Suppression of the neoplastic phenotype by transfection of phospholipase C β 3 to neuroendocrine tumor cells

The expression of phospholipase C β 3 (PLCB3) is low or absent in several neuroendocrine neoplasias. To investigate the role of PLCB3 in the neuroendocrine tumorigenesis, we transfected a PLCB3 construct to three neuroendocrine tumor cell lines with a low PLCB3 expression. The growth rate and tumorigenicity were assessed in vitro by [³H]thymidine incorporation and cell counting, in vivo, by xenografting to nude mice. In vitro, PLCB3 expressing clones showed a significant growth inhibition. The tumor weight was reduced for one of the two xenografted PLCB3-transfected cell lines and in both, a reduced number of proliferating (Ki-67 positive) cells was observed. This study implies an essential role for PLCB3 in the neuroendocrine tumorigenesis.     

Differentially expressed cDNAs in PLCbeta3-induced tumor suppression in a human endocrine pancreatic tumor cell line: activation of the human mismatch repair protein 3 gene

Phospholipase Cbeta3 (PLCB3) is located to chromosome 11q13 in the vicinity of the multiple endocrine neoplasia type1 (MEN1) gene and shows loss of expression in some neuroendocrine tumors. Transfection of PLCB3 to neuroendocrine cell lines induces growth suppression and phenotypic alterations, but the mechanisms remain unclear. To investigate the underlying events behind this tumor suppression, we performed an RT-Differential cDNA Display of total RNA from BON-1 (human endocrine pancreatic tumor cell line) transfected with PLCB3 and compared to wild type and BON-1 transfected with vector without insert. PLCB3 transfection resulted in increased expression of 4 genes and decreased of 2. The two inhibited were homologous to S100A3 and Chromogranin A. One of the four activated cDNAs could be identified as human mismatch repair protein 3 mRNA (hMSH3), and another was homologous to TIS/MA-3 mRNA (mouse topoisomerase suppressor inhibited gene/mouse apoptosis gene-3). Differential expression of these genes may contribute to the PLCB3-induced tumor suppression of neuroendocrine tumor cell lines.     

Tumor-associated antigen expression and growth requirements predict tumorigenesis in squamous cell carcinoma

Squamous cell carcinomas (SCCs) are the most common malignancies in man. While clinical specimens are theoretically ideal to study tumor development and progression, practical difficulties such as normal cell contamination, the presence of different cell types. and limited material make preclinical studies of model systems involving a homogeneous population of normal or transformed cells preferable. Tumor-associated antigens (TAAs) found on the cell surface, including integrins, mucins, cadherins, growth factor receptors, membrane bound antigens, and glycoproteins are known to play an important role in squamous carcinogenesis. We hypothesized that (1) alterations in TAA expression in vitro predict in vivo alterations, (2) analysis of a group of TAAs would provide a better indication of SCC tumorigenesis than any single marker, and (3) SCCs with independence from exogenous growth factors in vitro would demonstrate the most aggressive growth in vivo. The cell line which grew best in vitro without serum or other supplements demonstrated the most rapid tumor growth. whereas cell lines which grew only with supplements rarely formed tumors. Normal keratinocytes. eight SCC and two immortal keratinocyte cell lines were evaluated by flow cytometry for the expression of 10 cell surface markers, including alpha and beta integrins, minor blood group-related carbohydrate determinants. carcinoembryonic antigen-related proteins, E-cadherin, and GA733 (epithelial glycoprotein. epithelial cell adhesion molecule). None of the cell lines with abnormal expression of < or = 2 markers formed tumors, whereas all lines with altered expression of > or = 3 markers formed tumors. Using GA733 expression as an example, we found that altered TAA expression in vitro predicted the presence of TAA alterations in clinical specimens. In summary, in vitro independence from supplements for optimal growth and altered expression of > or = 3 cell surface markers were good predictors of SCC tumorigenesis. These findings may be useful in decreasing the need for whole animal tumorigenicity experiments.     

Silencing LINC00491 inhibits prostate cancer development through the miR-384/TRIM44 axis

Accumulating evidence has demonstrated the key role of long noncoding (lnc)RNAs in tumorigenesis. Prostate cancer (PCa) is a cancer with high mortality that requires further exploration of the underlying molecular mechanisms. In the present study, we aimed to discover novel potential biomarkers for diagnosing PCa and targeting treatment. Overexpression of the lncRNA, LINC00491, was verified in PCa tumor tissues and cell lines using the real-time polymerase chain reaction. Cell proliferation and invasion were then analyzed via the Cell Counting Kit-8, colony formation, and transwell assays in vitro, and tumor growth in vivo. The interaction of miR-384 with LINC00491, as well as TRIM44, was investigated via bioinformatics analyses, subcellular fractionation, luciferase reporter gene assays, radioimmunoprecipitation, pull-down, and western blot analyses. LINC00491 was overexpressed in PCa tissues and cell lines. LINC00491 knockdown resulted in impaired cell proliferation and invasion in vitro and decreased tumor growth in vivo. Moreover, LINC00491 acted as a sponge for miR-384 and its downstream target, TRIM44. Additionally, miR-384 expression was downregulated in PCa tissues and cell lines, and its expression was negatively correlated with LINC00491. A miR-384 inhibitor restored the inhibitory effects of LINC00491 silencing on PCa cell proliferation and invasion. LINC00491 is a tumor promoter in PCa via enhancing TRIM44 expression by sponging miR-384 to facilitate the development of PCa. LINC00491 plays a significant role in PCa and could serve as both a biomarker for early diagnosis and a novel treatment target.     

CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration

CXCL5 (epithelial neutrophil activating peptide-78) which acts as a potent chemoattractant and activator of neutrophil function was reported to play a multifaceted role in tumorigenesis. To investigate the role of CXCL5 in bladder cancer progression, we examined the CXCL5 expression in bladder cancer tissues by real-time PCR and Western blot, additionally, we used shRNA-mediated silencing to generate stable CXCL5 silenced bladder cancer T24 cells and defined its biological functions. Our results demonstrated that mRNA and protein of CXCL5 is increased in human bladder tumor tissues and cell lines, down-regulation of CXCL5 in T24 cells resulted in significantly decreased cell proliferation, migration and increased cell apoptosis in vitro through Snail, PI3K-AKT and ERK1/2 signaling pathways. These data suggest that CXCL5 is critical for bladder tumor growth and progression, it may represent a potential application in cancer diagnosis and therapy.     

Modulation of glucose transporter 1 (GLUT1) expression levels alters mouse mammary tumor cell growth in vitro and in vivo

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential.Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1.These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo.     

miR-122 Regulates Tumorigenesis in Hepatocellular Carcinoma by Targeting AKT3

MicroRNAs (miRNAs) have been implicated in the orchestration of diverse cellular processes including differentiation, proliferation, and apoptosis and are believed to play pivotal roles as oncogenes and tumor suppressors. miR-122, a liver specific miRNA, is significantly down-regulated in most hepatocellular carcinomas (HCCs) but its role in tumorigenesis remains poorly understood. Here we identify AKT3 as a novel and direct target of miR-122. Restoration of miR-122 expression in HCC cell lines decreases AKT3 levels, inhibits cell migration and proliferation, and induces apoptosis. These anti-tumor phenotypes can be rescued by reconstitution of AKT3 expression indicating the essential role of AKT3 in miR-122 mediated HCC transformation. In vivo, restoration of miR-122 completely inhibited xenograft growth of HCC tumor in mice. Our data strongly suggest that miR-122 is a tumor suppressor that targets AKT3 to regulate tumorigenesis in HCCs and a potential therapeutic candidate for liver cancer.     

Targeted Knockdown of IQGAP1 Inhibits the Progression of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo

IQGAP1 is a scaffolding protein that can regulate several distinct signaling pathways. The accumulating evidence has demonstrated that IQGAP1 plays an important role in tumorigenesis and tumor progression. However, the function of IQGAP1 in esophageal squamous cell carcinoma (ESCC) has not been thoroughly investigated. In the present study, we showed that IQGAP1 was overexpressed in ESCC tumor tissues, and its overexpression was correlated with the invasion depth of ESCC. Importantly, by using RNA interference (RNAi) technology we successfully silenced IQGAP1 gene in two ESCC cell lines, EC9706 and KYSE150, and for the first time found that suppressing IQGAP1 expression not only obviously reduced the tumor cell growth, migration and invasion in vitro but also markedly inhibited the tumor growth, invasion, lymph node and lung metastasis in xenograft mice. Furthermore, Knockdown of IQGAP1 expression in ESCC cell lines led to a reversion of epithelial to mesenchymal transition (EMT) progress. These results suggest that IQGAP1 plays crucial roles in regulating ESCC occurrence and progression. IQGAP1 silencing may therefore develop into a promising novel anticancer therapy.     

Knockdown of FRAT1 Expression by RNA Interference Inhibits Human Glioblastoma Cell Growth,Migration and Invasion

Background

FRAT1 positively regulates the Wnt/β-catenin signaling pathway by inhibiting GSK-3-mediated phosphorylation of β-catenin. It was originally characterized as a protein frequently rearranged in advanced T cell lymphoma, but has recently also been identified as a proto-oncogene involved in tumorigenesis. Our previous studies showed that FRAT1 was dramatically overexpressed in gliomas and its expression level was significantly increased along with clinicopathological grades.

Methods

In the current study, we used RT-PCR and Western blotting to assess the mRNA and protein levels of FRAT1 in three glioma cell lines. In addition, to evaluate its functional role in gliomas, we examined the effects of FRAT1 knockdown on proliferation, migration and invasion in vitro and tumor growth in vivo using glioblastoma U251 cells and RNAi.

Results

FRAT1 was highly expressed in all three glioma cell lines. RNAi-mediated down-regulation of endogenous FRAT1 in human glioblastoma U251 cells resulted in suppression of cell proliferation, arrest of cell cycle, inhibition of cell migration and invasion in vitro. Moreover, FRAT1 depletion significantly impaired tumor xenograft growth in nude mice.

Conclusions

Our results highlight the potential role of FRAT1 in tumorigenesis and progression of glioblastoma. These findings provide a biological basis for FRAT1 as a potential molecular marker for improved pathological grading and as a novel candidate therapeutic target for glioblastoma management.     

Glucocorticoids stimulate the release of a viral tumor marker (MMTV gp52) while inhibiting tumor cell growth

The influence of glucocorticoid treatments on the release of mouse mammary tumor virus (MMTV) envelope antigen (gp52) has been studied in C3H mammary tumor cell cultures and compared to treatment-mediated effects on tumor cell growth. Simultaneous assessment of extracellular viral antigen levels and tumor cell growth has indicated that both are coordinately affected by glucocorticoid treatment. While gp52 release is stimulated by treatment, this effect is accompanied by an inhibition of tumor cell growth. These stimulatory and inhibitory effects are mediated by dexamethasone (DEX) in a dose-dependent fashion, and both effects are more pronounced with the synthetic glucocorticoids DEX or triamcinolone acetonide (TA). Quantitation of media gp52 levels by RIA revealed the following hierarchy of glucocorticoid enhancement: TA greater than DEX greater than prednisolone greater than hydrocortisone greater than triamcinolone. A similar order of activity was observed in terms of inhibition of cell growth. The ability of TA to enhance gp52 release was 2.4-2.7 times greater than DEX, a previously proven stimulator of MMTV expression. Cell density of B9 mammary tumor cells was reduced 73% following 72 h of 10(-8) MTA treatment while C3H Mm5mt/cl mammary tumor cells were reduced by 53%. Hormone-mediated changes in in vitro gp52 release suggest that hormones might also influence plasma levels of MMTV gp52 as a systemic marker for the presence and status of murine mammary tumors. Coordinate stimulatory and inhibitory effects suggest that glucocorticoids may play a complex role in murine mammary tumorigenesis and subsequent mammary disease.     

T-cadherin-mediated cell growth regulation involves G2 phase arrest and requires p21(CIP1/WAF1) expression

Members of the cadherin family have been implicated as growth regulators in multiple tumor types. Based on recent studies from our laboratory implicating T-cadherin expression in mouse brain tumorigenesis, we examined the role of T-cadherin in astrocytoma growth regulation. In this report, we show that T-cadherin expression increased during primary astrocyte physiologic growth arrest in response to contact inhibition and serum starvation in vitro, suggesting a function for T-cadherin in astrocyte growth regulation. We further demonstrate that transient and stable reexpression of T-cadherin in deficient C6 glioma cell lines results in growth suppression. In addition, T-cadherin-expressing C6 cell lines demonstrated increased homophilic cell aggregation, increased cell attachment to fibronectin, and decreased cell motility. Cell cycle flow cytometry demonstrated that T-cadherin reexpression resulted in G2 phase arrest, which was confirmed by mitotic index analysis. This growth arrest was p53 independent, as T-cadherin could still mediate growth suppression in p53(-/-) mouse embryonic fibroblasts. T-cadherin-expressing C6 cell lines exhibited increased p21(CIP1/WAF1), but not p27(Kip1), expression. Lastly, T-cadherin-mediated growth arrest was dependent on p21(CIP1/WAF1) expression and was eliminated in p21(CIP1/WAF1)-deficient fibroblasts. Collectively, these observations suggest a novel mechanism of growth regulation for T-cadherin involving p21(CIP1/WAF1) expression and G2 arrest.     

Connexins as targets for cancer chemoprevention and chemotherapy

Cells within a tissue continuously interact to coordinate normal tissue functions and maintain homeostasis. Gap junctional communication (GJC), mediated by the connexin protein family, allows this type of intercellular crosstalk resulting in synchronized and cooperative tissue behavior such as cardiac contraction. In cancer, loss of these types of cell:cell interactions has been shown to facilitate tumorigenesis and enable the autonomous cell behavior associated with transformed cells. Indeed, many human tumor lines demonstrate deficient or aberrant GJC and/or loss of connexin expression. Restoration of exogenous connexin expression/GJC function is correlated with increased cell growth control both in vitro and in vivo. In support of this growth regulatory hypothesis, decreased connexin expression has been observed in situ in early human neoplasia of various organs. Additionally, genetically engineered mice lacking particular connexins (Connexins 32 or 43) exhibit increased susceptibility to radiation and chemically-induced liver and/or lung tumorigenesis. These studies strongly suggest that connexins and GJC serve a tumor suppressor role. Consistent with this proposed role, in a model cell culture system, retinoids and carotenoids up-regulate Connexin43 (Cx43) expression in direct proportion to their ability to suppress carcinogen-induced neoplastic transformation. Here, we discuss the important role of connexins and GJC in tumorigenesis and suggest the possibility of connexins as potential anti-oncogenic targets for chemoprevention and/or chemotherapy.     

Neuropeptide Y inhibits cholangiocarcinoma cell growth and invasion

No information exists on the role of neuropeptide Y (NPY) in cholangiocarcinoma growth. Therefore, we evaluated the expression and secretion of NPY and its subsequent effects on cholangiocarcinoma growth and invasion. Cholangiocarcinoma cell lines and nonmalignant cholangiocytes were used to assess NPY mRNA expression and protein secretion. NPY expression was assessed by immunohistochemistry in human liver biopsies. Cell proliferation and migration were evaluated in vitro by MTS assays and matrigel invasion chambers, respectively, after treatment with NPY or a neutralizing NPY antibody. The effect of NPY or NPY depletion on tumor growth was assessed in vivo after treatment with NPY or the neutralizing NPY antibody in a xenograft model of cholangiocarcinoma. NPY secretion was upregulated in cholangiocarcinoma compared with normal cholangiocytes. Administration of exogenous NPY decreased proliferation and cell invasion in all cholangiocarcinoma cell lines studied and reduced tumor cell growth in vivo. In vitro, the effects of NPY on proliferation were blocked by specific inhibitors for NPY receptor Y2, but not Y1 or Y5, and were associated with an increase in intracellular d-myo-inositol 1,4,5-trisphosphate and PKCα activation. Blocking of NPY activity using a neutralizing antibody promoted cholangiocarcinoma growth in vitro and in vivo and increased the invasiveness of cholangiocarcinoma in vitro. Increased NPY immunoreactivity in human tumor tissue occurred predominantly in the center of the tumor, with less expression toward the invasion front of the tumor. We demonstrated that NPY expression is upregulated in cholangiocarcinoma, which exerts local control on tumor cell proliferation and invasion. Modulation of NPY secretion may be important for the management of cholangiocarcinoma.     

microRNA-203 suppresses bladder cancer development by repressing bcl-w expression

It is increasingly clear that microRNAs (miRNAs) play an important role in many diseases, including tumorigenesis. However, the mechanisms by which miRNAs regulate bladder cancer development remain poorly understood. Here, we evaluated the expression of microRNA-203 (miR-203) in bladder cancer tissues using real-time PCR, and defined the target genes and biologically functional effect using luciferase reporter assay, flow cytometry and western blot analysis. We first verified that the expression of miR-203 was decreased in bladder cancer tissues. Moreover, ectopic expression of miR-203 promoted the apoptosis of human bladder cancer cell lines and inhibited cell proliferation, whereas its depletion increased cell growth. We further verified that miR-203 directly targeted 3'-untranslated region of the bcl-w gene, and decreased its expression in vitro and in vivo. Western blot analysis also showed that the expression level of miR-203 was negatively correlated with bcl-w level in tumor tissues. These data suggest an important role for miR-203 in the molecular etiology of bladder cancer and implicate the potential application of miR-203 in bladder cancer therapy.