Pathology & Oncology Research

Comparison of CDH1 Gene Hypermethylation Status in Blood and Serum among Gastric Cancer Patients Abstract Hypermethylation is epigenetic alteration, well known for gene silencing. CHD1 gene is known as invasion and tumor suppressor gene, decreased expression due to hypermethylation could promote tumor cell invasion and metastasis. Present study designed to investigate the CDH1 gene promoter hypermethylation status by methylation specific polymerase chain reaction in 100 newly diagnosed gastric cancer patients. 53% of hypermethylation was observed in DNA extracted from blood in Gastric cancer patients while 66% was observed in serum based DNA. Significant differences in CDH1gene promoter hypermethylation was observed in serum based DNA extracted from Gastric cancer patients. Patients in early stage (I & II) vs advanced stage (III & IV), distant organ metastases vs no metastases had 60% vs 7% and 42% 24% of CDH1 promoter hypermethylation in serum DNA (p = 0.006, 0.001) respectively. Patients who were with lymph node invasion, loss of appetite, loss of weight had 55%, 47%, 61% CDH1 gene promoter hypermethylation compare to who were not with lymph node invasion, loss of appetite, loss of weight had 11%, 19%, 5% of hypermethylation and these differences was found to be significant. Strong association was observed with overall median survival of patients (p < 0.0001). Patients who had CDH1 gene promoter hypermethylation in serum based DNA showed poor overall median survival (14.3 months) and unmethylated patients had better overall median survival (33.2 months). CDH1 hypermethylation status was found to be associated with advancement of disease, distant organ metastases and lymph node invasion in Gastric cancer patients.

Prognostic Significance of Serum PSA Level and Telomerase, VEGF and GLUT-1 Protein Expression for the Biochemical Recurrence in Prostate Cancer Patients after Radical Prostatectomy Abstract The aim of the study was to evaluate prognosis for biochemical recurrence (BR) by analysing the pathological and biological characteristics of prostate cancer (PCa) after radical prostatectomy (RP). There were 130 men with clinically localized PCa in whom pretreatment serum PSA level and Ki-67, prostate specific membrane antigen (PSMA), glucose transporter-1 (GLUT-1), vascular endothelial growth factor (VEGF), microvessel density (MVD) and human telomerase reverse transcriptase (hTERT) proteins expression, based on number of immunohistochemically positive cells (labelling index), were retrospectively studied. In order to assess the prognostic significance of analysed variables in univariate and multivariate Cox analysis, patients were dichotomized based on cut-off points chosen by receiver operating characteristic (ROC) curves. There were 83 males (63.8%) at pT stage 1–2 and 47 (36.1%) at pT stage 3–4, respectively, with median (range) age of 62.8 years (49–77), and median follow-up of 78.5 months (12–148). In 42 (32.3%) men BR was found. In univariate analysis, tumour biological features: PSA ≤ 8 ng/mL (p = 0.006), Ki-67LI ≤ 12.7% (p = 0.015), VEGFLI>11.0% (p = 0.030), and hTERTLI>6.7% (p = 0.016), but not clinicopathological parameters, appeared to be positive prognosticators for BRFS. In the Cox analysis, Ki-67 lost its significance, and clinicopathological parameters appeared to be nonsignificant. The independent negative prognostic factors for BRFS were: PSA > 8.0 ng/mL, (Hazard ratio = 2.75, p = 0.003), GLUT-1 > 19.1% (HR = 2.1, p = 0.032), VEGF≤11.0% (HR = 1, p = 0.024) and hTERT≤6.7% (HR = 1, p = 0.017). High PSA level, and GLUT-1 expression and lower VEGF and nuclear hTERT expression may indicate the great role of hypoxia in BR induction in PCa.

Identification of Pathogenic Genes and Transcription Factors in Osteosarcoma Abstract Osteosarcoma (OS) is an aggressive malignant tumor of the bones. Our study intended to identify and analyze potential pathogenic genes and upstream regulators for OS. We performed an integrated analysis to identify candidate pathogenic genes of OS by using three Gene Expression Omnibus (GEO) databases (GSE66673, GSE49003 and GSE37552). GO and KEGG enrichment analysis were utilized to predict the functional annotation and potential pathways of differentially expressed genes (DEGs). The OS-specific transcriptional regulatory network was established to study the crucial transcriptional factors (TFs) which target the DEGs in OS. From the three GEO datasets, we identified 759 DEGs between metastasis OS samples and non-metastasis OS samples. After GO and KEGG analysis, 'cell adhesion' (FDR = 1.27E-08), 'protein binding' (FDR = 1.13E-22), 'cytoplasm' (FDR = 5.63E-32) and 'osteoclast differentiation' (FDR = 0.000992221) were significantly enriched pathways for DEGs. HSP90AA1 exhibited a highest degree (degree = 32) and was enriched in 'pathways in cancer' and 'signal transduction'. BMP6, regulated by Pax-6, was enriched in the 'TGF-beta signaling pathway'. We indicated that BMP6 may be downregulated by Pax-6 in the non-metastasis OS samples. The up-regulated HSP90AA1 and down-regulated BMP6 and 'pathways in cancer' and 'signal transduction' were deduced to be involved in the pathogenesis of OS. The identified biomarkers and biological process in OS may provide foundation for further study.

KIBRA Team Up with Partners to Promote Breast Cancer Metastasis Abstract Among women, breast cancer is the most frequently diagnosed cancer. Most of the breast cancers represent metastasis to distant organs at the time of diagnosis and accounts for the majority of deaths. Metastasis is characterized by many genetic aberrations including mutations, overexpression of oncogenes etc. KIBRA (KIdney/BRAin protein), a scaffolding protein is recently described as an important player in the process of invasion and metastasis. The Kidney/BRAin protein through its different domains interacts with various proteins to couple cytoskeleton arrangement, cell polarity and migration. N terminal and C terminal of the protein contains the WW, Internal C2 & putative class III PDZ domain that interacts with DDR1, DLC1 & PKCζ. These protein-protein interactions equip the breast cancer cells to invade and metastasize. Here, we discuss a comprehensive knowledge about the KIBRA protein, its domains and the interacting partners involved in metastasis of breast cancer.

lncRNA SNHG16 Exerts Oncogenic Functions in Promoting Proliferation of Glioma Through Suppressing p21 Abstract Glioma is a malignant brain tumor that accounts for 30% of all brain tumors and 80% of malignant brain tumors. This poor clinical outcome makes the study of molecular mechanisms in glioma as an urgent subject. However, the certain mechanism remains unclear. Long non-coding RNAs (lncRNAs) plays a key role in glioma development and progression. In the present study, we aimed to explore the potential mechanisms of lncRNA SNHG16 in glioma. The levels of lncRNA SNHG16 were qualified in both glioma tissues and cell lines using qRT-PCR assay. The ability of cell proliferation was tested via CCK-8 and colony formation assays. Transfections were performed to knockdown SNHG16 and its target gene p21. The cell cycles and cell apoptosis were evaluated using flow cytometry, and the expression of SNHG16, p21 and apoptosis biomarkers were qualified with qRT-PCR and western blot assays. The expression of SNHG16 were up-regulated in both glioma tissues and cell lines. Knockdown of SNHG16 was associated with poor proliferation, decreased monoclonal formation rates, but increased apoptosis rates, which also caused the high expression of p21. Moreover, p21 could mediate cell proliferation and monoclonal formation, promote cell apoptosis in glioma, which was negatively correlated with lncRNA SNHG16. The molecule mechanism experiments revealed that SNHG16 could not only inhibit the expression of p21 but also suppressed the level of caspase 3 and 9, while promoted cyclinD1 and cyclinB1 expression. lncRNA SNHG16 could promote the cell proliferation and inhibit the apoptosis of glioma through suppressing p21, indicating that lncRNA SNHG16 might be quite vital for the diagnosis and progression of glioma and could even be a novel therapeutic target for glioma.

Differential Analysis of lncRNA, miRNA and mRNA Expression Profiles and the Prognostic Value of lncRNA in Esophageal Cancer Abstract Integrative central axis of lncRNA-miRNA-mRNA plays pivotal roles in tumor development and progression. However, the regulatory role of lncRNA-miRNA-mRNA in esophageal cancer remains elusive. TCGA database was utilized to investigate the differential expression of lncRNA, miRNA and mRNA in esophageal cancer (ESCA) and normal esophageal tissues, and GEO database was used to further validate the expression profile of key genes. Differential lncRNAs in TCGA database were submitted to Starbase, and lncRNAs related to overall survival were analyzed using Kaplan-Meier and log-rank test. We found 145 lncRNAs, 112 miRNAs and 2000 protein coding mRNAs were differentially expressed in ESCA samples, which were tightly involved in chromosome segregation, extracellular matrix assembly by GO assay, and KEGG assay revealed the correlation of differentially expressed genes with cell cycle, apoptosis and cGMP-PKG signaling pathway. Furthermore, there were 291 nodes in ceRNA network, which consisted of 40 lncRNAs, 28 miRNAs and 233 mRNAs, and formed 677 relations. Furthermore, 6 of 10 lncRNAs in TCGA database were consistent with GEO database, and expressions of 10 mRNAs in TCGA database all exhibited the same tendency with GEO database. Notably, we found 8 lncRNAs (WDFY3-AS2, CASC8, UGDH-AS1, RAP2C-AS1, AC007128.1, AC016205.1, AC092803.2 and AC079949.2) were correlated with overall survival of the patients with ESCA. The key differentially expressed genes participate in the development and progression of ESCA, and thus the elucidation of functions of lncRNA-miRNA-mRNA will provide new novel therapeutic target for the patients with ESCA.

C-X-C Chemokine Receptor Type 7 (CXCR-7) Expression in Invasive Ductal Carcinoma of Breast in Association with Clinicopathological Features Abstract C-X-C chemokine receptor type 7 (CXCR-7) is an atypical receptor for chemokines whose role in different stages of carcinogenesis has been evaluated in breast cancer cell lines and animal models. Moreover, it has been demonstrated to be a target of regulation by the tumor suppressor microRNA (miR)-100. In the present study, we assessed CXCR-7 expression in 60 breast cancer patients in association with clinicopathological and demographic data of patients. We also extracted the results of our previous work on miR-100 expression in the same cohort of patients to assess the correlation between miR-100 and CXCR-7 expression levels. Transcript levels of CXCR-7 were significantly higher in tumoral tissues compared with adjacent non-cancerous tissues (ANCTs) (Tumoral vs. ANCTs: 3.64 ± 1.8 vs. 0.73 ± 1.3, P = 0.000). A significant negative correlation was detected between CXCR-7 protein and miR-100 transcript levels (r = −0.526, P < 0.05). High CXCR-7 mRNA levels were significantly associated with tumor size (P = 0.01). Besides, high protein levels were more prevalent in higher TNM stages (P = 0.000). Moreover, high CXCR-7 protein levels were significantly associated with ER (P = 0.005) and PR (P = 0.02) status. The present work provides further evidence for the role of CXCR-7 in breast cancer and proposes the elimination of inhibitory effects of miR-100 on CXCR-7 expression as a mechanism for its up-regulation in breast cancer tissues.

MicroRNA-182-5p Modulates Oral Squamous Cell Carcinoma Migration and Invasion Via Targeting MTSS1 Gene Abstract Oral squamous cell carcinoma (OSCC) characterized with invasive growth, local metastasis and later stage diagnosis was a common malignancy in head and neck region. The aim of this study was to explore the relationship between miR-182-5p and OSCC, which will contribute to find potential biomarker for OSCC metastasis. MiR-182-5p expression level was detected by the quantitative real-time PCR (qRT-PCR). Cell migration and invasion ability were examined by scratch and transwell assay. Loss-of function together with luciferase reporter assay were used to verify the miR-182-5p modulated OSCC cells migration and metastasis was mediated by MTSS1. The expression of MTSS1 protein was examined by western blotting. MiR-182-5p up-regulated in OSCC, was involved in the migration and invasion of OSCC and the increased miR-182-5p expression was correlated with lower OSCC differentiation grade, higher T and N stage. Bioinformatics analysis predicted MTSS1 gene was a potential target of miR-182-5p. Following co-transfection, qRT-PCR, luciferase activities assay and western blotting confirmed that MTSS1 gene was a direct target of miR-182-5p and silence of MTSS1 could reverse the effects of miR-182-5p on OSCC migration and invasion. MiR-182-5p was up-regulated in OSCC and the ability of miR-182-5p to promote MTSS1 repression may precipitate in the OSCC through bypassing cell migration and invasion control.

Dynamic FDG-PET/CT in the Initial Staging of Primary Breast Cancer: Clinicopathological Correlations Abstract Our aim was to evaluate correlation between clinicopathological features (clinical T and clinical N stages; histological type; nuclear grade; hormone-receptor and HER2 status, proliferation activity and tumor subtypes) of breast cancer and kinetic parameters measured by staging dynamic FDG-PET/CT examinations. Following ethical approval and patients' informed consent we included 34 patients with 35 primary breast cancers in our prospective study. We performed dynamic PET imaging, and assessed plasma activity noninvasively. To delineate primary tumors we applied a frame-by-frame semi-automatic software-based correction of motion artefacts. FDG two-compartment kinetic modelling was applied to assess K1, k2, k3 rate coefficients and to calculate Ki (tracer flux constant) and MRFDG (FDG metabolic rate). We found that k3, Ki and MRFDG were significantly higher in higher grade (p = 0.0246, 0.0089 and 0.0076, respectively), progesterone-receptor negative (p = 0.0344, 0.0217 and 0.0132) and highly-proliferating (p = 0.0414, 0.0193 and 0.0271) tumors as well as in triple-negative and hormone-receptor negative/HER2-positive subtypes (p = 0.0310, 0.0280 and 0.0186). Ki and MRFDG were significantly higher in estrogen-receptor negative tumors (p = 0.0300 and 0.0247, respectively). Ki was significantly higher in node-positive than in node-negative disease (p = 0.0315). None of the assessed FDG-kinetic parameters showed significant correlation with stromal TIL. In conclusion, we confirmed a significant relationship between kinetic parameters measured by dynamic PET and the routinely assessed clinicopathological factors of breast cancer: high-grade, hormone-receptor negative tumors with high proliferation rate are characterized by higher cellular FDG-uptake and FDG-phosphorylation rate. Furthermore, we found that kinetic parameters based on the dynamic examinations are probably not influenced by stromal TIL infiltration.

The Investigation of Lipoxygenases as Therapeutic Targets in Malignant Pleural Mesothelioma Abstract Advanced malignant pleural mesothelioma (MPM) has an extremely poor prognosis with limited chemotherapy options, therefore the identification of new therapeutic targets would aid in disease management. Arachidonic acid is metabolised by cyclooxygenase and lipoxygenase enzymes. The lipoxygenase isoenzymes 5-LOX and 12-LOX have been implicated in carcinogenesis. We aimed to examine 5-LOX and 12-LOX protein expression in a large retrospective series of mesothelioma samples. Further to this, the in vitro cytotoxic effects of lipoxygenase pathway inhibitors were investigated in mesothelioma cells. Archival samples from 83 patients with MPM were examined by immunohistochemistry for expression of the 5-LOX and 12-LOX proteins. The MTS assay was used to assess cell viability following 72 h treatment with the lipoxygenase pathway inhibitors baicalein, licofelone, MK-886 and zileuton in the MPM cell lines NCI-H2052, NCI-H2452 and MSTO-211H. Positive 12-LOX protein expression was recorded in 69/83 (83%) and positive 5-LOX expression was observed in 56/77 (73%) of MPM tissue samples. Co-expression of 5-LOX with 12-LOX was seen in 46/78 (58%) of MPM samples. Positive expression of 5-LOX, 12-LOX and COX-2 proteins was identified in the NCI-H2052, NCI-H2452 and MSTO-211H MPM cell lines. Baicalein (12-LOX and 15-LOX inhibitor) was effective in 3/3 MPM cell lines at low concentrations with an IC50 range of 9.6 μM to 20.7 μM. We have demonstrated that the 5-LOX and 12-LOX proteins are expressed in a significant proportion of MPM samples (73% and 83% respectively) and may represent novel therapeutic targets in this disease. We have demonstrated that the inhibition of the LOX pathway using baicalein may be effective as a novel treatment for MPM, however further human pharmacokinetic studies are required in order to establish whether the concentration used in vitro is clinically achievable.