Leptin induces ovarian cancer cell invasion via overexpression of MMP7, MMP9, and upA. In addition, the key role of ERα in leptin-increased cell growth was indicated. However, the influence of ER on leptin-mediated cell invasion remains still unknown. The present study was designed to evaluate the E2-independent effect of ERα/β on leptin-mediated cell invasion and cell proliferation in ovarian cancer. We utilized SKOV3 cancer (expressing OB-Rb and ERα/β, insensitive to estrogen) and OVCAR3 (expressing OB-Rb) cell lines to show the involvement of ER in leptin-mediated effects in an E2-independent manner. MTT, BrdU, and BD matrigel invasion assays were applied to analyze cell growth, proliferation, and invasion. The siRNA approach was used to confirm the role of ERα/β in leptin effects. Moreover, western blotting and Real-time PCR were employed to detect the OB-Rb, ER, MMP9/7, and upA proteins and mRNAs. Leptin, in the absence of E2, increased ERα expression in SKOV3 cells, which was attenuated using knockdown of OB-Rb gene by siRNA. The effect of leptin on the cell growth was promoted in the presence of PPT, but not in the presence of DNP and E2, which was lost when OB-Rb siRNA was transfected. Furthermore, ERα gene silencing and/or pre-incubation with ER antagonist (ICI 182,780, 10 nM) significantly reduced cell invasion and MMP9 expression stimulated by leptin. In conclusion, our findings demonstrated that ERα, but not ERβ, is involved in leptin-induced ovarian cancer in an E2-independent manner, providing new evidence for cancer progression in obesity-associated ovarian cancer.

Carnosine is an endogenous dipeptide found in the vertebrate skeletal muscles that is usually obtained through the diet. To investigate the mechanism by which carnosine regulates the migration and intravasation of human colorectal cancer (CRC) cells, we used cultured HCT-116 cells as an experimental model in this study. We examined HCT-116 cell migratory and intravasive abilities and expression of epithelial-mesenchymal transition (EMT)-associated molecules and matrix metalloproteinases (MMPs) after carnosine treatment. The results showed that both migration and invasion were inhibited in cells treated with carnosine. We found significant decreases in Twist-1 protein levels and increases in E-cadherin protein levels in HCT-116 cells after carnosine exposure. Although plasminogen activator (uPA) and MMP-9 mRNA and protein levels were decreased, TIMP-1 mRNA and protein levels were increased. Furthermore, the cytosolic levels of phosphorylated I

OBJECTIVE: To characterize the effect of ulipristal acetate (UPA) treatment on transforming growth factor (TGF) canonical and noncanonical signaling pathways in uterine leiomyoma tissue and cells. UPA decreased extracellular matrix in surgical specimens; we characterize the mechanism in this study.
DESIGN: Laboratory study.
SETTING: University.
INTERVENTION(S): Exposure of leiomyoma cell lines to UPA.
MAIN OUTCOME MEASURE(S): RNAseq was performed on matched myometrium and leiomyoma surgical specimens of placebo- and UPA-treated patients. Changes in gene expression and protein were measured using quantitative polymerase chain reaction and western immunoblot analysis, respectively.
RESULT(S): In surgical specimen, mRNA for TGF-β3 was elevated 3.75-fold and TGFR2 was decreased 0.50-fold in placebo leiomyomas compared with myometrium. Analysis of leiomyomas from UPA-treated women by western blot revealed significant reductions of active TGF-β3 (0.64 ± 0.12-fold), p-TGFR2 (0.56 ± 0.23-fold), pSmad 2 (0.54 ± 0.04-fold), and pSmad 3 (0.65 ± 0.09-fold) compared with untreated leiomyomas. UPA treatment demonstrated statistically significant reduction in collagen 1, fibronectin, and versican proteins. Notably, there was a statistically significant increase of the extracellular matrix protein fibrillin in leiomyoma treated with UPA (1.48 ± 0.41-fold). Data from in vitro assays with physiologic concentrations of UPA supported the in vivo findings.
CONCLUSION(S): TGF-β pathway is highly up-regulated in leiomyoma and is directly responsible for development of the fibrotic phenotype. UPA attenuates this pathway by reducing TGF-β3 message and protein expression, resulting in a reduction in TGF-β canonical signaling. In addition, UPA significantly increased fibrillin protein expression, which can serve to bind inactive TGF-β complexes. Therefore, UPA inhibits leiomyoma fibrosis by decreasing active TGF-β3 and diminishing signaling through the canonical pathway.
CLINICAL TRIAL REGISTRATION NUMBER: NCT00290251.

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is an important risk factor for hepatocellular carcinoma (HCC). Despite effective antiviral therapies, the risk for HCC is decreased but not eliminated after a sustained virologic response (SVR) to direct-acting antiviral (DAA) agents, and the risk is higher in patients with advanced fibrosis. We investigated HCV-induced epigenetic alterations that might affect risk for HCC after DAA treatment in patients and mice with humanized livers.
METHODS: We performed genome-wide ChIPmentation-based ChIP-Seq and RNA-seq analyses of liver tissues from 6 patients without HCV infection (controls), 18 patients with chronic HCV infection, 8 patients with chronic HCV infection cured by DAA treatment, 13 patients with chronic HCV infection cured by interferon therapy, 4 patients with chronic hepatitis B virus infection, and 7 patients with nonalcoholic steatohepatitis in Europe and Japan. HCV-induced epigenetic modifications were mapped by comparative analyses with modifications associated with other liver disease etiologies. uPA/SCID mice were engrafted with human hepatocytes to create mice with humanized livers and given injections of HCV-infected serum samples from patients; mice were given DAAs to eradicate the virus. Pathways associated with HCC risk were identified by integrative pathway analyses and validated in analyses of paired HCC tissues from 8 patients with an SVR to DAA treatment of HCV infection.
RESULTS: We found chronic HCV infection to induce specific genome-wide changes in H3K27ac, which correlated with changes in expression of mRNAs and proteins. These changes persisted after an SVR to DAAs or interferon-based therapies. Integrative pathway analyses of liver tissues from patients and mice with humanized livers demonstrated that HCV-induced epigenetic alterations were associated with liver cancer risk. Computational analyses associated increased expression of SPHK1 with HCC risk. We validated these findings in an independent cohort of patients with HCV-related cirrhosis (n = 216), a subset of which (n = 21) achieved viral clearance.
CONCLUSIONS: In an analysis of liver tissues from patients with and without an SVR to DAA therapy, we identified epigenetic and gene expression alterations associated with risk for HCC. These alterations might be targeted to prevent liver cancer in patients treated for HCV infection.

Previous research showed that the 4 genes of matrix metallopeptidase 9 (MMP9), cyto-keratin 20 (CK20), cyto-keratin 19 (CK19) and urokinase type plasminogen activator (uPA) are detectable in the peripheral blood. All the 4 genes are related to tumor invasion and metastasis. However, whether their expression is associated with clinicopathologic factors and the prognosis of patients with esophageal squamous cell carcinoma (ESCC) is still confused. Expression levels of MMP9, CK20, CK19, and uPA were evaluated by quantificational real-time polymerase chain reaction (qRT-PCR) in peripheral blood of 205 ESCC patients who received radical resection. The cut-off value was 1000 copy numbers. Their impacts on clinicopathologic factors and survival were investigated. The uPA expression positively correlated with gender (P = .046) and tumor size (P = .046). Meanwhile, CK19 expression positively correlated with tumor size (P = .029), vascular invasion (P = .024), and CK20 expression positively correlated with tumor size (P = .035) and degrees of differentiation (P = .032). Moreover, the overexpression of MMP9 has a correlation with postoperative radiotherapy (P = .041) and chemotherapy (P = .012). Among the 4 genes, only uPA is a prognostic indicator for disease-free survival and overall survival both in univariate analysis and multivariate analysis (P = .015). This study suggests that circulating uPA mRNA in peripheral blood can serve as a potential unfavorable prognosis biomarker in ESCC. Further perspective, multi-center and large-scale study is still needed.

BACKGROUND: The aim of this study was to evaluate the prognostic potential of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) tumor tissue levels and examine the association between these biomarkers and classical prognostic factors in early node-negative luminal breast cancer patients. The clinical value of 4G/5G variants of PAI-1 gene was evaluated.
PATIENTS AND METHODS: This study involved 81 node-negative, estrogen receptor-positive and/or progesterone receptor-positive and human epidermal growth factor receptor 2-negative operable breast cancer patients who underwent radical surgical resection and received adjuvant endocrine therapy. Determination of uPA and PAI-1 concentrations in the breast cancer tissue extracts was performed using FEMTELLE® uPA/PAI-1 ELISA. An insertion (5G)/deletion (4G) polymorphism at position - 675 of the PAI-1 gene was detected by PCR-RFLP analysis.
RESULTS: Our research showed that patients with uPA tumor tissue levels higher than 3 ng/mg of protein had significantly reduced disease-free survival (DFS) and overall survival (OS) when compared to patients with uPA tumor tissue levels lower or equal to 3 ng/mg of protein. Patients with PAI-1 tumor tissue levels higher than 14 ng/mg of protein had significantly decreased OS in comparison with patients with PAI-1 tumor tissue levels lower or equal to 14 ng/mg of protein. ROC analysis confirmed the uPA and PAI-1 discriminative potential for the presence/absence of relevant events in these patients and resulted in higher cut-off values (5.65 ng/mg of protein for uPA and 27.10 ng/mg of protein for PAI-1) than standard reference cut-off values for both biomarkers. The prognostic importance of uPA and PAI-1 ROC cut-off values was confirmed by the impact of uPA higher than 5.65 ng/mg of protein and PAI-1 higher than 27.10 ng/mg of protein on poorer DFS, OS and event-free survival (EFS). We observed that patients with dominant allele in PAI-1 genotype (heterozygote and dominant homozygote, - 675 4G/5G and - 675 5G/5G) had significantly increased DFS, OS and EFS when compared with patients with recessive homozygote genotype (- 675 4G/4G).
CONCLUSION: Our study indicates that uPA and PAI-1 tumor tissue levels and 4G/5G variants of PAI-1 gene might be of prognostic significance in early node-negative luminal HER2-negative breast cancer patients treated with adjuvant endocrine therapy.

BACKGROUND: BRAF inhibitor (BRAF-I) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms behind BRAF-I responsiveness and acquired resistance is therefore an important issue. Here we assessed the role of urokinase type plasminogen activator receptor (uPAR) as a potentially valuable biomarker in the acquisition of BRAF-I resistance in V600E mutant melanoma cells.
METHODS: We examined uPAR and EGFR levels by real time PCR and western blot analysis. uPAR loss of function was realized by knocking down uPAR by RNAi or using M25, a peptide that uncouples uPAR-integrin interaction. We investigated uPAR-β1integrin-EGFR association by co-immunoprecipitation and confocal immuno-fluorescence analysis. Acquired resistance to BRAF-I was generated by chronic exposure of cells to vemurafenib.
FINDINGS: We proved that uPAR knockdown in combination with vemurafenib inhibits melanoma cell proliferation to greater extent than either treatment alone causing a decrease in AKT and ERK1/2 phosphorylation. Conversely, we demonstrated that uPAR enforced over-expression results in reduced sensitivity to BRAF inhibition. Moreover, by targeting uPAR and EGFR interaction with an integrin antagonist peptide we restored vemurafenib responsiveness in melanoma resistant cells. Furthermore, we found significant detectable uPAR and EGFR levels in tumor biopsies of 4 relapsed patients.
INTERPRETATION: We disclosed an unpredicted mechanism of reduced sensitiveness to BRAF inhibition, driven by elevated levels of uPAR and identified a potential therapeutic strategy to overcome acquired resistance.
FUNDS: Associazione Italiana Ricerca sul Cancro (AIRC); Ente Cassa di Risparmio di Firenze.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI2) has been shown to be associated with invasive phenotypes and prognosis in hepatocellular carcinoma (HCC). However, its biological roles and underlying mechanisms in invasion of HCC have not been explored. The present study aimed to address the issues.
METHODS: First, sub-lines in that PAI2 was stably overexpressed and silenced were established based on MHCC97H and BEL7402 cell lines, respectively. Wound-healing and transwell assays were applied to evaluate cell migration and invasion. Urokinase-type plasminogen activator (uPA) activity was measured using an ELISA kit. Real-time RT-PCR and western blotting were used to show gene expression at mRNA and protein levels. E2F1 expression in human specimens was determined by tissue microarray-based immunohistochemical staining.
RESULTS: The sub-lines, MHCC97H-PAI2 and BEL7402-siPAI2, were successfully established. The two sub-lines carried much lower and higher migration and invasion powers, respectively, in contrast to the controls. In MHCC97H-PAI2 sub-line, intra-medium uPA activity was significantly decreased, while RB expression was obviously elevated, compared with the controls. The BEL7402-siPAI2 sub-line presented the opposite trend. To identify the role of RB/E2F1 pathway, we transiently overexpressed E2F1 in MHCC97H-PAI2 sub-line, and largely reversed the inhibitory effects of PAI2 on cell migration and invasion, through regulating multiple matrix metalloproteinases and epithelial-mesenchymal transition. In HCC specimens, E2F1 expression was much higher in tumor than in non-tumor tissues, and was significantly related to Edmondson-Steiner grade, overall as well as tumor-free survival.
CONCLUSIONS: Our data suggest that PAI2 inhibits invasive potential of HCC cells via uPA- and RB/E2F1-related mechanisms.

Aim: The aim of this study is to identify a gene prognostic signature for the head-and-neck squamous cell carcinoma (HNSCC). HNSCC is one of the most common malignancies worldwide; however, the molecular mechanisms underlying the malignancy are unclear.
Materials and Methods: We analyzed the gene expression profiles of GSE2379, GSE53819, and GSE59102 derived from the gene expression omnibus, and the cancer genome atlas (TCGA) HNSC databases. The R software was used to identify the differentially expressed genes (DEGs) between HNSCC tissues and normal controls. Gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway, protein-protein interactions network, and survival analyses of common DEGs were also performed.
Results: A total of 52 upregulated and 31 downregulated DEGs were identified. Functional analyses demonstrated that these DEGs were mainly enriched in extracellular matrix-receptor interaction, focal adhesion, tyrosine metabolism, and cytokine-cytokine receptor interaction. According to the survival analyses, PLAU and SERPINE1 could predict the overall survival of HNSCC patients from the TCGA cohort. Multivariable Cox regression analyses showed that the PLAU and SERPINE1 were independent prognostic factors for HNSCC patients. The prediction power of this two-gene signature was evaluated through receiver operating characteristic curve analysis and achieved a better prognostic value than PLAU (area under curve 0.613 [95% confidence interval 0.569-0.656] vs. 0.577 [0.533-0.621]; P = 0.008) or SERPINE1 (0.613 [0.569-0.656] vs. 0.586 [0.541-0.629]; P = 0.043) when considered alone.
Conclusions: The study has identified a set of novel genes and pathways that play significant roles in the carcinogenesis and progression of HNSCC. This two-gene signature may prove to be a useful therapeutic target for HNSCC.

AIM: To identify and predict the competing endogenous RNA (ceRNA) networks in colorectal cancer (CRC) by bioinformatics analysis.
METHODS: In the present study, we obtained CRC tissue and normal tissue gene expression profiles from The Cancer Genome Atlas project. Differentially expressed (DE) genes (DEGs) were identified. Then, upregulated and downregulated miRNA-centered ceRNA networks were constructed by analyzing the DEGs using multiple bioinformatics approaches. DEmRNAs in the ceRNA networks were identified in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using KEGG Orthology Based Annotation System 3.0. The interactions between proteins were analyzed using the STRING database. Kaplan-Meier survival analysis was conducted for DEGs and real time quantitative polymerase chain reaction (RT-qPCR) was also performed to validate the prognosis-associated lncRNAs in CRC cell lines.
RESULTS: Eighty-one DElncRNAs, 20 DEmiRNAs, and 54 DEmRNAs were identified to construct the ceRNA networks of CRC. The KEGG pathway analysis indicated that nine out of top ten pathways were related with cancer and the most significant pathway was "colorectal cancer". Kaplan-Meier survival analysis showed that the overall survival was positively associated with five DEGs (IGF2-AS, POU6F2-AS2, hsa-miR-32, hsa-miR-141, and SERPINE1) and it was negatively related to three DEGs (LINC00488, hsa-miR-375, and PHLPP2). Based on the STRING protein database, it was found that SERPINE1 and PHLPP2 interact with AKT1. Besides, SERPINE1 can interact with VEGFA, VTN, TGFB1, PLAU, PLAUR, PLG, and PLAT. PHLPP2 can interact with AKT2 and AKT3. RT-qPCR revealed that the expression of IGF2-AS, POU6F2-AS2, and LINC00488 in CRC cell lines was consistent with the
CONCLUSION: CeRNA networks play an important role in CRC. Multiple DEGs are related with clinical prognosis, suggesting that they may be potential targets in tumor diagnosis and treatment.

Metastasis of hepatocellular carcinoma (HCC) is usually unrecognized before any pathological examination, resulting in time-taking treatment and poor prognosis. As a consequence, HCC patients usually show symptoms of depression. In order to suppress such psychiatric disorders and to facilitate better treatment outcome, antidepressants are prescribed. Up to present, information about the effect of antidepressants on HCC is still lacking. Therefore, we chose fluoxetine (FXT), one of the top five psychiatric prescriptions in the United States, together with the HepG2 cell model to explore its effect on HCC. Our study found that FXT (5 µM) increased the migratory distance of HepG2 cells by a factor of nearly 1.7 compared to control. In addition, our study also investigated the effect of genipin (GNP), which is an active compound from Gardenia jasminoides Ellis fruit (family Rubiaceae), on the FXT-induced HepG2 cells. Our study found that 30 and 60 µM GNP reduced the migratory distance by 42% and 74% respectively, compared to FXT treatment alone. Furthermore, we also found that FXT upregulated matrix metalloproteinases (MMPs) genes, increased the protein expression of MMPs, urokinase-type plasminogen activator (uPA), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), activator protein 1 (AP-1), phosphorylated mitogen-activated protein kinase (P-p38), phosphorylated protein kinase B (P-Akt), downregulated tissue inhibitor metalloproteinases (TIMPs) genes and decreased the TIMPs proteins expression whereas, GNP fully counteracted the action of FXT. Conclusively, this study has provided valuable information regarding the possible molecular mechanisms through which FXT affects the metastatic invasiveness of HepG2 cells and evidences to support that GNP counteracts such effect via the same molecular mechanisms.

Introduction: Breast cancer is the most common malignant tumor in women. On the list of causes of death immediately after lung cancer. It is a heterogeneous disease, considering the differences in morphological, cytogenetic, molecular, clinical and therapeutic aspects, so that the prognosis in a patient with the same histological grade and pathological status may vary.
Aim: In this paper we wanted to identify the correlation between the assay of the serum values of uPA-PAI-1 complexes and individual prognostic-predictive parameters, primarily with the status of estrogenic (Er), progesterogenic (PgR) and Her-2 receptors ("human epidermal growth factor).
Material and methods: The study was conducted at the Clinic for General and Abdominal Surgery, University Clinical Center of Sarajevo (CCUS), from September 2016 to April 2017. The study included 66 patients, ages 18 to 75, in whom by the needle biopsy preoperatively was pathohistologically verified primary invasive breast cancer.
Results: Two thirds of the sample were classified as invasive ductal carcinoma, similar to the percentage (68.2%) of pT2 size, and almost half in the grade G3. Lymph node status was negative in 54.5% of respondents, and positive in 31.8% of respondents. Most patients had positive estrogenic (83.3%) and progesterone receptors (62.1%). Almost 80% was Her-2 negative. The blood vessel invasion was present in 56.1%, while the neural invasion was present in less than a third of the sample (30.3%). Median values of uPA-PAI-1 complexes were 1.4 (interquartile range 0.9); almost 70% of the sample was negative for the status analysis of uPA-PAI-1 complex (<1).
Discussion: A statistically significant difference was determined in the mean values of uPA-PAI-1 complexes in subgroups according to menopausal status, tumor size, histological grade, histological type (invasive ductal carcinoma vs. invasive lobular cancer versus invasive ductal carcinoma vs. invasive lobular cancer), status axillary lymph nodes, Ki67 status (as binary variables), invasion of the blood vessels and neural invasion, as well as subgroups according to the status of expression of hormonal (estrogen and progesterone) receptors.
Conclusion: There is a statistically significant difference in the mean values of the uPA-PAI-1 complex and Her-2 receptor expression. Generally, in perspective, this would be the role played by the uPA/PAI-1 complex in breast cancer, which is that the elevated complex values have a negative prognosis and effect on survival, similar to the negative Her-2 receptor status. Complex uPA/PAI-1 is not a specific serum protein in breast cancer patients and cannot be taken as an individual prognostic-predictive marker for mass pre- or post treatment screening and prediction. Unfortunately, none of the biomarkers are able to independently and fully identify patients of the unknown stage of the disease with better or worse prognosis or to identify cases of more aggressive tumor behavior of the same stage for timely inclusion of adjuvant therapy and reduction of the risk of metastatic disease. The decision on treatment and prognosis should be the result of a combination of all diagnostic, therapeutic, pathohistological and molecular-genetic variables.

BACKGROUND: Waixenicin A, a bioactive extract of soft coral Sarcothelia edmondsoni, has been shown to be anti-neoplastic. However, its mechanisms of action remain unclear. Cancer stem cells (CSCs) and associated stemness factors are implicated in lung cancer. Here, we investigated the role of Waixenicin A on CSCs-like and metastatic lung cancer cells.
METHODS: We demonstrated and compared TRPM7 expression in the non-tumor lung tissues or bronchial epithelial 16-HBE cell line. TRPM7 was aberrantly expressed in the cancer tissues and SPCA-1, NCI-H520, SK-MES-1, A549 and 95D cell lines.
RESULTS: Increased TRPM7 expression was associated with enhanced SOX2, KLF4, and CD133, Hsp90α, uPA, and MMP2 expression in lung cancer cells. TRPM7-silencing inhibited epithelial-to-mesenchymal transition (EMT), suppressed stemness markers and phenotypes, concomitantly suppressed Hsp90α/uPA/MMP2 axis. Coincidently, Waixenicin A treatment downregulated TRPM7 and oncogenic markers; Waixenicin A also attenuated the ability of lung cancer cells to form tumorspheres, in vitro. In validation, our clinicopathological analyses showed that a higher TRPM7 expression was positively correlated with the larger tumor size (p = 0.007), positive lymph node metastasis (p = 0.005) and disease grade (p = 0.003).
CONCLUSIONS: Through its ability to inhibit Hsp90α/uPA/MMP2 signaling and suppress TRPM7 expression, we showed that Waixenicin A is a potential anticancer therapeutic agent for treating malignant lung cancer.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant cancers with a high incidence and high mortality in East Asia. Identifying biomarkers and clarifying the regulatory mechanisms of HCC are of great importance. Herein, we report the role and mechanism of activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element-binding protein family of transcription factors in HCC.
METHODS: ATF3 overexpression vector and shRNAs were transfected into HCC cancer cells to upregulate or downregulate ATF3 expression. In vitro and in vivo assays were performed to investigate the functional role of ATF3 in hepatocellular carcinoma. RNA-Seq was performed to screen the differentially expressed genes downstream of ATF3. The dual-luciferase reporter assay, chromatin immunoprecipitation (Ch-IP) analysis and functional rescue experiments were used to confirm the target gene regulated by ATF3. Tissue microarrays (TMAs) comprising 236 human primary HCC tissues were obtained and immunohistochemical staining were carried out to analyze the clinical significance of ATF3.
RESULTS: The results indicate that ATF3 significantly inhibited the proliferation and mobility of HCC cells both in vitro and in vivo. Cysteine-rich angiogenic inducer 61 (CYR61) is a key target for transcriptional regulation by ATF3. Both ATF3 and CYR61 were consistently downregulated in human HCC tissues, and their expression levels were significantly and positively correlated with each other.
CONCLUSIONS: Our findings indicate that ATF3 functions as a tumor suppressor in HCC through targeting and regulating CYR61.

Gastric cancer is one of the most fatal cancers worldwide. The incidence and death rates are still increasing for gastric cancer. Increasing studies have shown that proviral insertion in murine lymphomas 2 (PIM2) functions as critical regulator of multiple cancers. However, it remains unknown whether and how PIM2 regulates gastric cancer progression. In this study, PIM2 was increased in the gastric cancer tissues of patients. Patients with high PIM2 expression levels had significantly shorter survival than those with low PIM2 expression. PIM2 knockdown reduced proliferation, migration and invasion in vitro by up-regulating E-cadherin, and down-regulating N-cadherin and Vimentin. Knockdown of PIM2 induced apoptosis in gastric cancer cells, which was regulated by endoplasmic reticulum (ER) stress, as evidenced by the increased expression levels of Activating transcription factor (ATF) 6, ATF4, X-box- binding protein-1 (XBP-1) and C/EBP homologous protein (CHOP). In addition, our data showed that PIM2 silence induced reactive oxygen species (ROS) production, leading to the activation of c-Jun N-terminal kinase (JNK). Importantly, we found that PIM2 knockdown-induced apoptosis and ER stress could be abolished by reducing reactive oxygen species (ROS) generation. In vivo, PIM2 knockdown showed a significant reduction in SGC-7901 xenograft tumor size. In summary, our findings provided experimental evidence that PIM2 might function as an important oncogene in gastric cancer, which supplied promising target for developing new therapeutic strategy in gastric cancer.

BACKGROUND: It has been reported that enhancing the cellular levels of miR-193b as well as breast cancer-metastasis-suppressor-1 (BRMS1) protein is associated with diminished metastatic characteristics in breast cancer. In view of these facts, as a new therapeutic intervention, we employed a restoration-based strategy using both miR-193b-3p mimic and optimized BRMS1 in the context of a chimeric construct.
METHODS: miR-193b-3p and BRMS1 genes were cloned and the resulting plasmids were transfected into the MDA-MB231, MCF-7 and MCF-10A cell lines. microRNA expression levels were assessed by rea time PCR using LNA-primer and protein expression was confirmed by western blot method. Then, apoptosis, MTT, colony formation and invasion assays were carried out.
RESULTS: The expression levels of miR-146a, miR-146b and miR-373 were up-regulated, while the miR-520c, miR-335 and miR-10b were down-regulated following the exogenous BRMS1 expression. The exogenous over-expression of BRMS1 was associated with higher amounts of endogenous miR-193b-3p expression and enabled more efficient targeting of the 3'UTR of uPA. Although, miR-193b-3p and BRMS1 are individually capable of suppressing breast cancer cell growth, migration and invasion abilities, their cistronic expression was capable of enhancing the ability to repress the breast cancer cells invasion.
CONCLUSIONS: Our results collectively indicated the existence of an additive anti-metastatic effect between miR-193b-3p and BRMS1. Moreover, it has been hypothesized that the exogenous expression of a protein can effect endogenous expression of non-relevant microRNA. Our findings provide new grounds for miR-restoration therapy applications as an amenable anti-metastatic strategy.

Kaempferia parviflora (KP) is a famous medicinal plant from Thailand, and is a rich source of various kinds of methoxyflavones (MFs). Many kinds of food products such as tea, capsule, and liquor are manufactured from the rhizomes of KP. In this study, KP infusions were prepared with different brewing conditions, and the amounts of three major methoxylflavones, 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), and 3,5,7,3',4'-pentamethoxyflavone (PMF), were analyzed. The antiproliferative activities of DMF, TMF, and PMF isolated from the brewed tea samples were evaluated. TMF was discovered to be significantly effective at inhibiting proliferation of SNU-16 human gastric cancer cells in a concentration dependent manner. TMF induced apoptosis, as evidenced by increments of sub-G1 phase, DNA fragmentation, annexin-V/PI staining, the Bax/Bcl-xL ratio, proteolytic activation of caspase-3,-7,-8, and degradation of poly (ADP-ribose) polymerase (PARP) protein. Furthermore, it was found that TMF induced apoptosis via ER stress, verified by an increase in the level of C/EBP homologous protein (CHOP), glucose regulated protein 78 (GRP78), inositol-requiring enzyme 1 α (IRE1α), activating transcription factor-4 (ATF-4), and the splice isoform of X-box-binding protein-1 (XBP-1) mRNA.

BACKGROUND: In breast cancer (BC), deregulation of DNA methylation leads to aberrant expressions and functions of key regulatory genes. In our study, we investigated the relationship between the methylation profiles of genes associated with cancer invasivity and clinico-pathological parameters. In detail, we studied differences in the methylation levels between BC patients with haematogenous and lymphogenous cancer dissemination.
METHODS: We analysed samples of primary tumours (PTs), lymph node metastases (LNMs) and peripheral blood cells (PBCs) from 59 patients with sporadic disseminated BC. Evaluation of the DNA methylation levels of six genes related to invasivity, ADAM23, uPA, CXCL12, TWIST1, SNAI1 and SNAI2, was performed by pyrosequencing.
RESULTS: Among the cancer-specific methylated genes, we found lower methylation levels of the SNAI2 gene in histologic grade 3 tumours (OR = 0.61; 95% CI, 0.39-0.97; P = 0.038) than in fully or moderately differentiated cancers. We also evaluated the methylation profiles in patients with different cancer cell dissemination statuses (positivity for circulating tumour cells (CTCs) and/or LNMs). We detected the significant association between reduced DNA methylation of ADAM23 in PTs and presence of CTCs in the peripheral blood of patients (OR = 0.45; 95% CI, 0.23-0.90; P = 0.023).
CONCLUSION: The relationships between the decreased methylation levels of the SNAI2 and ADAM23 genes and cancer de-differentiation and haematogenous dissemination, respectively, indicate novel functions of those genes in the invasive processes. After experimental validation of the association between the lower values of SNAI2 and ADAM23 methylation and clinical features of aggressive BCs, these methylation profiles could improve the management of metastatic disease.

Colorectal cancer (CRC) is one of the leading causes of death by cancer worldwide. Bowel cancer screening programs enable us to detect early lesions and improve the prognosis of patients with CRC. However, they also generate a significant number of problematic polyps, e.g., adenomas with epithelial misplacement (pseudoinvasion) which can mimic early adenocarcinoma. Therefore, biomarkers that would enable us to distinguish between adenoma with epithelial misplacement (pseudoinvasion) and adenoma with early adenocarcinomas (true invasion) are needed. We hypothesized that the former are genetically similar to adenoma and the latter to adenocarcinoma and we used bioinformatics approach to search for candidate genes that might be potentially used to distinguish between the two lesions. We used publicly available data from Gene Expression Omnibus database and we analyzed gene expression profiles of 252 samples of normal mucosa, colorectal adenoma, and carcinoma. In total, we analyzed 122 colorectal adenomas, 59 colorectal carcinomas, and 62 normal mucosa samples. We have identified 16 genes with differential expression in carcinoma compared to adenoma:

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers in the world, and more molecular mechanisms should be illuminated to meet the urgent need of developing novel detection and therapeutic strategies. We analyzed the related microarray data to find the possible hub genes and analyzed their prognostic values using bioinformatics methods. The mRNA microarray datasets GSE62452, GSE15471, GSE102238, GSE16515, and GSE62165 were finally chosen and analyzed using GEO2R. The overlapping genes were found by Venn Diagrams, functional and pathway enrichment analyses were performed using the DAVID database, and the protein-protein interaction (PPI) network was constructed by STRING and Cytoscape. OncoLnc, which was linked to TCGA survival data, was used to investigate the prognostic values. In total, 179 differentially expressed genes (DEGs) were found in PDAC, among which, 130 were up-regulated genes and 49 were down-regulated. DAVID showed that the up-regulated genes were significantly enriched in extracellular matrix and structure organization, collagen catabolic and metabolic process, while the down-regulated genes were mainly involved in proteolysis, reactive oxygen species metabolic process, homeostatic process and cellular response to starvation. From the PPI network, the 21 nodes with the highest degree were screened as hub genes. Based on Molecular Complex Detection (MCODE) plug-in, the top module was formed by ALB, TGM, PLAT, PLAU, EGF, MMP7, MMP1, LAMC2, LAMA3, LAMB3, COLA1, FAP, CDH11, COL3A1, ITGA2, and VCAN. OncoLnc survival analysis showed that, high expression of ITGA2, MMP7, ITGB4, ITGA3, VCAN and PLAU may predict poor survival results in PDAC. The present study identified hub genes and pathways in PDAC, which may be potential targets for its diagnosis, treatment, and prognostic prediction.

BACKGROUND: Conventional parameters including Ki67, hormone receptor and Her2/neu status are used for risk stratification for breast cancer. The serine protease urokinase plasminogen activator (uPA) and the plasminogen activator inhibitor type-1 (PAI-1) play an important role in tumour invasion and metastasis. Increased concentrations in tumour tissue are associated with more aggressive potential of the disease. Multigene tests provide detailed insights into tumour biology by simultaneously testing several prognostically relevant genes. With OncotypeDX®, a panel of 21 genes is tested by means of quantitative real-time polymerase chain reaction. The purpose of this pilot study was to analyse whether a combination of Ki67 and uPA/PAI-1 supplies indications of the result of the multigene test.
METHODS: The results of Ki67, uPA/PAI-1 and OncotypeDX® were analysed in 25 breast carcinomas (luminal type, pT1/2, max pN1a, G2). A statistical and descriptive analysis was performed.
RESULTS: With a proliferation index Ki67 of < 14%, the recurrence score (RS) from the multigene test was on average in the low risk range, with an intermediate RS usually resulting if Ki67 was > 14%. Not elevated values of uPA and PAI-1 showed a lower rate of proliferation (average 8.5%) than carcinomas with an increase of uPA and/or PAI-1 (average 13.9%); p = 0.054, Student's t-test. When Ki67 was > 14% and uPA and/or PAI-1 was raised, an intermediate RS resulted. These differences were significant when compared to cases with Ki67 < 14% with non-raised uPA/PAI-1 (p < 0.03, Student's t-test). Without taking into account the proliferative activity, an intermediate RS was also verifiable if both uPA and PAI-1 showed raised values.
CONCLUSION: A combination of the values Ki67 and uPA/PAI-1 tended to depict the RS to be expected. From this it can be deduced that an appropriate analysis of this parameter combination may be undertaken before the multigene test in routine clinical practice. The increasing cost pressure makes it necessary to base the implementation of a multigene test on ancillary variables and to potentially leave it out if not required in the event of a certain constellation of results (Ki67 raised, uPA and PAI-1 raised).

Endoplasmic reticulum (ER) stress is an adaptive response to various stress conditions and plays emerging roles in cancer. Activating transcription factor 6 (ATF6), one of the three major ER stress transducers, has been shown to contribute to chemoresistance by altering cancer cell survival. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogene, and its expression has been correlated with the prognosis of patients with cancer. In this study, we aimed to explore the relationship between ER stress-related ATF signaling and CIP2A. We found that CIP2A expression was positively correlated with ATF6 expression by analyzing publicly available RNA sequence data of patients with colorectal cancer (The Cancer Genome Atlas, TCGA). In addition, we demonstrated that tunicamycin-induced ER stress in vitro upregulated ATF6 and CIP2A. Mechanistically, we found that ATF6 directly bound to the CIP2A promoter and induced CIP2A gene expression, which contributed to colon cancer cell survival. Furthermore, knockdown of CIP2A reduced the viability of cells under ER stress. Most importantly, immunohistochemical analysis of a tissue microarray from a colon cancer patient cohort showed that higher expression levels of ATF6 and CIP2A were associated with a trend toward poor prognosis. Taken together, our results show that ER stress-related ATF6 upregulates CIP2A and contributes to the prognosis of colon cancer. Targeting CIP2A may disrupt ER stress-mediated colon cancer cell survival and thus improve the prognosis of patients with colon cancer.

Uterine leiomyoma is a benign tumor that grows within the muscle tissue of the uterus. Ulipristal acetate (UPA) is a pre‑operative drug used to reduce the size of leiomyoma. The aim of the present study was to examine the in vitro mechanistic details of action of UPA on uterine leiomyomas. Primary cultures of leiomyoma cells were isolated from patient myomectomy specimens and incubated in the presence or absence of UPA at various concentrations. The proliferation, cell viability and doubling time properties of the treated cells were analyzed. In addition, the mRNA and protein expression levels of p21, p27, cyclin E, cyclin‑dependent kinase 2 (CDK2), matrix metalloproteinase (MMP)‑2 and MMP‑9 were examined, as well as the structure of F‑actin in the primary‑cultured leiomyoma cells. The results demonstrated that UPA exerted inhibitory effects on proliferation of primary‑cultured leiomyoma cells. Expression of p21 and p27 was upregulated, while cyclin E and CDK2 were downregulated in UPA‑treated primary‑cultured leiomyoma cells. An increased expression of MMP‑2 was observed in primary‑cultured leiomyoma cells and a leiomyoma tissue sample of a patient with previous history of UPA treatment. Furthermore, a pronounced formation of F‑actin stress fibers was observed in leiomyoma cells of the UPA‑treated patient. These data suggest that UPA treatment attenuated the proliferation of uterine fibroid cells via upregulation of p21 and p27, resulting in cell cycle delay. The findings in the current study also suggest that UPA may cause extracellular matrix constriction, leading to the shrinkage in size of the leiomyoma possibly via stimulation of MMP‑2 expression and induction of actin stress fibers.

Tetrahydroindolocarbazoles (THICZs) with versatile substituents, have been designed, synthesized, structure characterized, then investigated for their in-vitro anticancer screening, urokinase inhibition (uPA) evaluated, DNA-damage determination was further explored. Compounds 5, 8, 10 and 17 displayed the most promising antitumor activities against the breast cancer cell line as compared to the standard drug, doxorubicin with IC

BACKGROUND/AIM: Bisdemethoxycurcumin (BDMC) exhibits biological activities including anticancer and anti-metastasis in human cancer cell lines, but there is no available information to show whether BDMC suppresses cell migration and invasion of human cervical cancer cells.
MATERIALS AND METHODS: Wound-healing, migration, invasion, zymography, and western blotting assays were used to investigate the effects of BDMC on HeLa cells in vitro.
RESULTS: BDMC reduced the total viable cell number in a dose-dependent manner. The wound-healing assay show BDMC suppressed the movement of HeLa cells. Furthermore, the trans-well chamber assays showed that BDMC suppressed the cell migration and invasion. Gelatin zymograph assay showed that BDMC did not inhibit matrix metalloproteinase-2 (MMP-2) and -9 activities in vitro. However, western blotting assay showed that BDMC significantly reduced protein levels of growth factor receptor-bound protein 2 (GRB2), Ras homolog gene family, member A (Rho A), urokinase-type plasminogen activator (uPA), RAS, MMP-2, and N-cadherin but increased those of phosphor-extracellular-signal related kinase (p-ERK1/2), E-cadherin and nuclear factor-ĸB (NF-ĸB) in HeLa cells. Confocal laser microscopy assay was used to further confirm BDMC increased NF-ĸB when compared to controls.
CONCLUSION: BDMC may have potential as a novel anti-metastasis agent for the treatment of human cervical cancer.

The urokinase-type plasminogen activator receptor (uPAR), a glycoprotein localized on the cell surface with a glycosylphosphatidylinositol anchor, plays a crucial role in cell invasion, and the metastasis of several cancers, including bladder cancer, and its expression are significantly negatively correlated with patient survival rates. Apigenin, a naturally produced phytochemical compound found in fruits, vegetables, and plant leaves, has been shown to mediate a variety of cancer-metastasis-related molecules in various cancers. The effect of apigenin on uPAR expression is still unknown. In this study, we examined the effects of apigenin on IL-1β-induced uPAR expression and investigated its potential mechanisms. We discovered in this study that IL-1β could remarkably induce uPAR expression in bladder cancer T24 cells and that apigenin-inhibited IL-1β could induce uPAR expression concentration-dependently. Interestingly, NF-κB and AP-1 transcription factors were critically required for IL-1β-induced high uPAR expression. Apigenin suppressed the transcriptional activity of both AP-1 and NF-κB by inhibiting ERK1/2 and JNK signaling pathways. These results suggest that apigenin can exert anti-invasion effects by inhibiting uPAR expression via mediating (ERK1/2, JNK)/AP-1 and (ERK1/2, JNK)/NF-κB signaling pathways in human T24 cells. Our present study generated novel and valuable biological insight into anti-invasion through treatment with a small native compound.

X‑box‑binding protein 1 (XBP1) contributes to various types of cancer including breast, bladder cancer and esophageal squamous cell carcinoma. The aim of the study was to examine the metastatic role of XBP1 in oral squamous cell carcinoma (OSCC), and identify possible downstream molecules. Immunohistochemical staining was conducted on tissue microarrays comprising 96 OSCC cases to determine the expression level of XBP1 and analyze its association with metastasis, clinicopathological characteristics and survival prognosis. Compared with the adjacent normal tissues of OSCC, the expression of XBP1 was significantly increased in the tumor center and front area, and lymph nodes metastases (P<0.05). A relatively high XBP1 expression was associated with histological grades (P<0.05), advanced clinical stages (P<0.05), unfavorable 5‑year survival (P=0.027). Suppressed XBP1 expression caused a significant reduction of cell invasion capability (P<0.05). AXL and the downstream molecules, such as PI3K, MMP1, MMP3, and uPA were significantly suppressed when XBP1 expression was inhibited in OSCC cells. Once XBP1 was activated by Thapsigargin, AXL expression was restored. Moreover, aberrant AXL expression was associated with XBP1 overexpression in OSCC tissues (P<0.05). In conclusion, XBP1 is a potential target that is relevant to suppressing cell invasion and is associated with patient prognosis in OSCC.

[Despite as a major inhibitor of urokinase (uPA), paradoxically,] Plasminogen activator inhibitor-1 (PAI-1) has been validated to be highly expressed in various types of tumor biopsy tissues or plasma compared with controls based on huge clinical data bases analysis, more importantly, PAI-1 alone or in conjunction with uPA have been identified as prognostic for disease progression and relapse in certain cancer types. particularly in breast cancer. In addition to play important roles in cell adhesion, migration and invasion, PAI-1 has been reported to induce tumor vascularization and thus promote cell dissemination and tumor metastasis. Furthermore, there are many tumor promoting factors involved in the modulation of PAI-1 expression and activity, which will strengthen the pro-tumorigenic roles of PAI-1. Undoubtedly, PAI-1 may be a promising target for therapeutic intervention of specific cancer treatment. In fact, some PAI-1 inhibitors are currently being evaluated in cancer therapy, which may be developed to new antitumor agents in the future.

Serglycin is an intracellular proteoglycan that is expressed and constitutively secreted by numerous malignant cells, especially prominent in the highly-invasive, triple-negative MDA-MB-231 breast carcinoma cells. Notably, de novo expression of serglycin in low aggressive estrogen receptor α (ERα)-positive MCF7 breast cancer cells promotes an aggressive phenotype. In this study, we discovered that serglycin promoted epithelial to mesenchymal transition (EMT) in MCF7 cells as shown by increased expression of mesenchymal markers vimentin, fibronectin and EMT-related transcription factor Snail2. These phenotypic traits were also associated with the development of drug resistance toward various chemotherapy agents and induction of their proteolytic potential as shown by the increased expression of matrix metalloproteinases, including MMP-1, MMP-2, MMP-9, MT1-MMP and up-regulation of urokinase-type plasminogen activator. Knockdown of serglycin markedly reduced the expression of these proteolytic enzymes in MDA-MB-231 cells. In addition, serglycin expression was closely linked to a pro-inflammatory gene signature including the chemokine IL-8 in ERα-negative breast cancer cells and tumors. Notably, serglycin regulated the secretion of IL-8 in breast cancer cells independently of their ERα status and promoted their proliferation, migration and invasion by triggering IL-8/CXCR2 downstream signaling cascades including PI3K, Src and Rac activation. Thus, serglycin promotes the establishment of a pro-inflammatory milieu in breast cancer cells that evokes an invasive mesenchymal phenotype via autocrine activation of IL-8/CXCR2 signaling axis.

RESEARCH QUESTION: Does ulipristal acetate (UPA) modify the expression of genes related to apoptosis or the extracellular matrix in uterine myomas and are any modifications associated with a clinical response?
DESIGN: Targeted analysis of 176 apoptosis- or extracellular-matrix-related genes was conducted using polymerase chain reaction (PCR) arrays. Relevant results were validated by quantitative PCR. Four groups were established: responsive short-term (one course, n = 9), responsive long-term (two to four courses, n = 9), non-responsive (n = 9), and the control group who was not given any hormone therapy (n = 9). The clinical response was monitored by medical imagery and considered significant when volume reduction was greater than 25%.
RESULTS: Compared with untreated myomas, significant changes in expression of four genes were found in UPA-treated myomas. Gene expression of integrin subunit beta 4 was repressed by UPA treatment (fold change [FC] = -12.50, P < 0.001, q < 0.001), tenascin-C expression was downregulated in UPA-responsive patients (FC = -2.50, P = 0.010, q = 0.090), survivin was repressed in short-term UPA-responsive tumours (FC = -7.69, P < 0.001, q = 0.010), and catenin delta 2 gene expression was upregulated in non-responsive myomas (FC = +7.36, P < 0.001, q = 0.010).
CONCLUSION: This characterization provides the first molecular distinction between myomas responsive or non-responsive to UPA treatment.