships between secreted frizzled-related proteins (sFRPs), WIF1 genes and the invasion of NFPAs by tissue microassays (TMAs) of samples from 163 patients. Significantly weaker staining of WIF1 and sFRP4 were detected in the invasive group compared with the non-invasive group by TMAs (p = 0.002, p < 0.001). Univariate analysis showed a significant correlation between tumor invasion and low expression of WIF1 and sFRP4 (p = 0.002, p < 0.001). A similar trend was observed when analyzing the mRNA and protein levels through RT-PCR and western blot experiments. Methylation of the WIF1 promoter was significantly increased in invasive NFPAs compared with the noninvasive group (p = 0.004). The average progression free survival time in the high WIF1 group was longer than that in the low WIF1 group (p = 0.025). Furthermore, RT-PCR measured the levels of 11 miRNAs targeting WIF1 according to the Targetscan database and PubMed. The levels of miRNA-137, miRNA-374a-5p and miRNA-374b-5p in the invasive group were 0.037-fold, 0.577-fold and 0.44-fold that of the noninvasive group (p = 0.003, p = 0.049 and p = 0.047). Overexpression of miRNA-137 could inhibit the proliferation and invasion of GH3 cells through cell viability and Transwell experiments (p < 0.05). Furthermore, the WIF1 level was upregulated after overexpression of miRNA-137 compared with miRNA-137-NC (control miRNA) in GH3 cells. Our data suggest that WIF1 may be potential biomarker for the aggressiveness of NFPAs. miRNA-137 plays an important role in the Wnt signaling pathway by affecting promoter methylation of WIF1.

Malignant transformation in non-functioning pituitary adenomas (pituitary carcinoma).

Non-functioning pituitary carcinomas (NFPC) are defined as tumours of adenophyseal origin with craniospinal or systemic dissemination, with the absence of a hormonal hypersecretion syndrome. These are a histologically heterogenous group of tumours, comprising gonadotroph, null cell, "silent" tumours of corticotroph, somatotroph or lactotroph cell lineages as well as plurihormonal Pit-1 tumours. NFPC are exceedingly rare, and hence few cases have been described. This review has identified 38 patients with NFPC reported in the literature. Recurrent invasive non-functioning pituitary adenomas (NFPA) were observed in a majority of patients. Various factors have been identified as markers of the potential for aggressive behaviour, including rapid tumour growth, growth after radiotherapy, gain or shift of hormone secretion and raised proliferative markers. Typically, there is a latency of several years from the original presentation with an NFPA to identification of metastases and only 5cases reported with rapidly progressive malignant disease within 1month of presentation. Therapeutic options include debulking surgery, radiation therapy and chemotherapy with temozolomide recommended as first line systemic treatment. Although long-term survivors are described, prognosis remains generally very poor (median survival 8months). Improvements in molecular tumour profiling may assist in predicting tumour behaviour, guide therapeutic choices and identify novel therapies.

A Novel Invasive-Related Biomarker in Three Subtypes of Nonfunctioning Pituitary Adenomas.

To identify biomarkers key to invasiveness of the 3 subtypes of nonfunctioning pituitary adenomas (NFPAs) and provide a guidance for therapeutic decision making and identification of potential adjuvant drugs. Fifty NFPA tumor tissues obtained from transsphenoidal surgery were used in the study. Three invasive NFPAs and 4 noninvasive NFPAs were used for gene expression microarray analyses. In addition, there are 5 invasive NFPAs and 4 noninvasive NFPAs used for proteomic analyses. Invasive-related biomarkers were identified by bioinformatics analysis by integrating the transcriptomics and proteomics data sets. All 3 subtypes of NFPAs (null cell adenomas, oncocytomas, and gonadotroph adenomas) were used to validate differentially expressed candidate biomarkers by means of quantitative real-time reverse transcription polymerase chain reaction and Western blot. The level of EZR was downregulated in pituitary adenoma cell line GH3 to investigate the invasive effect of EZR on GH3 cells by using the RNA interference technique. Eight genes involved in the invasion function were found by bioinformatics analysis, and the EZR gene was identified as a novel invasive-related biomarker in the 3 subtypes of NFPAs. The expression level of EZR was found higher in terms of invasiveness than the noninvasive ones of the 3 subtypes of NFPAs. Moreover, the knockdown of EZR inhibited the invasion of GH3 cells invitro. EZR is a novel biomarker in terms of invasion among the 3 subtypes of NFPAs, and it is a promising guide for therapeutic decision making as well.

Smad3 and phospho-Smad3 are potential markers of invasive nonfunctioning pituitary adenomas

BackgroundTransforming growth factor-β (TGF-β) signaling plays important roles in tumor development. Nevertheless, the roles of TGF-β/Smad signaling in nonfunctioning pituitary adenomas (NFPAs) have not been fully studied.MethodsTumor samples were obtained from patients who had NFPAs and underwent endoscopic transsphenoidal surgery or craniotomy at Beijing Tiantan Hospital from March 2008 to December 2012. Immunohistochemistry was performed to determine the expression of Smad transducer proteins in NFPAs. Ki-67 was evaluated as an indicator of the proliferative activity of NFPAs.ResultsA total of 161 patients with NFPAs were identified; 59 (36.6%) had invasive NFPAs and 102 (63.4%) had noninvasive NFPAs. Protein levels of Smad3 and phospho-Smad3 (p-Smad3) were significantly lower in patients with invasive NFPAs than in patients with noninvasive NFPAs (P<0.05 and P<0.01, respectively). The Ki-67 index was markedly greater in invasive NFPAs than in noninvasive NFPAs (P<0.05) and was significant correlated with p-Smad3 levels (P<0.05, r=−0.702).ConclusionA low level of Smad3 and p-Smad3 proteins was associated with the invasion of NFPAs.

Integrative proteomics and transcriptomics identify novel invasive-related biomarkers of non-functioning pituitary adenomas.

Non-functioning pituitary adenomas (NFPAs) are usually macroadenomas and display invasion into surrounding tissues. The treatment for invasive NFPAs is still challenging. This study describes the differential patterns of gene expression between invasive and non-invasive NFPAs and identifies novel biomarkers involved in invasion of NFPAs for diagnosis and treatment. Using gene microarray technology, we examined the gene expression profile and found 1160 differentially expressed messenger RNA (mRNA) between invasive and non-invasive NFPAs. Then, we examined the protein profile by liquid chromatography tandem mass spectrometry (LC-MS/MS) and found 433 differentially expressed proteins between invasive and non-invasive NFPAs. Subsequently, we integrated the proteomics and transcriptomics datasets and identified 29 common changed molecules. Through bioinformatics analysis using Ingenuity Pathway Analysis (IPA) software, we showed that the 29 molecules were enriched in 25 canonical signaling pathways, 25 molecular and cellular functions, and 2 networks. Eight genes were identified involved in the invasion function by the molecular and cellular functions analysis, including CAT, CLU, CHGA, EZR, KRT8, LIMA1, SH3GLB2 and SLC2A1. Furthermore, we validated the decreased CHGA expression and increased CLU expression in invasive NFPAs by qRT-PCR and Western blot. Our study demonstrated that integration of proteomics and transcriptomics could prove advantageous for accelerating tumor biomarker discovery and CHGA and CLU might be important novel biomarkers and therapeutic targets for invasion of NFPAs.