Shorter Survival Times Of Patients Research Articles

Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice

Inflammation regulated by interleukin (IL) 8 promotes metastasis of hepatocellular carcinoma (HCC). The transcription factor forkhead box C1 (FOXC1) promotes metastasis by activating the epithelial to mesenchymal transition; its levels in liver tumors have been associated with shorter survival times of patients. We investigated whether FOXC1 activates inflammation signaling pathways in HCC cell lines. We performed studies in the human HCC cell lines Huh-7 and SMMC7721, as well as the metastatic cell lines MHCC97H and HCCLM3. Cell lines were incubated with IL8 and transcription of reporter genes was measured; cells were also incubated with kinase inhibitors. Levels of FOXC1 or IL8 were knocked down with small interfering messenger RNAs in Huh7 cells; cells were analyzed invitro in migration and invasion assays. To study metastasis, HCC cells were injected into flanks of BALB/C nude mice; 4 weeks later, the subcutaneous tumor fragments were collected and implanted into livers of the nude mice, and number and size tumors formed were measured. Chromatin immunoprecipitation assays were used to measure binding of transcription factors promoter regions of genes. We measured levels of FOXC1, IL8, CXCR1, and CCL2 in 2 groups of human HCC tissues collected from the Xijing or Tongji Hospitals in China (n= 690 and n= 312 samples, respectively) using immunohistochemistry. Incubation of HCC cells with IL8 led to increased expression of FOXC1, via activation of phosphoinositide 3-kinase signaling to AKT and hypoxia-inducible factor 1α. Knockdown of FOXC1 in HCC cells that overexpressed IL8 reduced the numbers of metastases formed in mice, compared with cells without FOXC1 knockdown. Transgenic overexpression of FOXC1 in HCC cells with IL8 knockdown increased the numbers of metastases formed in mice compared with cells without FOXC1 overexpression. CXCR1 and CCL2 were direct transcriptional targets of FOXC1. Knockdown of the combination of CXCR1 and CCL2 reduced the invasive activities of HCC cells that overexpress FOXC1 and formation of lung metastases in mice, and transgenic overexpression of CXCR1 increased cell's invasive and metastatic abilities after knockdown of FOXC1. Liver metastases grown from cells that overexpressed FOXC1 were infiltrated by tumor-associated macrophages, and CCL2 knockdown decreased tumor-associated macrophage infiltration; depletion of macrophages from mice significantly reduced growth of metastases by cells that overexpressed FOXC1. In human HCC tissues, level of FOXC1 correlated with levels of IL8 and CXCR1 and CCL2 and infiltration of tumors by macrophage. In multivariate analysis, detection of FOXC1 and CCL2 were independent predictors forpostoperative recurrence of HCC and overall survival. In HCC cell lines, IL8 activates expression of FOXC1 via the phosphoinositide 3-kinase signaling to AKT and hypoxia-inducible factor 1α. FOXC1 expression leads to transactivation of CXCR1 and CCL2, promoting inflammation and the invasive and metastatic abilities of HCC cells.

RNA interference against TMEM97 inhibits cell proliferation, migration, and invasion in glioma cells

Gliomas are the most common form of primary brain tumor in the adult central nervous system. Altered expression and prognostic value of transmembrane protein 97 (TMEM97) has been recently reported in different types of human tumors. However, the association of TMEM97and glioma is poorly defined. Here, we reported that TMEM97 was significantly increased in glioma tissues compared to non-tumorous brain tissues. Furthermore, TMEM97 levels were progressively increased with increasing histologic tumor grade in glioma. Higher TMEM97 expression level was correlated with shorter survival time of patients with glioma. Downregulation of TMEM97 through RNA interference inhibited cell proliferation and G1/S transition in two glioma cell lines, U87 and U373. More importantly, TMEM97 silencing induced a significant decrease in the expression of G1/S transition key regulators, cyclin D1, cyclin E, CDK2, and CDK4. Additionally, downregulation of TMEM97 in glioma cells notably repressed cell migration and cell invasion. Further analysis suggested that the decreased invasion was associated with alterations in EMT markers, including E-cadherin, β-catenin, and Twist. Since expression of TMEM97 seems to be associated with the oncogenic potential of glioma, and suppression of its expression can inhibit cancer cell growth and metastasis, TMEM97 may be a potential therapeutic target in human glioma.

CUEDC2 down-regulation is associated with tumor growth and poor prognosis in lung adenocarcinoma.

CUE domain-containing 2 (CUEDC2) is a multi-functional protein, which regulates cell cycle, growth factor signaling and inflammation. We found that CUEDC2 was low in lung adenocarcinoma cell lines and lung adenocarcinoma tissues at both mRNA and protein levels. Low levels of CUEDC2 were correlated with a shorter survival time in patients with lung adenocarcinoma (p = 0.004). CUEDC2 expression was correlated with tumor T classification (P = 0.001) at clinical stage (P = 0.001) and tumor size (P = 0.033). Multivariate analysis suggested that CUEDC2 expression is an independent prognostic indicator for patients with lung adenocarcinoma. Ectopic expression of CUEDC2 decreased cell proliferation in vitro and inhibited tumor growth in nude mice in vivo. Knockdown of endogenous CUEDC2 by short hairpin RNAs (shRNAs) increased tumor growth. Inhibition of proliferation by CUEDC2 was associated with inactivation of the PI3K/Akt pathway, induction of p21 and down-regulation of cyclin D1. Our results suggest that decreased expression of CUEDC2 contributes to tumor growth in lung adenocarcinoma, leading to a poor clinical outcome.

Abstract 891: Urinary ADAM12 levels detect the presence of pancreatic cancer

Abstract Pancreatic malignancies are the fourth leading cause of all cancer-related deaths of both men and women in the United States. Each year, approximately 32,000 new patients are diagnosed with this disease and nearly the same number die from it. A majority of patients with pancreatic malignancies, including both pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumors (pNET) present with advanced disease due to a lack of specific symptoms and current diagnostic limitations, making this disease extremely difficult to detect. The combination of poor prognosis and late presentation of pancreatic cancer patients highlights the urgent need for the development of effective, early detection strategies for this disease. Our laboratory has established a comprehensive biomarker discovery initiative whose objective is to identify proteins present in urine of cancer patients, to determine whether their presence might be relevant to disease status and stage and to validate their diagnostic and prognostic efficacy in large scale clinical trials. In the current study, our goal was to determine whether ADAM12 (a disintegrin and metalloprotease 12) could be detected in the urine of patients with pancreatic malignancies and whether ADAM12 levels might serve as an independent predictor of disease status. Retrospective analyses of urine samples (n=130) from PDAC and pNET patients as well as age- and sex-matched controls were conducted. Urinary ADAM12 levels were determined using a monospecific ELISA system. In addition, ADAM12 protein expression in tumor and normal pancreatic tissues was analyzed via immunohistochemistry (IHC). Multivariable logistic regression analyses indicated that, when controlling for age and sex, urinary ADAM12 could serve as significant independent predictor for distinguishing PDAC (P<0.001) and pNET (P<0.008) patients from healthy controls. Kaplan-Meier analysis of estimated patient survival stratified by urinary ADAM12 levels indicated a significantly shorter patient survival time for PDAC patients with high ADAM12 levels (P=0.015) compared to patients with lower urinary ADAM12. In addition, IHC analysis indicated that ADAM12 protein expression was upregulated ∼4-fold in Grade I-III PDAC compared to normal pancreatic tissue. Taken together, our results suggest that the measurement of ADAM12 levels may have diagnostic value in detection and/or clinical monitoring of disease status in patients with pancreatic malignancies. [Supported by: The Advanced Medical Foundation] Citation Format: Roopali Roy, David Zurakowski, Matthew Kulke, Marsha A. Moses. Urinary ADAM12 levels detect the presence of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 891. doi:10.1158/1538-7445.AM2014-891

Genomic Instability Causes HGF Gene Activation in Colon Cancer Cells, Promoting Their Resistance to Necroptosis

Genomic instability promotes colon carcinogenesis by inducing genetic mutations, but not all genes affected by this process have been identified. We investigated whether genomic instability in human colorectal cancer (CRC) cells produces mutations in the hepatocyte growth factor (HGF) gene. We genotyped human colon tumor tissues and adjacent nontumor tissues collected from 78 patients University of Pittsburgh Health Sciences and Veterans Hospital, along with 40 human CRC and adjacent nontumor tissues in a commercial microarray. We used cellular, biochemical, and molecular biological techniques to investigate the factors that alter HGF signaling in colon cancer cells and its effects on cell proliferation and survival. All tested human CRC tissues and cell lines that had microsatellite instability contained truncations in the regulatory deoxyadenosine tract element (DATE) of the HGF gene promoter. The DATE was unstable in 14% (11 of 78) of CRC samples; DATE truncation was also polymorphic and detected in 18% (13 of 78) of CRC tissues without microsatellite instability. In CRC cell lines, truncation of DATE activated expression of HGF, resulting in its autocrine signaling via MET. This promoted cell proliferation and resistance to necroptosis. HGF signaling via MET reduced levels of the receptor-interacting serine-threonine kinase 1, a mediator of necroptosis, in CRC cells. High levels of HGF protein in tumor tissues correlated with lower levels of receptor-interacting serine-threonine kinase 1 and shorter survival times of patients. Thirty-one percent of CRC samples contain alterations in the DATE of the HGF promoter. Disruption of the DATE increased HGF signaling via MET and reduced levels of receptor-interacting serine-threonine kinase 1 and CRC cell necroptosis. DATE alteration might be used as a prognostic factor or to select patients for therapies that target HGF-MET signaling.

TRIM59 Is Up-regulated in Gastric Tumors, Promoting Ubiquitination and Degradation of p53

Little is known about factors that promote gastric carcinogenesis. We analyzed multiple microarray data sets for messenger RNAs (mRNAs) that were increased significantly in human gastric tumor samples, compared with the adjacent normal gastric tissue. We foundexpression of tripartite motif 59 (TRIM59), which encodes a putative ubiquitin ligase, to be increased, and investigated itseffects in gastric cancer cell lines. We analyzed microarray data sets from the Oncomine database. We used quantitative polymerase chain reaction and immunoblotting to measure levels of TRIM59 mRNA and protein in50 human gastric cancer and paired normal tissues, obtained from Renji Hospital and the First Affiliated Hospital of Nanchang University, in China. We also measured protein levels in the gastric epithelial cell line GES-1; the cancer celllines MKN45, AGS, SGC7901, BGC823, Snu5, N87, and Snu1; and in tissue arrays of 108 human gastric tumors. TRIM59 was knocked down and overexpressed in gastric cancer cell lines, and the effects on proliferation, clone formation, migration, and growth of xenograft tumors in nude mice were assessed. TRIM59-related signaling pathways were examined by immunoblotting and quantitative polymerase chain reaction. We analyzed interactions among TRIM59, P53, and ubiquitin in immunoprecipitation studies. Levels of TRIM59 mRNA and protein were increased significantly in gastric tumors compared with nontumor tissues; increased levels were associated with advanced tumor stage and shorter patient survival times. TRIM59 knockdown reduced proliferation, clone formation, and migration of gastric cancer cell lines, as well as growth of xenograft tumors in nude mice; overexpression of TRIM59 had the opposite effects. TRIM59 interacted physically with P53, increasing its ubiquitination and degradation. Increased levels of TRIM59 in human gastric tumors correlated withreduced expression of P53 target genes. The putative ubiquitin ligase TRIM59 is up-regulated in human gastric tumors compared with nontumor tissues. Levels of TRIM59 correlate with tumor progression and patient survival times. TRIM59 interacts with P53, promoting its ubiquitination and degradation, and TRIM59 might promote gastric carcinogenesis via this mechanism.

Serine-arginine protein kinase 1 is associated with breast cancer progression and poor patient survival.

Breast cancer is the most common cancer and the second leading cause of mortality for women worldwide. It is necessary to identify valuable molecular markers to predict breast cancer progression in patients and treatment effect. Serine-arginine protein kinase 1 (SRPK1), a member of SR kinase family, phosphorylates the SR splicing factors which plays essential roles in normal cell development and multiple human diseases. In the current study, we wanted to explore if there are any relationships between SRPK1 expression in breast cancer and its clinical characteristics. The results showed that SRPK1 is upregulated in breast cancer cell lines and tissues at both mRNA and protein levels, measured by quantitative reverse transcriptase PCR and Western blotting. Immunohistochemical analysis showed a high expression of SRPK1 in 132 paraffin samples of patients with breast cancer; statistical analyses demonstrated that high expression of SRPK1 significantly correlated with clinical staging of patients with breast cancer (P < 0.001), TNM classification (P < 0.05). Low expression of SRPK1 leads to longer survival time, while high expression of SRPK1 leads to shorter survival time of patients. Multivariate analysis revealed that upregulation of SRPK1 might be an independent prognostic marker for the outcomes of patients with breast cancer. In conclusion, upregulation of SRPK1 might play an important role in the progression of breast cancer and might be considered as the potential diagnostic and therapeutic target to this malignancy.

ITF2 Prevents Activation of the β-Catenin–TCF4 Complex in Colon Cancer Cells and Levels Decrease With Tumor Progression

Immunoglobulin transcription factor 2 (ITF2) was believed to promote neoplastic transformation via activation of β-catenin. However, ITF2 recently was reported to suppress colon carcinogenesis. We investigated the roles of ITF2 in colorectal cancer cell lines and tumor formation and growth in mice. Levels of ITF2, β-catenin, and c-Myc were measured in 12 human colorectal tumor samples and by immunohistochemistry. ITF2 regulation of β-catenin and T-cell factor (TCF) were analyzed using luciferase reporter, reverse-transcription quantitative polymerase chain reaction, flow cytometry, and immunoblot analyses. Mice were given subcutaneous injections of human colorectal cancer cell lines that stably express ITF2, small hairpin RNAs to reduce levels of ITF2, or control plasmids; xenograft tumor growth was assessed. Human colorectal carcinoma tissue arrays were used to associate levels of ITF2 expression and clinical outcomes. Levels of β-catenin, cMyc, and ITF2 were increased in areas of human colon adenomas and carcinomas, compared with nontumor areas of the same tissues. ITF2 levels were reduced and cMyc levels were increased in areas of carcinoma, compared with adenoma. In human colorectal cancer cell lines, activation of the β-catenin-TCF4 complex and expression of its target genes were regulated negatively by ITF2. ITF2 inhibited formation ofthe β-catenin-TCF4 complex by competing with TCF4 for β-catenin binding. Stable transgenic expression of ITF2 in human colorectal cancer cell lines reduced their proliferation and tumorigenic potential in mice, whereas small hairpin RNA knockdown of ITF2 promoted growth of xenograft tumors in mice. In an analysis of colorectal tumor tissue arrays, loss of ITF2 from colorectal tumor tissues was associated with poor outcomes of patients. A gene set enrichment analysis supported the negative correlation between the level of ITF2 and activity of the β-catenin-TCF4 complex. In human colorectal cancer cell lines and tissue samples, ITF2 appears to prevent activation of the β-catenin-TCF4 complex and transcription of its gene targets. Loss of ITF2 promotes the ability of colorectal cancer cells to form xenograft tumors, and is associated with tumor progression and shorter survival times of patients.

Increased Expression of EIF5A2, Via Hypoxia or Gene Amplification, Contributes to Metastasis and Angiogenesis of Esophageal Squamous Cell Carcinoma

Solid tumors often become hypoxic, leading to activation of hypoxia-response genes. We investigated the effects of overexpression of the hypoxia response genes eIF5A2 in esophageal squamous cell carcinoma (ESCC). We used quantitative real-time polymerase chain reaction and immunohistochemistry analyses to compare expression of eIF5A2 between paired ESCC samples and nontumor esophageal tissues, and fluorescence in situ hybridization to detect gene copy-number alterations. Luciferase reporter and chromatin immunoprecipitation assays were used to study interactions between eIF5A2 and hypoxia-inducible factor-1α (HIF1α). We determined the effects of eIF5A2 overexpression and knockdown in ESCC cell lines and growth of ESCC xenograft tumors in nude mice. Levels of eIF5A2 messenger RNA and protein were increased in >40% of ESCC samples compared with matched nontumor tissues, along with levels of HIF1α and vascular endothelial growth factor. Increased levels of EIF5A2 were significantly associated with ESCC metastasis to lymph nodes (P < .001) and tissue invasion (P= .037), and shorter survival times of patients (P < .001). Amplification of eIF5A2 was detected in 35.14% of ESCC samples that overexpressed eIF5A2. Hypoxia increased expression of eIF5A2 4- to 8-fold in ESCC cell lines; we observed bidirectional regulation between eIF5A2 and HIF1α. Transient transfection of ESCC cell lines with eIF5A2 increased their migratory and invasive abilities and markers of the epithelial to mesenchymal transition, and eIF5A2 knockdown or HIFα inhibition reduced these. In mice, xenograft tumors grown from ESCC cells that expressed eIF5A2 formed tumors more rapidly than cells that expressed only vector (controls); they also expressed higher levels of HIF1α and vascular endothelial growth factor, and formed more microvessels than controls. Knockdown of eIF5A2 in ESCC cells with interfering RNAs reduced their growth as xenograft tumors in mice, particularly when mice were given docetaxel or cisplatin. eIF5A2 is overexpressed by gene amplification or hypoxia in ESCCs, and associated with up-regulation of HIF1α, metastasis, and shorter survival times of patients. Increased expression of eIF5A2 increases metastasis and angiogenesis in ESCC via the HIF1α-mediated signaling pathway.

Allele-Specific Imbalance of Oxidative Stress-Induced Growth Inhibitor 1 Associates With Progression of Hepatocellular Carcinoma

Although there are a few highly penetrant mutations that are linked directly to cancer initiation, more less-penetrant susceptibility alleles have been associated with cancer risk and progression. We used RNA sequence analysis to search for genetic variations associated with pathogenesis of hepatocellular carcinoma (HCC). We analyzed 400 paired HCC and adjacent nontumor tissues, along with clinical information, from patients who underwent surgery at Sun Yat-Sen University in Guangzhou, China. Total RNA was extracted from tissues and sequenced, and variations with allele imbalance were identified. Effects of variants on cell functions were investigated in HCC cell lines and tumor xenografts in mice. Variants were associated with patient outcomes. We found a high proportion of allele imbalance in genes related to cellular stress. A nucleotide variation in the Oxidative Stress-Induced Growth Inhibitor 1 (OSGIN1) gene (nt 1494: G-A) resulted in an amino acid substitution (codon 438: Arg-His). The variant form of OSGIN1 was specifically retained in the tumor tissues. Functional assays showed that the common form of OSGIN1 functioned as a tumor suppressor, sensitizing HCC cells to chemotherapeutic agents by inducing apoptosis. However, the variant form of OSGIN1 was less effective. It appeared to affect the translocation of OSGIN1 from the nucleus to mitochondria, which is important for its apoptotic function. The expression pattern and localization of OSGIN1 was altered in HCC specimens, compared with adjacent liver tissue. Levels of OSGIN1 messenger RNA were reduced in 24.7% of HCC specimens, and down-regulation was associated with shorter overall and disease-free survival times of patients. Patients with the OSGIN1 1494A variant had the shortest mean survival time (32.68 mo) among patient subgroups, and their tumor samples had the lowest apoptotic index. We identified OSGIN1 as a tumor suppressor that is down-regulated or altered in human HCCs. Variants of OSGIN1 detected in HCC samples reduce apoptosis and are associated with shorter survival times of patients.

Survival and Prognostic Factors for AIDS and Non-AIDS Patients with Non-Hodgkin’s Lymphoma in Bahia, Brazil: A Retrospective Cohort Study

Despite the benefits of HAART, HIV-infected patients are increasingly affected by different malignancies. We compared a 5-year-period survival time and prognostic factors for HIV-1-infected individuals diagnosed with non-Hodgkin lymphomas (NHL) in a nested case-control study, with non-HIV-infected individuals in Salvador, Brazil. Survival time and prognostic factors were compared to HIV-negative patients. 31 cases (versus 63 controls) had a significantly more advanced NHL at diagnosis and lower mean CD4 count (26 cells/mm3) than controls. Mean overall survival (OS) was 35.8 versus 75.4 months, for cases and controls, respectively (P < 0.001), while mean event-free survival time (EFS) was 34.5 months for cases, versus 68.8 for controls (P = 0.002). Higher IPI, increased LDH levels, bone marrow infiltration, lower absolute lymphocyte counts (<1,000 cells/mm3), and type B symptoms were associated with a shorter survival time for cases. Although patients without poorer prognostic factors at baseline had an OS comparable to controls, the mean CD4 cell count for cases was similar for patients with favorable and nonfavorable response to therapy. Our findings suggest that HIV-1 infection is significantly associated with a shorter survival time for patients with NHL, independently of other predictive factors and of disease stage.

Loss of DAXX and ATRX Are Associated With Chromosome Instability and Reduced Survival of Patients With Pancreatic Neuroendocrine Tumors

Sporadic pancreatic neuroendocrine tumors (pNETs) are rare and genetically heterogeneous. Chromosome instability (CIN) has been detected in pNETs from patients with poor outcomes, but no specific genetic factors have been associated with CIN. Mutations in death domain-associated protein gene (DAXX) or ATR-X gene (ATRX) (which both encode proteins involved in chromatin remodeling) have been detected in 40% of pNETs, in association with activation of alternative lengthening of telomeres. We investigated whether loss of DAXX or ATRX, and consequent alternative lengthening of telomeres, are related to CIN in pNETs. We also assessed whether loss of DAXX orATRX is associated with specific phenotypes of pNETs. We collected well-differentiated primary pNET samples from 142 patients at the University Hospital Zurich and from 101 patients at the University Hospital Bern (both located in Switzerland). Clinical follow-up data were obtained for 149 patients from general practitioners and tumor registries. The tumors were reclassified into 3 groups according to the 2010 World Health Organization classification. Samples were analyzed by immunohistochemistry and telomeric fluorescence in situ hybridization. We correlated loss of DAXX, or ATRX, expression, and activation of alternative lengthening of telomeres with data from comparative genomic hybridization array studies, as well as with clinical and pathological features of the tumors and relapse and survival data. Loss of DAXX or ATRX protein and alternative lengthening of telomeres were associated with CIN in pNETs. Furthermore, loss of DAXX or ATRX correlated with tumor stage and metastasis, reduced time of relapse-free survival, and decreased time of tumor-associated survival. Loss of DAXX or ATRX is associated with CIN in pNETs and shorter survival times of patients. These results support the hypothesis that DAXX- and ATRX-negative tumors are a more aggressive subtype of pNET, and could lead to identification of strategies to target CIN in pancreatic tumors.

MUC15 Inhibits Dimerization of EGFR and PI3K–AKT Signaling and Is Associated With Aggressive Hepatocellular Carcinomas in Patients

Aberrant expression of MUC15 correlates with development of colorectal adenocarcinoma, and MUC15 has been reported to prevent trophoblast invasion of human placenta. However, little is known about the role of MUC15 in pathogenesis of hepatocellular carcinoma (HCC). We analyzed HCC samples and matched nontumor liver tissues (controls) collected from 313 patients who underwent hepatectomy in Shanghai, China, from January 2006 through September 2009. Levels of messenger RNAs and proteins were determined by immunohistochemical, quantitative reverse transcription polymerase chain reaction, and immunoblot analyses. Statistical analyses were used to associate levels of MUC15 with tumor features and patient outcomes. Levels of MUC15 messenger RNA and protein were reduced in a greater percentage of HCC samples than control tissues. Tumors with reduced levels of MUC15 were more likely to have aggressive characteristics (eg, high levels of α-fetoprotein, vascular invasion, lack of encapsulation, and poor differentiation) than those with low levels. Patients whose tumors had reduced levels of MUC15 had shorter overall survival times (24 months vs 46 months for patients with tumors with high levels of MUC15) and time to disease recurrence. Stable expression of MUC15 in HCC cell lines (SMMC-7721 and HCC-LM3) reduced their proliferation and invasive features in vitro, and ability to form metastatic tumors in mice. MUC15 reduced transcription of the matrix metalloproteinases 2 and 7 increased expression of tissue inhibitor of metalloproteinase-2, which required phosphoinositide 3-kinase-v-akt murine thymoma viral oncogene homolog signaling. Physical interaction between MUC15 and epidermal growth factor receptor led to its relocation and degradation within early endosomes and was required for inactivation of phosphoinositide 3-kinase-v-akt murine thymoma viral oncogene homolog signaling. Reduced levels of MUC15 in HCCs are associated with shorter survival times of patients and reduced time to disease recurrence. Expression of MUC15 in HCC cells reduces their aggressive behavior in vitro and in mice by inducing dimerization of epidermal growth factor receptor and decreasing phosphoinositide 3-kinase signaling via v-akt murine thymoma viral oncogene homolog.

Identifying Ki-67 specific miRNA–mRNA interactions in malignant astrocytomas

Background: Ki-67 is an excellent indicator of glioma cell growth. However, limited information is available regarding the mechanisms underlying abnormal expression of Ki-67 in glioma tissue. The aim of this study is to identify Ki-67 specific miRNA–mRNA interactions on basis of miRNA and mRNA expression profilings. Methods: We performed a large-scale miRNA (n=829) and mRNA (n=29,421) expression profiling in primary glioblastoma multiforme (pGBM) and anaplastic astrocytoma (AA) tissues (with an aim to investigate Ki-67 related miRNAs and mRNAs). From target prediction databases, the targeting relationships between Ki-67 specific miRNAs and mRNAs were established, and functions of these mRNAs were analyzed by DAVID. The functional verifications of the candidate miRNA were also performed in LN229 cell line. Results: High expression level of Ki-67 protein predicted a shorter survival time for patients with AA. Integrated analysis of profiling data from pGBM and AA revealed 4 Ki-67 positively and 5 negatively correlated miRNAs, along with the top 12 Ki-67 positively and 2 negatively correlated mRNAs. By means of target prediction, we found that the target mRNAs employed by miR-218 were the most significant among Ki-67 specific mRNAs. Up-regulation of miR-218 was further demonstrated to reduce Ki-67 expression, promote apoptosis, and induce G0/G1 phase cell cycle arrest in LN229 cells. Conclusions: Ki-67 protein may be regulated by specific miRNA–mRNA interactions which may contribute to the proliferation of glioma cells.

Yes-Associated Protein Up-regulates Jagged-1 and Activates the NOTCH Pathway in Human Hepatocellular Carcinoma

Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear. By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients. Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer. The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).

Hypoxia-induced factor-1 alpha upregulates vascular endothelial growth factor C to promote lymphangiogenesis and angiogenesis in breast cancer patients

Hypoxia-induced factor-1 alpha (HIF-1α) affects many effector molecules and regulates tumor lymphangiogenesis and angiogenesis during hypoxia. The aim of this study was to investigate the role of HIF-1α in the regulation of vascular endothelial growth factor C (VEGF-C) expression and its effect on lymphangiogenesis and angiogenesis in breast cancer. Lymphatic vessel density (LVD), microvessel density (MVD) and the expressions of HIF-1α and VEGF-C proteins were evaluated by immunohistochemistry in 75 breast cancer samples. There was a significant correlation between HIF-1α and VEGF-C (P = 0.014, r = 0.273, Spearman's coefficient of correlation). HIF-1α and VEGF-C overexpression was significantly correlated with higher LVD (P = 0.003 and P = 0.017, respectively), regional lymph nodal involvement (P = 0.002 and P = 0.004, respectively) and advanced tumor, node, metastasis (TNM) classification (P = 0.001 and P = 0.01, respectively). Higher MVD was observed in the group expressing higher levels of HIF-1α and VEGF-C (P = 0.033 and P = 0.037, respectively). Univariate analysis showed shorter survival time in patients expressing higher levels of HIF-1α and VEGF-C. HIF-1α was also found to be an independent prognostic factor of overall survival in multivariate analysis. The results suggest that HIF-1α may affect VEGF-C expression, thus acting as a crucial regulator of lymphangiogenesis and angiogenesis in breast cancer. This study highlights promising potential of HIF-1α as a therapeutic target against tumor lymph node metastasis.

Helicobacter pylori Causes Epigenetic Dysregulation of FOXD3 to Promote Gastric Carcinogenesis

Deregulation of forkhead box (Fox) proteins, an evolutionarily conserved family of transcriptional regulators, leads to tumorigenesis. Little is known about their regulation or functions in the pathogenesis of gastric cancer. Promoter hypermethylation occurs during Helicobacter pylori-induced gastritis. We investigated whether the deregulated genes contribute to gastric tumorigenesis. We used integrative genome-wide scans to identify concomitant hypermethylated genes in mice infected with H pylori and human gastric cancer samples. We also analyzed epigenetic gene silencing in gastric tissues from patients with H pylori infection and gastritis, intestinal metaplasia, gastric tumors, or without disease (controls). Target genes were identified by chromatin immunoprecipitation microarrays and expression and luciferase reporter analyses. Methylation profile analyses identified the promoter of FOXD3 as the only genomic region with increased methylation in mice and humans during progression of H pylori-associated gastric tumors. FOXD3 methylation also correlated with shorter survival times of patients with gastric cancer. Genome demethylation reactivated FOXD3 expression in gastric cancer cell lines. Transgenic overexpression of FOXD3 significantly inhibited gastric cancer cell proliferation and invasion, and reduced growth of xenograft tumors in mice, at least partially, by promoting tumor cell apoptosis. FOXD3 bound directly to the promoters of, and activated transcription of, genes encoding the cell death regulators CYFIP2 and RARB. Levels of FOXD3, CYFIP2, and RARB messenger RNAs were reduced in human gastric tumor samples, compared with control tissues. FOXD3-mediated transcriptional control of tumor suppressors is deregulated by H pylori infection-induced hypermethylation; this could perturb the balance between cell death and survival. These findings identify a pathway by which epigenetic changes affect gastric tumor suppression.

Genomic DNA Copy-Number Alterations of the let-7 Family in Human Cancers

In human cancer, expression of the let-7 family is significantly reduced, and this is associated with shorter survival times in patients. However, the mechanisms leading to let-7 downregulation in cancer are still largely unclear. Since an alteration in copy-number is one of the causes of gene deregulation in cancer, we examined copy number alterations of the let-7 family in 2,969 cancer specimens from a high-resolution SNP array dataset. We found that there was a reduction in the copy number of let-7 genes in a cancer-type specific manner. Importantly, focal deletion of four let-7 family members was found in three cancer types: medulloblastoma (let-7a-2 and let-7e), breast cancer (let-7a-2), and ovarian cancer (let-7a-3/let-7b). For example, the genomic locus harboring let-7a-3/let-7b was deleted in 44% of the specimens from ovarian cancer patients. We also found a positive correlation between the copy number of let-7b and mature let-7b expression in ovarian cancer. Finally, we showed that restoration of let-7b expression dramatically reduced ovarian tumor growth in vitro and in vivo. Our results indicate that copy number deletion is an important mechanism leading to the downregulation of expression of specific let-7 family members in medulloblastoma, breast, and ovarian cancers. Restoration of let-7 expression in tumor cells could provide a novel therapeutic strategy for the treatment of cancer.

Compartment- and malignance-dependent up-regulation of γ-glutamyltranspeptidase and dipetidylpeptidase-IV activity in human brain gliomas.

γ-Glutamyltranspeptidase (GGT, syn. γ-Glutamyltransferase) and dipeptidylpeptidase-IV (DPP-IV) activity participates in metabolic and growth control of normal and tumor cells by processing biologically active peptides. Here, we report on up-regulation of these enzymes in human brain gliomas determined by catalytic enzyme histochemistry and immunocytochemistry. Higher activity of GGT was found in 50%, 68% and 81% of WHO grade II, III and IV tumors, respectively. The process started at/near the microvasculature, from where it spread to the parenchyma. On average, the enzyme activity in grade II, III and IV gliomas exceeded controls 2.0, 3.0 and 3.5-fold, respectively. Up-regulation of DPP-IV-like activity also started at the microvasculature, but mainly in pericytes and mononuclear-like cells around the vessels and dispersed in the parenchyma. Marked elevation of this enzyme activity, comprising also tumor parenchyma, occurred only in grade IV glioblastomas (65% patients; 3.6 times above controls) which can, therefore, help in their differentiation from grade III gliomas. The increase of total DPP-IV-like activity also included its two enzymatic homologs, the canonical DPP-IV/CD26 and FAP-1α. The increase in GGT is supposed to be a tumor grade dependent response of microvasculature and tumor astrocytes to stress induced by tissue hypoxia and/or the metabolic aberrancies. The increase in DPP-IV-like activity in high-grade tumors can be attributed to inflammatory/scavenging processes performed by the mononuclear-like cells and, in glioblastomas, also to regressive changes in the structure and function of the microvasculature and tumor parenchyma, including astrocyte stress response. The inverse relationship between DPP-IV-like activity and Ki67 in most glioblastomas and shorter survival time of patients with low activity of this enzyme also suggest its anti-oncogenic effects.

SURVIVAL AND RISK FACTORS ASSOCIATED WITH MORTALITY IN DIABETIC PATIENTS ON CONTINUOUS AMBULATORY PERITONEAL DIALYSIS IN SOUTHERN CHINA

This study aimed to analyze the survival and the risk factors associated with mortality in diabetic CAPD patients in Southern China. This single-center prospective cohort study enrolled all incident CAPD patients from Jan 2006 to Dec 2009 and followed-up until Dec 2011.Survival in diabetic and non-diabetic CAPD patients and the risk factors on mortality in the diabetic CAPD subjects were evaluated. Among 841 incident CAPD subjects in this study, 193 patients (22.9%) were diabetes. The mean vintage of CAPD was 29.4±15.5 months. Shorter patient survival time was found in diabetic patients compared to that of non-diabetic ones ( p <0.01). The 1-, 3- and 5-year patient survival rates were 90%, 64% and 40% in diabetes and 95%, 88% and 75% in non-diabetes, respectively ( p <0.01). There was no significant difference in the death-censored technique survival time between diabetic and non-diabetic patients. The 1-, 3- and 5-year technique survival rates were 96%, 88% and 86% in diabetic, while 99%, 93% and 87% in non-diabetic patients, respectively. Diabetic CAPD patients that died during the follow-up period had older age(63.4±10.5 vs. 58.6±10.4 yrs, p <0.01), higher proportion of cardiovascular diseases (CVD)(64.9% vs. 47.1%, p <0.05), higher level of hsCRP [5.1(1.5∼11.7) vs. 1.8 (0.8∼7.2)] mg/L, p <0.01), but lower levels of haemoglobin (95.5±20.2 vs. 103.4±19.2g/L, p <0.01), serum albumin (33.7±4.0 vs. 35.3±4.9g/L, p <0.05) and 24h urine output [600(300∼813) vs. 800(500∼1100]ml/d, p <0.01) compared to the survivors. The presence of CVD, advanced age, higher glycosylated haemoglobin, lower hemoglobin and serum albumin at the initiation of PD were independent predictors of death in diabetic patients.It was concluded that survival of diabetic CAPD patients is not as good as that of non-diabetic patients in Southern Chinese patients, but better than those reported by other previous studies. Better management of modifiable risk factors such as hyperglycaemia, anemia and hypoalbuminemia may improve outcomes of diabetic PD patients.