The oncogenic roles of GPR176 in ovarian cancer: a molecular target for aggressiveness and gene therapy.

BACKGROUND: At present, the discovery of new biomarkers is of great significance for the early diagnosis, treatment and prognosis assessment of ovarian cancer. Previous findings indicated that aberrant G-protein-coupled receptor 176 (GPR176) expression might contribute to tumorigenesis and subsequent progression. However, the expression of GPR176 and the molecular mechanisms in ovarian cancer had not been investigated. METHODS: GPR176 expression was compared with clinicopathological features of ovarian cancer using immunohistochemical and bioinformatics analyses. GPR176-related genes and pathways were analysed using bioinformatics analysis. Additionally, the effects of GPR176 on ovarian cancer cell phenotypes were investigated. RESULTS: GPR176 expression positively correlated with elder age, clinicopathological staging, tumour residual status, and unfavourable survival of ovarian cancer, but negatively with purity loss, infiltration of B cells, and CD8+ T cells. Gene Set Enrichment Analysis showed that differential expression of GPR176 was involved in focal adhesion, ECM-receptor interaction, cell adhesion molecules and so on. STRING and Cytoscape were used to determine the top 10 nodes. Kyoto Encyclopaedia of Genes and Genomes analysis indicated that GPR176-related genes were involved in the ECM structural constituent and organisation and so on. GPR176 overexpression promoted the proliferation, anti-apoptosis, anti-pyroptosis, migration and invasion of ovarian cancer cells with overexpression of N-cadherin, Zeb1, Snail, Twist1, and under-expression of gasdermin D, caspase 1, and E-cadherin. CONCLUSION: GPR176 might be involved in the progression of ovarian cancer. It might be used as a biomarker to indicate the aggressive behaviour and poor prognosis of ovarian cancer and a target of genetic therapy.

Ovarian cancer is a gynecological cancer with high mortality. Due to the limited screening tests and treatments available, most ovarian cancer patients are diagnosed at a late stage and the prognosis is poor. The addition of new cancer diagnostic biomarkers and new intervention targets may improve quality of life and survival for patients with ovarian cancer. Previous studies have revealed the aberrant GPR176 expression might contribute to tumorigenesis and subsequent progression in many other tumours. In our study, GPR176 was found to promote the proliferation, anti-apoptosis, anti-pyroptosis, migration and invasion, EMT, and weakening the cellular adhesion of ovarian cancer cells, and involved in the Bcl-2/Bax or the PI3K/Akt/mTOR pathway. Therefore, abnormal expression of GPR176 might be served as a biomarker for aggressive behaviour and poor prognosis of ovarian cancer and a target for gene therapy.

The relationship between pepsinogen C and gastric carcinogenesis: a transgene and population study.

BACKGROUND: Pepsinogen C (PGC) is expressed in chief cells, fundic mucous neck cells, and pyloric gland cells of gastric epithelium and also in breast, prostate, lung, and seminal vesicles. METHODS: We explored the clinicopathological and prognostic significances of PGC mRNA using pathological and bioinformatics analyses. We generated PGC knockout and PGC-cre transgenic mice to observe the effects of PGC deletion and PTEN abrogation in PGC-positive cells on gastric carcinogenesis. Finally, we observed the effects of altered PGC expression on aggressive phenotypes by CCK8, Annexin V staining, wound healing and transwell assays and analyzed the partner proteins of PGC using co-IP (co-immunoprecipitation) and double fluorescence staining. RESULTS: PGC mRNA level was inversely correlated with the T and G stage and a short survival of gastric cancer (p < 0.05). PGC protein expression was negatively linked to lymph node metastasis, dedifferentiation, and low Her-2 expression of gastric cancer (p < 0.05). No difference in body weight or length was evident between wild-type (WT) and PGC knockout (KO) mice (p > 0.05), but PGC KO mice had a shorter survival than WT mice (p < 0.05). No gastric lesions were observed in the mucosa of the granular stomach in PGC KO mice, which displayed lower frequency and severity of gastric lesion than in WT mice after treated with MNU. Transgenic PGC-cre mice showed high cre expression and activity in the lung, stomach, kidney, and breast. Gastric cancer and triple-negative lobular breast adenocarcinoma were found in PGC-cre/PTENf/f mice with two previous pregnancies and breast feeding, but breast cancer was not seen in transgenic mice exposed to either estrogen or progesterone, or those with two previous pregnancies and no breast feeding. PGC suppressed proliferation, migration, invasion, and induced apoptosis, and interacted with CCNT1, CNDP2 and CTSB. CONCLUSION: PGC downregulation was seen in gastric cancer, but PGC deletion resulted in resistance to chemically-induced gastric carcinogenesis. PGC expression suppressed the proliferation and invasion of gastric cancer cells possibly by interacting with CCNT1, CNDP2 and CTSB. Spontaneous triple-negative lobular adenocarcinoma and gastric cancer were seen in PGC-cre/PTENf/f mice, and the breast carcinogenesis was closely linked to pregnancy and breast feeding, but not to single exposure to estrogen or progesterone, or pregnancy. Limiting either pregnancy or breast feeding might help to prevent hereditary breast cancer.

The potential oncogenic effect of tissue-specific expression of JC polyoma T antigen in digestive epithelial cells.

JC polyoma virus (JCPyV), a ubiquitous polyoma virus that commonly infects people, is identified as the etiologic factor for progressive multifocal leukoencephalopathy and has been closely linked to various human cancers. Transgenic mice of CAG-loxp-Laz-loxp T antigen were established. T-antigen expression was specifically activated in gastroenterological target cells with a LacZ deletion using a cre-loxp system. Gastric poorly-differentiated carcinoma was observed in T antigen-activated mice using K19-cre (stem-like cells) and PGC-cre (chief cells), but not Atp4b-cre (parietal cells) or Capn8-cre (pit cells) mice. Spontaneous hepatocellular and colorectal cancers developed in Alb-cre (hepatocytes)/T antigen and villin-cre (intestinal cells)/T antigen transgenic mice respectively. Gastric, colorectal, and breast cancers were observed in PGC-cre/T antigen mice. Pancreatic insulinoma and ductal adenocarcinoma, gastric adenoma, and duodenal cancer were detected in Pdx1-cre/T antigen mice. Alternative splicing of T antigen mRNA occurred in all target organs of these transgenic mice. Our findings suggest that JCPyV T antigen might contribute to gastroenterological carcinogenesis with respect to cell specificity. Such spontaneous tumor models provide good tools for investigating the oncogenic roles of T antigen in cancers of the digestive system.

FAM64A aggravates proliferation, invasion, lipid droplet formation, and chemoresistance in gastric cancer: A biomarker for aggressiveness and a gene therapy target.

FAM64A is a mitogen-induced regulator of the metaphase and anaphase transition. Here, we found that FAM64A messenger RNA (mRNA) and protein expression levels were higher in gastric cancer tissue than in normal mucosa (p < .05). FAM64A methylation was negatively correlated with FAM64A mRNA expression (p < .05). The differentially expressed genes of FAM64A were mainly involved in digestion, potassium transporting or exchanging ATPase, contractile fibers, endopeptidase, and pancreatic secretion (p < .05). The FAM64A-related genes were principally categorized into ubiquitin-mediated proteolysis, cell cycle, chromosome segregation and mitosis, microtubule binding and organization, metabolism of amino acids, cytokine receptors, lipid droplet, central nervous system, and collagen trimer (p < .05). FAM64A protein expression was lower in normal gastric mucosa than intestinal metaplasia, adenoma, and primary cancer (p < .05), negatively correlated with older age, T stage, lymphatic and venous invasion, tumor, node, metastasis stage, and dedifferentiation (p < .05), and associated with a favorable overall survival of gastric cancer patients. FAM64A overexpression promoted proliferation, antiapoptosis, migration, invasion, and epithelial-mesenchymal transition via the EGFR/Akt/mTOR/NF-κB, while the opposite effect was observed for FAM64A knockdown. FAM64A also induced chemoresistance directly or indirectly through lipid droplet formation via ING5. These results suggested that upregulation of FAM64A expression might induce aggressive phenotypes, leading to gastric carcinogenesis and its subsequent progression. Thus, FAM64A could be regarded as a prognosis biomarker and a target for gene therapy.

A bioinformatics analysis of the clinicopathological and prognostic significance of FAM64A mRNA expression in gynecological cancers.

FAM64A is a mitotic regulator which promotes cell metaphase-anaphase transition and is highly expressed in a cell-cycle-dependent manner. In this study, we examined the clinicopathological and prognostic significance of FAM64A mRNA expression in gynecological cancers. We conducted a bioinformatics analysis of FAM64A mRNA expression using Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), xiantao, The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN), and Kaplan-Meier (KM) plotter databases. FAM64A expression was elevated in breast, cervical, endometrial, and ovarian cancers when compared with normal tissue. Expression was positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade and TP53 mutation, and endometrial cancer serous subtype. FAM64A expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. FAM64A functioned as an independent predictor of overall and disease-specific survival in breast cancer patients. FAM64A-correlated genes were involved in ligand-receptor interactions, and chromosomal, cell cycle, and DNA replication processes in breast, cervical, endometrial and ovarian cancers. Top hub genes primarily included cell cycle-related proteins in breast cancer, mucins and acetylgalactosaminyl transferases in cervical cancer, kinesin family members in endometrial cancer, and synovial sarcoma X and the cancer/testis antigen in ovarian cancer. FAM64A mRNA expression was positively related to Th2 cell infiltration, but negatively associated with neutrophil and Th17 cell infiltration in breast, cervical, endometrial, and ovarian cancers. FAM64A expression may be considered a potential biomarker reflecting carcinogenesis, histogenesis, aggressive behaviour, and prognosis in gynecological cancers.Impact statementWhat is already known on this subject? FAM64A is located in cell nucleolar and nucleoplasmic regions, and during mitosis it putatively controls metaphase-to-anaphase transition. FAM64A appears to regulate different physiological processes, including apoptosis, tumorigenesis, neural differentiation, stress responses, and the cell cycle.What the results of this study add? FAM64A expression was up-regulated in breast, cervical, endometrial, and ovarian cancers, and positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade, and TP53 mutation, and a serous subtype in endometrial cancer. FAM64A expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. FAM64A functioned as an independent predictor of overall and disease-specific survival in breast cancer. FAM64A-correlated genes were involved in ligand-receptor interactions, chromosomal, cell cycle, and DNA replication processes, while FAM64A mRNA expression was positively related to Th2 cell infiltration but negatively correlated with neutrophil and Th17 cell infiltration in four gynecological cancers.What the implications of these findings for clinical practice and/or further research? In the future, abnormal FAM64A mRNA expression may serve as a biomarker of carcinogenesis, histogenesis, aggressiveness, and prognosis in gynecological malignancies.

Bioinformatic analysis of the clinicopathological and prognostic significance of oocyte-arresting BTG4 mRNA expression in gynecological cancers.

BTG4 arrests the cell cycle and suppresses oocyte and embryonic development. We performed a bioinformatic analysis of BTG4 expression. BTG4 expression was downregulated in breast cancer compared with normal tissues (p < .05), but the opposite was observed in cervical, endometrial and ovarian cancers (p < .05). BTG4 methylation was negatively correlated with its mRNA expression in breast, cervical and endometrial cancers (p < .05). BTG4 mRNA expression was negatively correlated with T staging and distant metastasis of breast cancer; and with tumor invasion, clinical stage, low weight and BMI, low histological grade and no diabetes in endometrial cancer but positively with T stage and non-keratinizing squamous carcinoma in endometrial cancer. BTG4 expression was negatively correlated with the survival of ovarian cancer patients (p < .05), but positively for breast, cervical and endometrial cancers (p < .05). BTG4 expression is thus a potential marker reflecting the carcinogenesis, aggressiveness and prognosis in gynecological cancers.Impact StatementWhat is already known on this subject? Previous studies have revealed the structure and location of BTG4. BTG4 inhibit cell proliferative, promote apoptosis, induce G1 cell cycle arrest. BTG4 promotes the development of mouse embryos from cell stage 1 to 2. The methylation and biological function of BTG4 were clarified in gastric and/or colorectal cancer cells.What do the results of this study add? BTG4 is found to closely link to reflect the carcinogenesis, histogenesis, aggressive behaviors and prognosis of gynecological cancers, and involved in ligand-receptor interaction, microtubule motor activity, dynein light chain binding, cilium organization, assembly, and movement in endometrial and ovarian cancers.What are the implications of these finding for clinical practice and/or further research? Aberrant BTG4 mRNA expression can be employed as a marker of the tumorigenesis, histogenesis, aggressiveness and prognosis of gynecological cancers in the future practice and guide the investigation of BTG4-related signal pathways.

Bioinformatics analysis of the clinicopathological and prognostic significance of BAG3 mRNA in gynecological cancers.

BAG3 is a co-chaperone BAG family protein that plays important roles in protein homeostasis, cell survival, cell motility, and tumour metastasis. This study aimed to clarify the clinicopathological and prognostic implications of BAG3 mRNA expression in tumours. We performed bioinformatics analysis on BAG3 mRNA expression using TCGA, XIANTAO, UALCAN, and Kaplan-Meier plotter databases. BAG3 mRNA expression was downregulated in breast and endometrial cancers and positively correlated with favourable PAM50 subtyping in breast cancer，clinical stage and short overall survival in ovarian cancer and negatively correlated with T stage, clinical stage, and histological grade in cervical and endometrial cancers. The top BAG3-related pathways included ligand-receptor interactions and activity, DNA packaging and nucleosomes, hormonal responses, membrane regions, microdomains and rafts, and endosomes in breast cancer; ligand-receptor interactions, transmembrane transporters and channels, cell adhesion, and keratinisation in cervical cancer; ligand-receptor interactions, anion transmembrane transporters, lipoproteins, keratinisation, cell adhesion, and protein processing in endometrial cancer; metabolism of porphyrin, chlorophyll, pentose, uronic acid, ascorbate, and alternate and cell adhesion in ovarian cancer. BAG3 expression could represent a potential marker for carcinogenesis, histogenesis, aggressive behaviours, and prognosis in gynecological cancers.IMPACT STATEMENTWhat is already known on this subject? BAG3 regulates cell activity, autophagy, and resistance to apoptosis through multiple domains and plays an important role in tumour development. BAG3 positively regulates tumour cell invasion and migration in cervical and ovarian cancers.What do the results of this study add? BAG3 expression is closely associated with histogenesis, clinicopathology, and prognosis in gynecological cancers and is involved in signalling pathways associated with the control of cell proliferation, migration, invasion, and drug resistance in tumours.What are the implications of these findings for clinical practice and/or further research? Abnormal BAG3 expression can be employed as a possible marker of tumour development, invasion, and prognosis, providing new ideas for treating cancer.

The bioinformatics analysis of the clinicopathological and prognostic significances of REG4 mRNA in gynecological cancers.

To clarify the clinicopathological importance of REG4 mRNA expression, we used GEO, TCGA, xiantao, UALCAN, and Kaplan-Meier plotter for a bioinformatics analysis in breast, cervical, endometrial and ovarian cancers. Compared to normal tissues, REG4 expression was found to be upregulated in breast, cervical, endometrial, and ovarian cancers (p < 0.05). Breast cancer had a higher level of REG4 methylation than normal tissues (p < 0.05), which was negatively correlated with its mRNA expression. REG4 expression was positively correlated with oestrogen and progesterone receptor expression, and aggressiveness of PAM50 classification of breast cancer patients (p < 0.05). Breast infiltrating lobular carcinomas expressed more REG4 than ductal carcinomas (p < 0.05). The REG4-related signal pathways mainly included peptidase, keratinisation, brush border and digestion and so forth in gynecological cancers. Our results indicated that REG4 overexpression was associated with gynecological carcinogenesis and their histogenesis, and may be used as a marker for aggressive behaviour and prognosis of breast or cervical cancer.IMPACT STATEMENTWhat is already known on this subject? REG4 encodes a secretory c-type lectin and plays an essential role in inflammation, carcinogenesis, apoptotic and radiochemotherapeutic resistance.What do the results of this study add? As a standalone predictor, REG4 expression was positively correlated with progression-free survival. Expression of REG4 mRNA was positively associated with the T stage and adenosquamous cell carcinoma of cervical cancer. The top signal pathways related to REG4 included smell and chemical stimulus, peptidase, intermediate filament, and keratinisation in breast cancer; ligand-receptor interaction, metabolism of hormone, xenobiotic and retinol, peptidase, brush border and digestion in cervical and ovarian cancers; bile secretion, intermediate filament, chemical carcinogenesis, brush border and keratinisation in endometrial cancer. REG4 mRNA expression was positively correlated with DC cell infiltration in breast cancer, positively with Th17 cells, TFH, cytotoxic cells and T cells in cervical and endometrial cancers, and negatively with DC cell infiltration, cytotoxic cells and T cells in ovarian cancer. The top hub genes mainly included small proline rich protein 2B in breast cancer; fibrinogens and apoproteins in cervical, endometrial and ovarian cancers.What are the implications of these finding for clinical practice and/or further research? Our study has showed that REG4 mRNA expression is a potential biomarker or therapeutic target for gynaecologic cancers.

The effects of REG4 expression on chemoresistance of ovarian cancer.

Although ovarian cancer usually responds well to platinum- and taxane-based first-line chemotherapy, most patients develop recurrence and chemoresistance. Regenerating gene 4 (REG4) is a secretory protein involved in cell differentiation and proliferation. We found higher REG4 expression in ovarian cancer than in normal tissues (p < .05). Regenerating gene 4 expression was negatively associated with overall, progression-free or post-progression survival rates of patients with ovarian cancer receiving platinum or paclitaxel treatment (p < .05) according to a Kaplan-Meier plotter. Regenerating gene 4 overexpression resulted in either cisplatin or paclitaxel resistance, and apoptosis resistance in CAOV3 ovarian cancer cells (p < .05). REG4-transfected ovarian cancer cells showed stronger migration and invasion treated with cisplatin or paclitaxel (p < .05). Additionally, cisplatin or paclitaxel exposure led to the overexpression of phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, phosphorylated mammalian target of rapamycin (p-mTOR), glutathione S-transferase-π, survivin, and B-cell lymphoma 2 in REG4 transfectants compared with control cells (p < .05). These findings suggested that REG4 expression was up-regulated in ovarian cancer, and associated with poor survival and chemotherapy resistance. REG4 promoted the occurrence, development, and chemotherapy resistance of ovarian cancer by regulating cell proliferation, apoptosis, migration, and invasion, and PI3K/Akt/m-TOR signalling pathways. IMPACT STATEMENTWhat is already known on this subject? REG4 mRNA expression is up-regulated in many digestive cancers. High REG4 expression was associated with an adverse prognosis, high tumour and nodal stages, poor differentiation, and hepatic and peritoneal metastases of digestive cancers. REG4 expression conferred cancer cells with increased resistance to chemoradiotherapy, especially 5-FU-based treatment, by activating the MAPK/Erk/Bim signalling pathway.What do the results of this study add? REG4 was highly expressed in ovarian cancer. The expression of p-PI3K, p-AKT, p-mTOR, GST-π, survivin, and Bcl-2 was increased in REG4-overexpressing cells. High REG4 expression was significantly associated with inferior OS, PFS, and PPS rates in patients with ovarian cancer receiving platinum chemotherapy. REG4 mediated cisplatin and paclitaxel resistance in CAOV3 ovarian cancer cells. The percentage of apoptotic cells was markedly lower in REG4-transfected compared to mock-transfected cells after cisplatin or paclitaxel treatment.What are the implications of these findings for clinical practice and/or further research? This study aimed to evaluate the prognostic significance of REG4 expression in ovarian cancer treated with platinum and paclitaxel, to explore REG4 chemoresistance mechanisms to platinum and paclitaxel, and to provide a scientific experimental basis for the clinical treatment and outcome evaluation of ovarian cancer. In order to provide comprehensive clinical treatment of ovarian cancer, it is helpful to improve our understanding of multi-drug resistance and identify new cancer diagnostic biomarkers.

The upregulated α-catulin expression was involved in head-neck squamous cell carcinogenesis by promoting proliferation, migration, invasion, and epithelial to mesenchymal transition.

Rho signaling component, α-catulin, is a cytoskeletal linker protein and plays an important role in apoptotic and senescence resistance, cytoskeletal reorganization, mobility, invasion, and epithelial to mesenchymal transition (EMT) of cancer cells. Here, we transfected α-catulin-expressing plasmid into head and neck squamous cell carcinoma (HNSCC) cell and examined the phenotypes and relevant molecules. α-catulin expression was detected on tissue microarray containing squamous epithelium, dysplasia, and cancer of head and neck by immunohistochemistry. It was found that α-catulin overexpression resulted in faster growth, migration and invasion, lower apoptosis, G2/M progression, and EMT than the mock and control (P < 0.05). α-catulin overexpression increased the expression of Cyclin E1, cdc2, survivin, Bcl-2, MMP-2, MMP-9, and N-cadherin but decreased the expression of Caspase-3 and E-cadherin by real-time PCR (P < 0.05). α-catulin expression was stronger in primary cancers than those in normal squamous epithelium and dysplasia (P < 0.05), but not correlated with aggressive behaviors or adverse prognosis of HNSCC patients (P > 0.05). Multivariate survival analysis showed that distant metastasis and TNM staging were independent prognostic factors for overall survival of the HNSCC patients (P < 0.05). These data indicated that upregulated expression of α-catulin protein might have impact on the tumorigenesis of HNSCC possibly by reducing apoptosis, enhancing proliferation, cell cycle progression, migration, invasion, and EMT. It might be regarded as a potential marker for head and neck carcinogenesis or a target of gene therapy for HNSCCs.

The roles of parafibromin expression in ovarian epithelial carcinomas: a marker for differentiation and prognosis and a target for gene therapy.

Parafibromin is a protein encoded by hyperparathyroidism 2 (HRPT2) and its downregulated expression is involved in the pathogenesis of parathyroid, breast, gastric, colorectal, lung, head and neck cancers. We aimed to investigate the roles of parafibromin expression in tumorigenesis, progression, or prognostic evaluation of ovarian cancers. HRPT2-expressing plasmid was transfected into ovarian cancer cells with the phenotypes and related molecules examined. The messenger RNA (mRNA) and protein expression of parafibromin were also examined in ovarian normal tissue, benign and borderline tumors and cancers by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, or immunohistochemistry respectively. It was found that parafibromin overexpression caused a lower growth, migration and invasion, higher sensitivity to cisplatin and apoptosis than the mock and control (P < 0.05). The transfectants showed the hypoexpression of phosphoinositide 3-kinase (PI3K), Akt, p70 ribosomal protein S6 kinase (p70s6k), Wnt5a, B cell lymphoma-extra large (Bcl-xL), survivin, vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9) than the mock and control at both mRNA and protein levels (P < 0.05). According to real-time PCR, parafibromin mRNA level was lower in ovarian benign tumors and cancers than normal ovary (P < 0.05), while parafibromin was strongly expressed in metastatic cancers in omentum than primary cancers by Western blot. Immunohistochemically, parafibromin expression was stronger in primary cancers than that in ovarian normal tissue (P < 0.05) but weaker than the metastatic cancers (P < 0.05) with a positive correlation with dedifferentiation, ki-67 expression and the lower cumulative survival rate (P < 0.05). These findings indicate that parafibromin downregulation might promote the pathogenesis, dedifferentiation and metastasis of ovarian cancers possibly by suppressing aggressive phenotypes, such as proliferation, cell cycle, apoptosis, migration and invasion.

The clinicopathological significances and biological functions of parafibromin expression in head and neck squamous cell carcinomas.

Downregulated parafibromin expression is involved in the pathogenesis and progression of parathyroid, breast, gastric, colorectal, and lung cancers. To investigate the roles of parafibromin expression in tumorigenesis, progression, and prognostic evaluation of head and neck squamous cell carcinomas (HNSCCs), we transfected parafibromin-expressing plasmid into HNSCC cell and examined the phenotypes and their relevant molecules. Parafibromin expression was detected on tissue microarray containing squamous epithelium, dysplasia, and carcinoma of head and neck by immunohistochemistry. Parafibromin overexpression was found to suppress growth, migration, and invasion, and induce apoptosis, S arrest, and mesenchymal to epithelial transition (EMT), compared with the mock and control (P < 0.05). Both overexpression of Cyclin E1, Bax, and E-cadherin and hypoexpression of c-myc, Bcl-xL, and slug were detected in B88 transfectants, in comparison to mock and control by real-time PCR. Parafibromin expression was weaker in primary cancers than those in normal squamous tissue and dysplasia (P < 0.05), but stronger than the metastatic cancers in lymph node (P < 0.05). Parafibromin expression was negatively correlated with lymph node metastasis, tumor-node-metastasis (TNM) staging, but positively with human papillomavirus (HPV) positivity (P < 0.05). The HNSCCs in tongue showed more parafibromin expression than those in larynx (P < 0.05). There was stronger parafibromin expression in moderately-than poorly-differentiated carcinomas (P < 0.05). The significantly positive correlation was observed between parafibromin expression and relapse-free survival rate by Kaplan-Meier curves (P < 0.05). Cox's proportional hazard model indicated that distant metastasis and parafibromin expression were independent prognostic factors for overall and relapse-free survival of HNSCC, respectively (P < 0.05). These findings suggest that downregulated expression of parafibromin protein plays an important role in the pathogenesis, differentiation, and metastasis of HNSCCs possibly by inducing apoptosis, suppressing proliferation, cell cycle progression, migration, invasion, and EMT. Parafibromin expression is an independent factor for relapse-free survival of HNSCCs.

The role of the REG4 gene and its encoding product in ovarian epithelial carcinoma.

BACKGROUND: Although its biological function remains poorly understood, REG4 is reported to be a potent activator of the EGFR/Akt/AP-1 signaling pathway in colon cancer cells and closely linked with the inhibition of apoptosis. METHODS: SKOV3 cells were transfected with a REG4-expressing plasmid or treated with recombinant REG4. We then analyzed proliferation, cell cycle, apoptosis, invasion and metastasis or expression of related molecules. REG4 expression was examined in normal ovarian tissue, benign and borderline tumors, and cancers by immunohistochemistry or real-time PCR. RESULTS: REG4 overexpression and the recombinant protein inhibited cell apoptosis, enhanced G2/S progression, proliferation, migration and invasion. Furthermore, expression of Wnt5a, p70s6k, survivin and VEGF expression was increased, while Bax expression was decreased at both the mRNA and protein levels compared to control or mock cells (P<0.05). REG4 mRNA levels were higher in benign tumors and primary cancer compared to those in normal ovarian tissue (P<0.05) while, REG4 protein expression was higher in all three tumor types than that in normal ovarian tissue (P<0.05). Higher REG4 mRNA expression was observed in mucinous carcinomas than serous carcinomas (P<0.05), and in well- and moderately-differentiated carcinomas than poorly-differentiated carcinomas (P<0.05). Survival analysis revealed an inverse relationship between REG4 expression and cumulative or relapse-free survival rates of the patients with ovarian cancer as an independent factor (P<0.05). CONCLUSIONS: Our findings indicate that aberrant REG4 expression plays an essential role in early ovarian carcinogenesis and is closely linked to mucinous ovarian tumors, differentiation and adverse prognosis of ovarian cancer by modulating proliferation, apoptosis, migration and invasion.

Aberrant Beclin 1 expression is closely linked to carcinogenesis, differentiation, progression, and prognosis of ovarian epithelial carcinoma.

Beclin 1, an important autophagy-related protein in human cells, is involved in autophagy, differentiation, anti-apoptosis, and cancer suppression, which is increased during periods of cell stress and extinguished during cell cycle. Human ovarian tumors display allelic loss of Beclin 1 with high frequency. To clarify Beclin 1's role in ovarian carcinogenesis and subsequent progression, its expression was examined by immunostaining on tissue microarrays containing ovarian normal tissue, benign and borderline tumors, and carcinomas. Beclin 1 mRNA and protein expression was examined in ovarian normal tissue, benign and borderline tumors, carcinoma tissue, and cell lines by reverse transcription polymerase chain reaction or Western blot, respectively. The results demonstrated that the higher Beclin 1 mRNA was observed in ovarian benign tumor than normal ovary and ovarian carcinoma (P < 0.05) and negatively correlated with the differentiation of ovarian carcinoma (P < 0.05). Beclin 1 protein expression was stronger in ovarian carcinoma than that in normal ovary and inversely related to the differentiation of ovarian carcinoma (P < 0.05) by Western blot. Immunohistochemically, Beclin 1 expression was statistically higher in ovarian borderline tumor and carcinoma than normal ovary and benign tumor (P < 0.05) and inversely linked to differentiation, lower ki-67 expression, and higher cumulative or relapse-free survival rate of ovarian carcinoma (P < 0.05). Cox proportional hazard model indicated that age and International Federation of Gynecology and Obstetrics staging (P < 0.05), but not pathological classification differentiation degree or Beclin 1 expression, were independent prognostic factors for overall and relapse-free ovarian carcinomas (P > 0.05). It was suggested that the aberrant Beclin 1 expression is closely linked to tumorigenesis and differentiation of ovarian carcinoma. Beclin 1 expression might be employed to indicate the worse prognosis of ovarian carcinomas, albeit not an independent factor.

The role of RhoC in epithelial-to-mesenchymal transition of ovarian carcinoma cells.

BACKGROUND: RhoC is a small G protein/GTPase and involved in tumor mobility, invasion and metastasis. Previously, up-regulated RhoC expression is found to play an important role in ovarian carcinogenesis and subsequent progression by modulating proliferation, apoptosis, migration and invasion. METHODS: We transfected RhoC-expressing plasmid and RhoC siRNA into CAOV3 and OVCAR3 cells respectively. These cells and transfectants were exposed to vascular epithelial growth factor (VEGF), transforming growth factor (TGF)-ß1 or their receptor inhibitors with the phenotypes and their related-molecules examined. RESULTS: TGF-ß1R or VEGFR inhibitor suppressed the proliferation, migration, invasion and lamellipodia formation, the expression of N-cadherin, α-SMA, snail and Notch1 mRNA or protein, and enhanced E-cadherin mRNA and protein expression in CAOV3 and its RhoC-overexpressing transfectants, whereas both growth factors had the opposite effects in OVCAR3 cells and their RhoC-hypoexpressing transfectants. Ectopic RhoC expression enhanced migration, invasion, lamellipodia formation and the alteration in epithelial to mesenchymal transition (EMT) markers of CAOV3 cells regardless of the treatment of VEGFR or TGF-ß1R inhibitor, whereas RhoC knockdown resulted in the converse in OVCAR3 cells even with the exposure to VEGF or TGF-ß1. CONCLUSION: RhoC expression might be involved in EMT of ovarian epithelial carcinoma cells, stimulated by TGF-ß1 and VEGF.

Downregulated inhibitor of growth 3 (ING3) expression during colorectal carcinogenesis.

BACKGROUND & OBJECTIVES: ING3 (inhibitor of growth protein 3) overexpression decreased S-phase cell population and colony-forming efficiency, and induced apoptosis at a p53-mediated manner. The aim of this study was to investigate the clinicopathological and prognostic significance of ING3 expression in colorectal carcinogenesis and subsequent progression. METHODS: ING3 expression was examined by immunohistochemistry on tissue microarray containing colorectal non-neoplastic mucosa (NNM), adenoma and adenocarcinoma. Colorectal carcinoma tissue and cell lines were studied for ING3 expression by Western blot or RT-PCR. RESULTS: ING3 mRNA was differentially expressed in Colo201, Colo205, DLD-1, HCT-15, HCT-116, HT-29, KM-12, SW480, SW620 and WiDr cells. Carcinomas showed significantly lower ING3 expression than matched NNM at mRNA level (P< 0.05), but not at protein level. Immunohistochemically, ING3 expression was significantly decreased from NNM, adenoma to adenocarcinoma (P< 0.05). ING3 expression was not correlated with age, sex, tumour size, depth of invasion, lymphatic or venous invasion, lymph node metastasis, tumour- node- metastasis staging or differentiation. Kaplan-Meier analysis indicated that ING3 protein expression was not associated the prognosis of the patients with colorectal carcinoma (P< 0.05). INTERPRETATION & CONCLUSIONS: Our study showed that downregulated ING3 expression might play an important role in colorectal adenoma-adenocarcinoma sequence. Further studies are required to understand the mechanism.

Beclin 1 expression is closely linked to colorectal carcinogenesis and distant metastasis of colorectal carcinoma.

Beclin 1 participates in development, autophagy, differentiation, anti- apoptosis, neurodegeneration, tumorigenesis and cancer progression. The roles of Beclin 1 in colorectal carcinogenesis and its subsequent progression are still unclear. Here, the mRNA and protein expression of Beclin 1 were determined in colorectal carcinoma and matched mucosa by Reverse transcriptase-polymerase chain reaction and Western blot. Immunohistochemistry and in situ hybridization (ISH) were performed on tissue microarryer with colorectal carcinoma, adenoma and mucosa. The expression of Beclin 1 mRNA and protein was found to be higher in colorectal carcinoma than matched mucosa by real-time PCR and Western blot (p < 0.05). According to the ISH data, Beclin 1 expression was lower in colorectal non-neoplastic mucosa (NNM) than adenoma and carcinoma (p < 0.05). Immunohistochemically, primary carcinoma showed stronger Beclin 1 expression than NNM and metastatic carcinoma in the liver (p < 0.05). Beclin 1 protein expression was negatively related to liver and distant metastasis (p < 0.05), but not correlated with age, sex, depth of invasion, lymphatic or venous invasion, lymph node metastasis, tumor-node-metastasis (TNM) staging, differentiation or serum carcinoembryonic antigen (CEA) concentration (p > 0.05). Survival analysis indicated that Beclin 1 expression was not linked to favorable prognosis of the patients with colorectal carcinoma (p > 0.05). Cox's model indicated that depth of invasion and distant metastasis were independent prognostic factors for colorectal carcinomas (p < 0.05). It was suggested that Beclin 1 expression is closely linked to colorectal carcinogenesis and distant metastasis of colorectal carcinoma.

The Antitumor and Sorafenib-resistant Reversal Effects of Ursolic Acid on Hepatocellular Carcinoma via Targeting ING5.

Inhibitor of growth 5 (ING5) has been reported to be involved in the malignant progression of cancers. Ursolic acid (UA) has shown remarkable antitumor effects. However, its antitumor mechanisms regarding of ING5 in hepatocellular carcinoma (HCC) remain unclear. Herein, we found that UA significantly suppressed the proliferation, anti-apoptosis, migration and invasion of HCC cells. In addition, ING5 expression in HCC cells treated with UA was obviously downregulated in a concentration- and time-dependent manner. Additionally, the pro-oncogenic role of ING5 was confirmed in HCC cells. Further investigation revealed that UA exerted antitumor effects on HCC by inhibiting ING5-mediated activation of PI3K/Akt pathway. Notably, UA could also reverse sorafenib resistance of HCC cells by suppressing the ING5-ACC1/ACLY-lipid droplets (LDs) axis. UA abrogated ING5 transcription and downregulated its expression by reducing SRF and YY1 expression and the SRF-YY1 complex formation. Alb/JCPyV T antigen mice were used for in vivo experiments since T antigen upregulated ING5 expression by inhibiting the ubiquitin-mediated degradation and promoting the T antigen-SRF-YY1-ING5 complex-associated transcription. UA suppressed JCPyV T antigen-induced spontaneous HCC through inhibiting ING5-mediated PI3K/Akt signaling pathway. These findings suggest that UA has the dual antitumoral functions of inhibiting hepatocellular carcinogenesis and reversing sorafenib resistance of HCC cells through targeting ING5, which could serve as a potential therapeutic strategy for HCC.

KRT80 expression works as a biomarker and a target for differentiation in gastric cancer.

Keratin 80 (KRT80) is a filament protein that participates in cell differentiation and the integrity of the epithelial barrier. Here, KRT80 expression was higher in gastric cancer compared with normal mucosa at both mRNA and protein levels by bioinformatic analysis, qRT-PCR and Western blot (p<0.05), however, the methylation of KRT80 was lower than in normal mucosa (p<0.05). There was a negative relationship between promoter methylation and expression level of KRT80 gene in gastric cancer (p<0.05). KRT80 mRNA and protein expression was positively correlated with the differentiation of gastric cancer (p<0.05), while KRT80 methylation was negatively associated with gastric cancer differentiation and p53 mutation (p<0.05). The expression of KRT80 mRNA was positively linked to the short survival time of gastric cancers (p<0.05). The differential genes of KRT80 mRNA were involved in ligand-receptor interaction, estrogen signal pathway, peptidase, filament and cytoskeleton, keratinocyte differentiation, vitamin D receptor, muscle contraction, and B cell-mediated immunity (p<0.05). KRT80-related genes were classified into cell adhesion and junction, cadherin binding, skin and epidermis development, and so forth (p<0.05). KRT80 knockdown suppressed proliferation, anti-apoptosis, anti-pyroptosis, migration, invasion and epithelial-mesenchymal transition in gastric cancer cells (p<0.05). These findings indicated that up-regulated expression of KRT80 played a crucial part in gastric carcinogenesis, and might be considered as a biological marker for aggressive behaviors and poor prognosis. Its silencing might be used as an approach of target therapy for gastric cancer patients.

The facilitating effects of KRT80 on chemoresistance, lipogenesis, and invasion of esophageal cancer.

Keratin 80 (KRT80) is a filament protein that makes up one of the major structural fibers of epithelial cells, and involved in cell differentiation and epithelial barrier integrity. Here, KRT80 mRNA expression was found to be higher in esophageal cancer than normal epithelium by RT-PCR and bioinformatics analysis (p < .05), opposite to KRT80 methylation (p < .05). There was a negative relationship between promoter methylation and expression level of KRT80 gene in esophageal cancer (p < .05). KRT80 mRNA expression was positively correlated with the differentiation, infiltration of immune cells, and poor prognosis of esophageal cancer (p < .05). KRT80 mRNA expression was positively linked to no infiltration of immune cells, the short survival time of esophageal cancers (p < .05). The differential genes of KRT80 mRNA were involved in fat digestion and metabolism, peptidase inhibitor, and intermediate filament, desosome, keratinocyte differentiation, epidermis development, keratinization, ECM regulator, complement cascade, metabolism of vitamins and co-factor (p < .05). KRT-80-related genes were classified into endocytosis, cell adhesion molecule binding, cadherin binding, cell-cell junction, cell leading edge, epidermal cell differentiation and development, T cell differentiation and receptor complex, plasma membrane receptor complex, external side of plasma membrane, metabolism of amino acids and catabolism of small molecules, and so forth (p < .05). KRT80 knockdown suppressed anti-apoptosis, anti-pyroptosis, migration, invasion, chemoresistance, and lipogenesis in esophageal cancer cells (p < .05), while ACC1 and ACLY overexpression reversed the inhibitory effects of KRT80 on lipogenesis and chemoresistance. These findings indicated that up-regulated expression of KRT80 might be involved in esophageal carcinogenesis and subsequent progression, aggravate aggressive phenotypes, and induced chemoresistance by lipid droplet assembly and ACC1- and ACLY-mediated lipogenesis.