ZEB1-Mediated Transcriptional Upregulation of circWWC3 Promotes Breast Cancer Progression through Activating Ras Signaling Pathway.

Zinc finger E-box binding homeobox 1 (ZEB1) has been widely recognized as an important driver of tumor growth and metastasis. However, nothing is known about ZEB1-regulated circular (circ)RNAs in cancer. In the current study, we evaluated the function of a novel ZEB1-regulated circRNA derived from the WWC3 gene locus, circWWC3 in breast cancer progression. We found that ZEB1 upregulated circWWC3 expression but not the linear WWC3 mRNA expression. circWWC3 is highly expressed in breast cancer tissues and is associated with the poor prognosis of breast cancer patients. Silencing of circWWC3 significantly suppresses the proliferation, migration, and invasion of breast cancer cells. Mechanically, circWWC3 upregulates multiple oncogenes' expression of the Ras signaling pathway through acting as the sponge of microRNA (miR)-26b-3p and miR-660-3p. Moreover, short hairpin (sh)RNA-mediated knockdown of circWWC3 partially antagonized ZEB1-mediated breast cancer growth and metastasis in vivo. Our findings reveal that ZEB1-mediated upregulation of circWWC3 promotes breast cancer progression through activating Ras signaling pathway, which provides a potential therapeutic target and prognostic biomarker for breast cancer.

Identification of epithelial-mesenchymal transition-related circRNA-miRNA-mRNA ceRNA regulatory network in breast cancer.

BACKGROUND: Circular RNAs (circRNAs) have attracted lots of attention in tumorigenesis and progression. However, circRNAs as crucial regulators in epithelial-mesenchymal transition have not been systematically identified in breast cancer. The purpose of our research was to investigate the circRNA network associated with epithelial-mesenchymal transition in breast cancer. METHODS: Expression profiling data of circRNAs were identified by circRNA microarray in transfected ZEB1 and control breast cancer cells. The differentially expressed circRNAs, miRNAs, and mRNAs were determined via fold change filtering. The competing endogenous RNAs (ceRNAs) network was established on the foundation of the relationship between circular RNAs, miRNAs and mRNAs. The CytoHubba was used to determine the hub genes from the protein-protein interaction (PPI) regulatory network. The GEPIA database was used to observe the expression of the hub genes mRNA between breast cancer tissues and normal tissues. The HPA database was applied to investigate the expression of six hub genes at the protein level. Morever, we further used Kaplan-Meier plotter to perform survival analysis of these hub genes. RESULTS: The top three up-regulated differential expressed circRNAs were identified by circRNA microarray. Following the Real-time PCR validation of the three circRNAs, two circRNAs (hsa_circRNA_002082 and hsa_circRNA_400031) were selected for further analysis. After the predicted target miRNA, ten circRNA-miRNA interactions including two circRNAs and ten miRNAs were determined. Furthermore, the Venn diagram was used to intersect the predicted target genes and the differentially expressed genes, and screened 174 overlapped genes. Subsequently, we constructed a PPI network, and selecting six hub genes, containing KIF4A, CENPF, OIP5, ZWINT, DEPDC1, BUB1B. The mRNA expression levels of the six hub genes were obviously up-regulated in breast cancer. The protein expression levels of KIF4A, CENPF, OIP5, and DEPDC1 were significantly increased in breast cancer tissues. Moreover, the survival analysis results revealed that high expression of the six hub genes were obviously correlated with poor prognosis of breast cancer patients. CONCLUSIONS: Our study constructed and analyzed a circRNA-associated ceRNA regulatory network and discovered that hsa_circRNA_002082 and hsa_circRNA_400031 may mechanism as ceRNAs to serve key roles in breast cancer epithelial-mesenchymal transition.

Identification of three miRNAs signature as a prognostic biomarker in breast cancer using bioinformatics analysis.

BACKGROUND: Accumulating evidences indicated that some miRNAs are dysregulated in breast cancer and involved in cell growth, migration and invasion, differentiation, cell cycle arrest, apoptosis, and autophagy. Our study aims to identify a novel set of biomarkers for predicting the prognosis of breast cancer patients. METHODS: We downloaded clinical information and raw sequencing data from The Cancer Genome Atlas (TCGA) database. We selected samples with miRNA sequencing data and relevant clinical prognostic data for subsequent analysis. The association between miRNA and prognosis function was analyzed by Cox regression analysis. The potential biofunctions of target miRNAs were investigated through bioinformatic analysis. RESULTS: We identified 84 differentially expressed miRNAs (DEmiRNAs), among them, 17 were downregulated and 67 were upregulated. We used Kaplan-Meier survival analysis to evaluate the prognostic value of three miRNAs (mir-105-1, mir-301b and mir-1258). We also found that the three-miRNA signature is independent prognostic factors for breast cancer by using Cox regression analysis. It might be participated in different signaling pathways associated with cancer by using functional enrichment analysis, including adherens junction, autophagy, and TGF-beta signaling pathway, ErbB signaling pathway, FoxO signaling pathway. CONCLUSIONS: Taken together, three-miRNA signature might be used as a potential predicting prognostic biomarker in breast cancer.

Effect of AR antagonist combined with PARP1 inhibitor on sporadic triple-negative breast cancer bearing AR expression and methylation-mediated BRCA1 dysfunction.

Triple-negative breast cancer (TNBC) patients usually present worse clinical outcomes due to their high heterogeneity. The purpose of our study is to investigate the prognostic role of AR and BRCA1 expression in sporadic TNBC patients, and effect of AR blockade and PARP1 inhibitor for TNBC patients who characterized by positive-AR expression and BRCA1 inactivation or dysfunction. In our present study, we found that AR is expressed in 43.6% and 34.0% of TNBC tissues, when 1% or 10% staining was used as the threshold for AR positivity, respectively. When 1% staining was used as the threshold, AR expression indicates a poor disease-free survival (DFS) of TNBC patients. TNBC patients with negative BRCA1 show a poor DFS, and BRCA1 suppression is associated with the methylation status of its promoter. Interestingly, BRCA1-/AR + TNBC patients have shorter DFS than other TNBC patients regardless of the threshold for AR positivity. AR antagonists MDV3100 enhances the PARP1 inhibitor Olaparib-mediated decrease of cell viability in AR-positive/BRCA1-inactivated cells in vitro and in vivo. Our results suggested that combination of AR blockade and PARP1 inhibitor may be a potential strategy for sporadic TNBC patients who characterized by positive-AR expression and BRCA1 inactivation or dysfunction.

Impact of RUNX2 gene silencing on the gemcitabine sensitivity of p53­mutated pancreatic cancer MiaPaCa­2 spheres.

Recently, it has been well­recognized that the response toward anticancer drugs differs between two­ and three­dimensional (2D and 3D) in vitro cancer cell growth models. In the present study, we have demonstrated that, similar to the conventional 2D monolayer culture systems which often lack in vivo physiological insights, RUNX2 gene silencing increases the gemcitabine (GEM) sensitivity of the 3D spheres generated from p53­mutated pancreatic cancer MiaPaCa­2 cells. According to our results, MiaPaCa­2 cells, but not p53­wild­type pancreatic cancer SW1990 cells efficiently formed sphere structures in serum­free sphere­forming medium. Although GEM treatment caused a marked induction of TAp73/TAp63 in MiaPaCa­2 spheres accompanied by the transcriptional activation of p53 family­target genes such as p21WAF1 and NOXA, only 20% of cells underwent cell death. Under these experimental conditions, mutant p53 expression level was increased in response to GEM and RUNX2 remained unchanged at the protein level regardless of GEM exposure, which may suppress the pro­apoptotic activity of TAp73/TAp63. Notably, RUNX2 gene silencing markedly augmented GEM­mediated cell death of MiaPaCa­2 spheres compared to that of non­depleted ones. Expression analyses revealed that forced depletion of RUNX2 further stimulated GEM­induced upregulation of TAp63 as well as its downstream target genes such as p21WAF1 and NOXA. In summary, our observations strongly indicated that, similarly to 2D monolayer culture, RUNX2 gene silencing increased GEM sensitivity of MiaPaCa­2 spheres and highlighted the therapeutic potential of RUNX2 in pancreatic cancer with p53 mutation.

Depletion of runt-related transcription factor 2 (RUNX2) enhances SAHA sensitivity of p53-mutated pancreatic cancer cells through the regulation of mutant p53 and TAp63.

Suberoylanilide hydroxamic acid (SAHA) represents one of the new class of anti-cancer drugs. However, multiple lines of clinical evidence indicate that SAHA might be sometimes ineffective on certain solid tumors including pancreatic cancer. In this study, we have found for the first time that RUNX2/mutant p53/TAp63-regulatory axis has a pivotal role in the determination of SAHA sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells. According to our present results, MiaPaCa-2 cells responded poorly to SAHA. Forced depletion of mutant p53 stimulated SAHA-mediated cell death of MiaPaCa-2 cells, which was accomapanied by a further accumulation of γH2AX and cleaved PARP. Under these experimental conditions, pro-oncogenic RUNX2 was strongly down-regulated in mutant p53-depleted MiaPaCa-2 cells. Surprisingly, RUNX2 silencing augmented SAHA-dependent cell death of MiaPaCa-2 cells and caused a significant reduction of mutant p53. Consistent with these observations, overexpression of RUNX2 in MiaPaCa-2 cells restored SAHA-mediated decrease in cell viability and increased the amount of mutant p53. Thus, it is suggestive that there exists a positive auto-regulatory loop between RUNX2 and mutant p53, which might amplify their pro-oncogenic signals. Intriguingly, knockdown of mutant p53 or RUNX2 potentiated SAHA-induced up-regulation of TAp63. Indeed, SAHA-stimulated cell death of MiaPaCa-2 cells was partially attenuated by p63 depletion. Collectively, our present observations strongly suggest that RUNX2/mutant p53/TAp63-regulatory axis is one of the key determinants of SAHA sensitivity of p53-mutated pancreatic cancer cells.

MAGE-A family serves as poor prognostic markers and potential therapeutic targets for epithelial ovarian cancer patients: a retrospective clinical study.

OBJECTIVE: The aim of our study is to investigate the expression pattern and prognostic significance of melanoma-associated antigens-A (MAGE-A) family in primary epithelial ovarian cancer (EOC) patients. STUDY DESIGN: The expression of MAGE-A family members, including MAGE-A1, -A2, -A3, -A4, -A6, -A10 and -A12 was immunohistochemically detected in 82 cases of primary EOC and 10 cases of pericarcinoma ovarian tissues. The association between MAGE-A family expression and the clinicopathological parameters as well as the prognosis of primary EOC patients was analyzed. RESULTS: MAGE-A family expressed in 48.8% of primary EOC tissues, but not expressed in pericarcinoma ovarian tissues. MAGE-A expression was associated with the pathological types, FIGO stage, and pre-operative serum CA125 level. Overall survival of EOC patients with positive MAGE-A family expression was significantly shorter than those patients with negative MAGE-A expression. Multivariate analysis showed that although MAGE-A family expression can affect the overall survival, it was not an independent prognostic marker for EOC patients. CONCLUSIONS: Molecular assessment of MAGE-A family members could be helpful to improve the prognostic evaluation and to provide a new potential therapeutic target for primary EOC patients.

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53-mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63.

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations.

LIM-domain-only proteins: multifunctional nuclear transcription coregulators that interacts with diverse proteins.

The LIM-only subclass of LIM proteins is a family of nuclear transcription co-regulators that are characterized by the exclusive presence of two tandem LIM domains and no other functional domains. To date, four LIM-domain-only (LMO) proteins (LMO1-LMO4) have been identified. They regulate gene transcription by functioning as "linker" or "scaffolding" proteins with a remarkable potential to mediate protein-protein interactions. These proteins play important roles in cell fate determination, cell growth and differentiation, tissues patterning, and organ development. In this review, we briefly described the functions of LMO proteins in the organ development and diseases. We also summarized the interaction proteins of each LMO family member, which may contribute to elucidating the functions of these mysterious and important linker proteins.

Multiple MAGE-A genes as surveillance marker for the detection of circulating tumor cells in patients with ovarian cancer.

Ovarian cancer is a leading cause of death among gynecologic malignancies. In this study, we reported the expression of melanoma-associated antigens A (MAGE-A) genes in peripheral blood from 80 patients with ovarian cancer and 30 healthy donors. MAGE-As expression was associated with the factors indicating poor prognosis. The expressions of MAGE-As and each individual MAGE-A genes were also associated with low overall survival of patients with ovarian cancer. Our results suggested MAGE-A genes may have the potential to be surveillance markers for the detection of circulating tumor cells and represent a poor prognosis for patients with ovarian cancer.