Silenced Cell Lines Research Articles

Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

Ras-Association Domain Family 10 (RASSF10) is the last identified member of the RASSF family. The functional characteristics of this new gene in human cancers remain largely unclear. Here, we examined RASSF10 for the biological functions and related molecular mechanisms in hepatocellular carcinoma (HCC). We found that RASSF10 is expressed in normal human liver tissue, but is silenced or down-regulated in 62.5% (5/8) of HCC cell lines. The mean expression level of RASSF10 was significantly lower in primary HCCs compared with their adjacent normal tissues (P<0.005, n=52). The promoter methylation contributes to the inactivation of RASSF10 as demonstrated by bisulfite genomic sequencing and demethylation treatment analyses. Transgenic expression of RASSF10 in silenced HCC cell lines suppressed cell viability, colony formation and inhibited tumor growth in nude mice (QGY7703, P<0.01; HepG2, P<0.05). Furthermore, RASSF10 was shown to induce the cell accumulation in G1 phase with the increase of p27, as well as the decrease of cyclinD1 and CDK2/CDK4. Over-expression of RASSF10 also inhibited HCC cells migration (P<0.01) or invasion (P<0.05). Adhesion genes array revealed that Matrix Metalloproteinase 2 (MMP2) was a downstream effector of RASSF10. RASSF10 acting as a tumor suppressor to inhibit HCC invasion partially mediated by Focal Adhesion Kinase or p38 MAPK to decrease the accumulation of MMP2. Our study suggests that RASSF10 acts as a tumor suppressor for HCC.

Nuclear EpICD expression and its role in hepatocellular carcinoma.

Regulated intramembrane proteolysis of epithelial cell adhesion molecule (EpCAM) results in shedding of the extracellular domain (EpEX) and release of the intra-cellular domain (EpICD) into the cytoplasm. Released EpICD associates with FHL2, β-catenin and Lef-1 to form a nuclear complex and triggers oncogenic signaling. This study was conducted to examine the nuclear expression of EpICD in hepatocellular carcinoma (HCC) and to assess the role of EpICD in HCC. EpICD immunoexpression was examined in 100cases of HCC using tissue microarrays and correlated with clinicopathological parameters. We also examined the role of EpICD in HCC using EpICD cDNA transfected HCC cell line and EpCAM silenced HCC cell line by small interfering RNA (siRNA). Nuclear expression of EpICD was observed in 19 of 100 (19%) cases. Nuclear expression of EpICD significantly correlated with nuclear expression of β-catenin, and Ki-67 labeling index. In addition, nuclear expression of EpICD was associated with higher histologic grade and advanced Tcategory. Forced overexpression of EpICD in the HCC cell significantly increased the cell proliferation, migration and invasion. The overexpression of EpICD also increased the expression levels of the active form of β-catenin and c-myc and cyclinD1. In contrast, downregulation of EpCAM by siRNA decreased the cell proliferation, migration, invasion and the expression of active form of β-catenin, c-myc and cyclinD1. Our present data suggest that EpICD plays important roles in HCC progression by modulating expression of target genes of EpCAM.

Epithelial membrane protein 2 regulates sphingosylphosphorylcholine-induced keratin 8 phosphorylation and reorganization: Changes of PP2A expression by interaction with alpha4 and caveolin-1 in lung cancer cells

Sphingosylphosphorylcholine (SPC) is found at increased in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. However, the detailed mechanism of SPC-induced K8 phosphorylation and reorganization is not clear.We observed that SPC dose-dependently reduced the expression of epithelial membrane protein 2 (EMP2) in lung cancer cells. Then, we examined the role of EMP2 in SPC-induced phosphorylation and reorganization of K8 in lung cancer cells. We found that SPC concentration-dependently reduced EMP2 in A549, H1299, and other lung cancer cells. This was verified at the mRNA level by RT-PCR and real-time PCR (qPCR), and intracellular variation through confocal microscopy. EMP2 gene silencing and stable lung cancer cell lines established using EMP2 lentiviral shRNA induced K8 phosphorylation and reorganization. EMP2 overexpression reduced K8 phosphorylation and reorganization. We also observed that SPC-induced loss of EMP2 induces phosphorylation of JNK and ERK via reduced expression of protein phosphatase 2A (PP2A). Loss of EMP2 induces ubiquitination of protein phosphatase 2A (PP2A). SPC induced caveolin-1 (cav-1) expression and EEA1 endosome marker protein but not cav-2. SPC treatment enhanced the binding of cav-1 and PP2A and lowered binding of PP2A and alpha4. Gene silencing of EMP2 increased and gene silencing of cav-1 reduced migration of A549 lung cancer cells.Overall, these results suggest that SPC induces EMP2 down-regulation which reduces the PP2A via ubiquitination induced by cav-1, which sequestered alpha4, leading to the activation of ERK and JNK.

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering β‐catenin signalling and GADD45G expression

Recent studies suggest that paired box 5 (PAX5) is down‐regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non‐small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down‐regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non‐cancerous tissues (P = 0.0201). PAX5 down‐regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down‐regulation of β‐catenin and up‐regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down‐regulating the β‐catenin pathway and up‐regulating GADD45G expression.

Verteporfin can Reverse the Paclitaxel Resistance Induced by YAP Over-Expression in HCT-8/T Cells without Photoactivation through Inhibiting YAP Expression

Background/Aims: Paclitaxel (PTX) is one of the most effective anti-cancer drugs. However, multiple drug resistance is still the main factor that hinders the effective treatment of cancer with PTX. Several factors including YAP over-expression can cause PTX resistance. In this study, we aimed to verify the role YAP plays in PTX resistance, explore the reversal of PTX resistance by verteporfin (VP) and investigate the effect of combination therapy of PTX and VP on the PTX resistant colon cancer cells (HCT-8/T). Methods: To study the relationship between YAP and PTX resistance, a stable YAP-over-expression or YAP silencing cell line was generated by transfected with YAP-plasmids or siYAP-RNA. WST-1 assay was performed to detect the cytotoxicity of PTX on HCT-8 and HCT-8/T cells. Clone formation assay and Transwell assay was preformed to determine the cell proliferation and invasion ability respectively. Immunofluorescence and Western blot analysis was performed for protein detection. Results: YAP was stronger expressed in HCT-8/T than in HCT-8, and PTX resistance was positively correlated with the level of YAP expression. VP, a strongly YAP inhibitor, could reduce the PTX resistance on HCT-8/T cells without light activation by inhibiting YAP. Beside, VP and PTX combination therapy showed synergism on inhibition of YAP and cytotoxicity to HCT-8/T. Moreover, verteporfin and PTX combination therapy affect the invasion and colony formation ability and induce apoptosis of HCT-8/T cells. Conclusions: VP can reverse the PTX resistance induced by YAP over-expression in HCT-8/T cells without photoactivation through inhibiting YAP expression.

Investigation of the inflammatory cell migration process in familial Mediterranean fever

Familial Mediterranean fever (FMF) is one of the most common autoinflammatory disorders and is characterized by episodic attacks of fever, along with inflammation. FMF pathogenesis is associated with various mutations in the MEFV gene, which encodes pyrin. Pyrin is expressed predominantly in neutrophils that have an important role in the innate immune response. Several proteins related with actin machinery have been identified as pyrin-interacting proteins in our in vitro cell migration models. Thus, in this study, we hypothesized that pyrin may have a key role in neutrophil migration during inflammation and decided to do functional analysis on pyrin silenced cell lines and primary neutrophils.

P2X7 receptor as predictor gene for glioma radiosensitivity and median survival

Glioblastoma multiforme (GBM) is considered the most lethal intracranial tumor and the median survival time is approximately 14 months. Although some glioma cells present radioresistance, radiotherapy has been the mainstay of therapy for patients with malignant glioma. The activation of P2X7 receptor (P2X7R) is responsible for ATP-induced death in various cell types. In this study, we analyzed the importance of ATP-P2X7R pathway in the radiotherapy response P2X7R silenced cell lines, in vivo and human tumor samples. Both glioma cell lines used in this study present a functional P2X7R and the P2X7R silencing reduced P2X7R pore activity by ethidium bromide uptake. Gamma radiation (2Gy) treatment reduced cell number in a P2X7R-dependent way, since both P2X7R antagonist and P2X7R silencing blocked the cell cytotoxicity caused by irradiation after 24h. The activation of P2X7R is time-dependent, as EtBr uptake significantly increased after 24h of irradiation. The radiotherapy plus ATP incubation significantly increased annexin V incorporation, compared with radiotherapy alone, suggesting that ATP acts synergistically with radiotherapy. Of note, GL261 P2X7R silenced-bearing mice failed in respond to radiotherapy (8Gy) and GL261 WT-bearing mice, that constitutively express P2X7R, presented a significant reduction in tumor volume after radiotherapy, showing in vivo that functional P2X7R expression is essential for an efficient radiotherapy response in gliomas. We also showed that a high P2X7R expression is a good prognostic factor for glioma radiosensitivity and survival probability in humans. Our data revealed the relevance of P2X7R expression in glioma cells to a successful radiotherapy response, and shed new light on this receptor as a useful predictor of the sensitivity of cancer patients to radiotherapy and median survival.

Abstract 4063: The oncogenic cell cycle-related kinase promotes tumor immune escape via modulation of myeloid-derived suppressor cells in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a sexually dimorphic cancer significantly associated with elevated levels of male sex hormone androgen and pro-inflammatory cytokine interleukin-6 (IL-6) (ref 1). Our previous genome-wide location and functional analysis has pinpointed cell cycle-related kinase (CCRK) as a critical mediator of androgen receptor (AR) oncogenic activity in HCC through a self-reinforcing circuitry involving β-catenin and the Polycomb repressor enhancer of zeste 2 (EZH2) (ref 2-4). As these key circuit players have also been shown to trigger inflammatory response and aggressiveness of HCC cells (ref 5,6), we aimed to investigate whether CCRK modulates tumor microenvironment during hepatocarcinogenesis. Expression profiling of 16 pro-inflammatory cytokines and chemokines revealed that ectopic expression of wild-type but not kinase-defective CCRK significantly induced the production of IL-6, IL-1β, tumor necrosis factor-α and cyclooxygenase 2 in immortal hepatic and HCC cells. Rescue experiments showed that CCRK-induced IL-6 production was dependent on EZH2 up-regulation and subsequent nuclear factor-κB activation. Based on the causal link between IL-6 and myeloid-derived suppressor cells (MDSCs), we next examined the relationship between CCRK and MDSCs in vitro and in vivo. By co-culturing CCRK-expressed or silenced hepatic and HCC cell lines with peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, we found that CCRK stimulated the expansion of Lin-CD33+CD11b+HLA-DR-MDSCs from PBMCs by flow cytometry analysis. The induced MDSCs also showed higher suppressive activity against CD3+ T cell proliferation. In a high-fat diet-fed mouse model exposed to low-dose carcinogen diethylnitrosamine, administration of lentivirus expressing short-hairpin (sh)RNA against Ccrk significantly reduced &amp;gt;70% obesity-promoted tumor multiplicity and size compared to mice treated with control shRNA (p&amp;lt;0.01). Notably, Ccrk down-regulation also significantly reduced the population of circulating CD11b+Gr-1intMDSCs in the mice (p&amp;lt;0.05). In summary, our findings demonstrate that the oncogenic CCRK in hepatic cells can promote MDSC expansion and suppressive functions possibly via IL-6 production. Such tumor-stromal cell interaction provides survival advantage of cancer cells via immune escape. In conclusion, CCRK not only functions as a signaling hub but also an immunoregulator, thus representing a new cancer immunotherapy target.

Abstract 1233: Impairment of TFF3 induces cellular apoptosis in prostate cancer cell lines and overexpression of TFF3 may be a prognostic biomarker in human prostate cancers

Abstract Background: Human trefoil factor 3(TFF3)/ Intestinal trefoil factor(ITF) is originally known well as an essential regulator for epithelial restitution in gastrointestinal tract and also suggested as a potential cancer marker in serum. Furthermore, the aberrant overexpression of TFF3 has been reported in various human cancers, majorly colorectal cancers, suggesting that TFF3 may play a critical role in tumor growth. However, its molecular signaling pathway remains unclear. Objective: To clarify whether TFF3 is related to survival/anti-apoptotic cell signaling pathways, and to determine that tff3 could be a molecular marker in prostate cancers. Design, Setting, and Participants: TFF3 gene was silenced by using siRNA in prostate cancer two kinds of cell lines, LNCap and PC3, following investigation of genes related to apoptosis and cell migration. The expression of TFF3 was analyzed by immunohistochemistry in 109 prostate cancer and 106 normal prostate tissues. Results and Limitations: TFF3 silenced prostate cancer cell lines exhibited increased annexin V binding and the activation of caspase-9 and caspase-3 which are crucial for programmed cell death, consistent with an increased rate of apoptosis, reduced rate of proliferation and migration. In human prostate cancer tissue microarray, TFF3 was positive in 54.7% (49.1%, 2+; 5.6%, 1+) of cancer cases (59/108) and 2.8% (1.9%, 2+; 0.9%, 1+) of morphologically normal tissue cases (3/106), P&amp;lt;0.0001. Conclusions: The results suggest that TFF3 expression seems play a role in promoting cell survival and be a potential useful marker in prostate cancers. In these contexts, TFF3 in human prostate cancers deserves further investigation to exploit as a cancer marker and anticancer drug. Citation Format: So-Youn Kim, Ji-Ying Liu, Yeun-Jun Chung. Impairment of TFF3 induces cellular apoptosis in prostate cancer cell lines and overexpression of TFF3 may be a prognostic biomarker in human prostate cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1233. doi:10.1158/1538-7445.AM2015-1233

Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target.

This study investigated the epigenetic alteration and biological function of the pro-apoptotic gene ASC/TMS1 in renal cell carcinoma. ASC/TMS1 was downregulated in five out of six RCC cell lines. A significant downregulation was also detected in sixty-seven paired renal tumors compared with adjacent non-cancerous tissues. The downregulation of ASC/TMS1 was correlated with promoter hypermethylation and could be restored with demethylation treatment. Re-expression of ASC/TMS1 in silenced RCC cell lines inhibited cell viability, colony formation, arrested cell cycle, induced apoptosis, suppressed cell invasion and repressed tumorigenicity in SCID mice. The antitumorigenic function of ASC/TMS1 in renal cancer was partially regulated by activation of p53 and p21 signaling. In addition, restoration of ASC/TMS1 sensitizes RCC cells to DNA damaging agents. Knockdown of ASC/TMS1 reduced DNA damaging agents-induced p53 activation and cell apoptosis. Moreover, ASC/TMS1 hypermethylation was further detected in 41.1% (83/202) of RCC tumors, but only 12% in adjacent non-cancerous tissues. ASC/TMS1 methylation was significantly correlated with higher tumor nuclear grade. In conclusion, ASC/TMS1 is a novel functional tumor suppressor in renal carcinogenesis. ASC/TMS1 tumor specific methylation may be a useful biomarker for designing improved diagnostic and therapeutic strategies for RCC.

LRG1 modulates invasion and migration of glioma cell lines through TGF-β signaling pathway

Studies have shown that the abnormal expression of leucine-rich α2 glycoprotein 1 (LRG1) is associated with multiple malignancies, yet its role in glioma pathology remains to be elucidated. In this study, we investigated the role of LRG1 in regulating proliferation, migration and invasion of glioma cells by establishing glioma cell strains with constitutively silenced or elevated LRG1 expression. LRG1 overexpression and silenced cell lines demonstrated modulation of glioma cellular proliferation, migration and invasion through MTT, cell scratching and Transwell assays. Furthermore, overexpression of LRG1 led to augmented activation of transforming growth factor-β (TGF-β) signaling pathway as well as downregulation of E-cadherin and resultant enhanced invasiveness, which was reversed by TGF-β signaling pathway inhibitor SB431542. In summary, our findings suggest that LRG1 promotes invasion and migration of glioma cells through TGF-β signaling pathway.

Downregulation of ARNT2 promotes tumor growth and predicts poor prognosis in human hepatocellular carcinoma.

Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is a transcriptional regulator and member of the basic helix-loop-helix/Per-ARNT-SIM (bHLH/PAS) superfamily. Recently, evidence of that ARNT is involved in carcinogenesis and cancer progression has emerged. The aim of current study was to investigate the role of ARNT2, a homolog of ARNT, in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC). Tissue microarray and immunohistochemical staining were used to examine the expression of ARNT2 in 195 HCC tissues. Factors associated with ARNT2 levels were assessed by univariate and multivariate Cox regression analyses. Cell proliferation, migration, and invasion assays were performed by using ARNT2 silencing and overexpressing HCCLM6 cell line. Orthotopic xenograft HCC model was used to elucidate the effects of ARNT2 on HCC progression in vivo. High intratumoral of ARNT2 level was well correlated with longer overall survival (OS) and lower tumor to recurrence (TTR) of HCC patients after resection. Multivariate analysis revealed that intratumoral ARNT2 overexpression was an independent prognostic factor for both OS and TTR. Knockdown of ARNT2 in HCCLM6 cells was significantly enhanced while overexpression of ARNT2 significantly inhibited the ability of cell proliferation, invasion, and migration. In animal studies, downregulation of ARNT2 in HCCLM6 cells promoted, whereas upregulation of ARNT2 in HCCLM6 cells reduced HCCLM6 growth in vivo. Our data demonstrate that ARNT2 plays an inhibitory role in HCC progression and suggest that ARNT2 may be a potential prognostic predictor and therapeutic target for HCC.

Effect of miR-20b on Apoptosis, Differentiation, the BMP Signaling Pathway and Mitochondrial Function in the P19 Cell Model of Cardiac Differentiation In Vitro

ObjectiveTo explore the effect of miR-20b on apoptosis, differentiation, the BMP signaling pathway and mitochondrial function in the P19 cell model of cardiac differentiation in vitro.MethodsA miR-20b over-expression vector, a miR-20b silencing vector and their corresponding empty vectors were constructed and transfected into P19 cells, separately. Stably miR-20b overexpressing and silenced P19 cell lines were successfully selected by blasticidin and puromycin, separately. The cells were induced to undergo apoptosis in FBS-free-α-MEM. The induced cells were examined by flow cytometry and measurement of their caspase-3 activities. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the relative expression of marker genes of cardiomyocytes during differentiation, such as cTnT, GATA4 and ANP. QRT-PCR was also used to detect the mitochondrial DNA (mtDNA) copy number. We investigated the cellular ATP production using a luciferase-based luminescence assay. The reactive oxygen species (ROS) was determined by DCFDA (2’, 7’-Dichlorofluorescein diacetate) and the mitochondrial membrane potential (MMP) was elucidated by a JC-1 fluorescent probe, both using fluorescence microscopy and flow cytometer. The expression of BMP signaling pathway-related proteins were analyzed by Western blotting.ResultsStably miR-20b overexpressing and silenced P19 cell lines were successfully obtained. MiR-20b overexpression increased apoptosis and promoted differentiation in P19 cells by promoting the activation of the BMP signaling pathway. In addition, miR-20b overexpression induced mitochondrial impairment in P19 cells during differentiation, which was characterized by lower MMP, raised ATP synthesis and increased ROS levels. The effects of miR-20b silencing were the exact opposite to those of overexpression.ConclusionCollectively, these results suggested that miR-20b was very important in apoptosis, differentiation and mitochondrial function of P19 cells. MiR-20b may represent a new therapeutic target for congenital heart diseases and provide new insights into the mechanisms of cardiac diseases.

Catechol-O-methyltransferase, a new target for pancreatic cancer therapy.

Catechol-O-methyltransferase (COMT) is an important molecule in different types of cancers. Its biological effect and therapeutic significance, however, rarely been investigated fully in pancreatic cancer. Immunohistologically, high COMT expression was significantly correlated with the longer overall survival of patients (P < 0.05), indicating its protective nature. The effects of COMT on cell growth, apoptosis, and invasion were evaluated using overexpression and silencing methods. In detail, we carried out experiments using one stably transduced and two transiently transfected pancreatic cancer cell lines in vitro, and one stably transduced cell line in vivo mice xenograft models. In vitro experiments showed that COMT inhibited cell proliferation, enhanced gemcitabine-induced apoptosis, and inhibited cell invasion in stably transduced and transiently transfected cell lines by regulating the PI3K/Akt pathway, p53, and E-cadherin. The COMT overexpressed and silenced cell lines showed significantly inhibited and enhanced growth capacities in in vivo xenograft models, respectively. In conclusion, COMT suppressed pancreatic cancer and its high expression predicted longer survival time. The interaction of COMT with the PI3K/Akt pathway makes it a potential target for therapy.

ECRG4 acts as a tumor suppressor and as a determinant of chemotherapy resistance in human nasopharyngeal carcinoma.

Human nasopharyngeal carcinoma (NPC) is a malignant type of cancer with an increasing incidence. As yet, however, molecular biomarkers with a strong diagnostic impact and a major therapeutic promise have remained elusive. Here, we identified the esophageal carcinoma related gene 4 (ECRG4) as a novel candidate tumor suppressor gene and a promising therapeutic target for NPC. RT-PCR, Western blotting, methylation-specific PCR and bisulfite sequencing were performed to assess the expression and methylation status of the ECRG4 gene in primary NPC samples, NPC-derived cell lines and patient-derived peripheral blood samples. The NPC-derived cell line CNE1 was selected for treatment with a methylation inhibitor to restore ECRG4 expression. In addition, cell proliferation, invasion and colony formation assays were performed to assess the inhibitory effects of exogenous ECRG4 expression in CNE1 cells. Down-regulated ECRG4 expression was found to occur in 82.5% (33/40) of the primary NPC biopsies tested. This down-regulation was significantly correlated with its tumor-specific promoter methylation status (72.5%, 29/40) and was also observed in the matching peripheral blood samples from the NPC patients (57.5%, 23/40). Pharmacologic demethylation through 5-aza-dC treatment led to gene reactivation in ECRG4 methylated and silenced NPC cell lines. Moreover, exogenous expression of ECRG4 in the CNE1 cell line strongly inhibited its growth and invasive capacities, as well as its enhanced chemosensitivity to cisplatin through autophagy induction. Our data suggest that methylation-mediated suppression of the ECRG4 gene occurs frequently in NPC and that restoration of its expression may have therapeutic benefits.

Mucin1 mediates autocrine transforming growth factor beta signaling through activating the c-Jun N-terminal kinase/activator protein 1 pathway in human hepatocellular carcinoma cells

In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-β) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-β signaling. To investigate the effect of MUC1 on TGF-β signaling, we determined TGF-β secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-β1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-β promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-β receptor (TβR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-β signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-β secretion and exogenous TGF-β1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TβR inhibitor or small interfering RNA silencing of TGF-β1 gene in HCC cells. The high correlation between MUC1 and TGF-β1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-β signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy.

Abstract B58: The novel MDM2 inhibitor RG7388 is highly effective against neuroblastoma in vitro and in vivo

Abstract Introduction: Novel less toxic therapeutic approaches to neuroblastoma therapy are urgently needed to improve survival and reduce treatment related side effects. As neuroblastoma is nearly uniformly p53 wild-type, research efforts have focused on activating innate p53-mediated apoptotic mechanisms, specifically through the inhibition of MDM2, the primary inhibitor of p53. The compound RG7388 has proven effective against adult tumors and is under consideration for clinical trials in pediatrics. Here we demonstrate the efficacy of this new class of MDM2 inhibitors against p53 wild-type neuroblastoma in vitro and in vivo orthotopic models of neuroblastoma. Methods: The human neuroblastoma cell lines, NGP, SH-SY5Y, LAN5 and LAN5 si-p53 (p53 silenced by shRNA), were utilized for in vitro and in vivo studies. Using graded concentrations of RG7388 (0 nM - 1500 nM), cell viability was measured by MTS assay. Apoptosis with RG7388 (0 nM, 100 nM, 500 nM) was measured using a flow cytometric annexin assay. In vivo effect of RG7388 was assessed in an orthotopic mouse model of neuroblastoma, in which neuroblastoma cells were intrarenally implanted into 80 female NCr nude mice (NGP n=22, SH-SY5Y n=18, LAN5 n=20, LAN5 si-p53 n=20). The mice were randomly divided into a control group which received vehicle (hydroxypropylcellulose/Tween 80) and a treatment group which received RG7388. RG7388 was initiated three weeks post-implantation by intraperitoneal injection once daily for 14 days at a concentration of 25 mg/kg for mice implanted with NGP and SH-SY5Y, and 35 mg/kg for mice implanted with LAN5 and LAN5 si-p53. At five weeks post-implantation, all mice were sacrificed, xenograft tumors resected and weighed. Xenograft tumor specimens underwent immunohistochemistry for apoptosis. Results: Using MTS assay and annexin apoptosis assay to measure cell viability and apoptosis in several cell lines of neuroblastoma (NGP, SH-SY5Y, LAN5) treated with RG7388, we found robust decrease in cell proliferation and increase in apoptosis compared to vehicle control in all p53 wild-type cell lines. The p53 silenced cell line, LAN5 si-p53, was found to be resistant to RG7388 with no decrease in cell proliferation and no increase in apoptosis. In vivo, all mice tolerated RG7388 well without morbidity. RG7388 significantly inhibited tumor growth by 59% in NGP (p=0.003), 67% in SH-SY5Y (p=0.006), and 75% in LAN5 (p=0.0019) xenograft tumors. However, RG7388 had no inhibitory effect on LAN5 si-p53 xenograft tumors compared to vehicle treatment (p=0.57). Tumor apoptosis was analyzed by cleaved caspase-3 staining, which showed xenografts treated with RG7388 had significantly increased apoptotic cells per high power field in NGP xenografts (p&amp;lt;0.0001) and SH-SY5Y xenografts (p=0.0014). Conclusion: The MDM2 small molecule inhibitor RG7388 significantly inhibits p53 wild-type neuroblastoma tumor growth but has no effect on p53-silenced neuroblastoma xenografts in an orthotopic mouse model. Our studies suggest that RG7388 inhibits tumor growth by p53-mediated apoptosis. Ongoing studies include assessment of the effect of RG7388 on tumor angiogenesis and metastasis. In vitro studies are ongoing to verify the molecular mechanism of RG7388 and confirm increase in p53 levels as well as downstream p53 pathways. Further studies are also being performed in several other p53 mutant neuroblastoma cell lines to elucidate the role of p53 for RG7388 treatment. Citation Format: Anna Lakoma, Eveline Barbieri, Matthew C. McVay, Zaowen Chen, Charitra Adhikari, Jason M. Shohet, Eugene S. Kim. The novel MDM2 inhibitor RG7388 is highly effective against neuroblastoma in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B58.

Abstract 4995: Role of MICAL1 in NEDD9 invasive signaling

Abstract Metastasis is the final step leading to patient death in most solid tumors, including head and neck squamous cell carcinoma (HNSCC). For metastasis to proceed, tumor cells must become mobile and invasive, losing their communal behavior. A goal of our work is to delineate the downstream effectors of growth factor signaling pathways that contribute to invasion. Using a phosphotyrosine proteomics screen we previously identified neural precursor cell expressed, developmentally down-regulated 9 (NEDD9; CasL) as a mediator of VEGF and IGF-1 signaling to invasion in HNSCC cells. NEDD9 tyrosine phosphorylation led to matrix metalloproteinase9 (MMP9) expression and secretion, invadopodia formation and enhanced invasion. To determine the molecular details of NEDD9 protein:protein interactions leading to invasion, we have generated a series of NEDD9 substrate domain tyrosine (Y to F, YXXP) and SH3 domain mutants. Mutation of all 13 YXXP motifs to FXXP blocked NEDD9 tyrosine phosphorylation, MMP9 expression and invadopodia formation. Of note, we found that NEDD9 co-immunoprecipitated with Molecule Interacting with CasL 1 (MICAL1). To further define this interaction and its downstream impact, stable NEDD9 or MICAL1 silenced HNSCC cell lines expressing select shRNAs were generated, with each exhibiting reduced migration and invasion. MICAL shRNA transfected cell lines also exhibited decreased MMP9 secretion, consistent with our findings that lack of MICAL1 decreases cell invasion and with findings of other labs that MICALs interact with Rab proteins altering intracellular trafficking. Further, we have verified that MICAL1 and NEDD9 form complexes in cells based on coimmunoprecipitation analyses. Better understanding of this pathway will undoubtedly lead to the identification of novel therapeutics for the prevention of invasion and metastasis. Supported by NIH R01CA134845 (SAR), P30 CA138313 and NIH/NCRR UL1 TR000062/TL1 TR000061 from the South Carolina Clinical &amp; Translational Research Institute (COH). Citation Format: Casey O. Holmes, Amanda M. Brock, Philip T. Sobash, Jessica A. Tiedeken, Steven A. Rosenzweig. Role of MICAL1 in NEDD9 invasive signaling. [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 4995. doi:10.1158/1538-7445.AM2014-4995

In vitro screen of prion disease susceptibility genes using the scrapie cell assay.

Prion diseases (transmissible spongiform encephalopathies) are fatal neurodegenerative diseases, including Creutzfeldt-Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. While genome-wide association studies in human and quantitative trait loci mapping in mice have provided evidence for multiple susceptibility genes, few of these have been confirmed functionally. Phenotyping mouse models is generally the method of choice. However, this is not a feasible option where many novel genes, without pre-existing models, would need to be tested. We have therefore developed and applied an in-vitro screen to triage and prioritize candidate modifier genes for more detailed future studies which is faster, far more cost effective and ethical relative to mouse bioassay models. An in vitro prion bioassay, the scrapie cell assay, uses a neuroblastoma-derived cell line (PK1) that is susceptible to RML prions and able to propagate prions at high levels. In this study, we have generated stable gene silencing and/or overexpressing PK1-derived cell lines to test whether perturbation of 14 candidate genes affects prion susceptibility. While no consistent differences were determined for seven genes, highly significant changes were detected for Zbtb38, Sorcs1, Stmn2, Hspa13, Fkbp9, Actr10 and Plg, suggesting that they play key roles in the fundamental processes of prion propagation or clearance. Many neurodegenerative diseases involve the accumulation of misfolded protein aggregates and ‘prion-like’ seeding and spread has been implicated in their pathogenesis. It is therefore expected that some of these prion-modifier genes may be of wider relevance in neurodegeneration.

稳定高表达和干扰 Nodal 基因黑色素瘤细胞株构建及EMT表型鉴定

Objective: To generate two B16 melanoma cell lines,stably overexpress or silence Nodal gene and identified their EMT markers for study of phenotypic changes and molecular mechanisms of Nodal induced EMT in melanoma. Methods: The mouse melanoma B16 cells were transfected with recombinant plasmid pLtdTomato-mNodal or shRNA plasmid pGFP-V-RS-Nodal respectively. The stable cell lines B16 / dT-mNodal and B16 / sh-Nodal were obtained after resistance screening and enrichment,positive clone selection and expasion. The intracellular gene and protein levels of Nodal and EMT markers in these two cell lines were detected by qRTPCR and Western blotting. Results: Both stable cell lines were constructed successfully. B16 / dT-mNoda cell line exhibited strong red fluorescence with obvious up-regulated of Nodal mRNA and protein and Showed the characteristics of mesenchymal cell. While B16 / sh-Nodal cell line displayed strong green fluorescence,as well as notable down-regulated Nodal mRNA and protein levels and Showed the characteristics of epithelial cell. Conclusion: The Nodal stably overexpressing or silencing cell lines B16 / dT-mNodal and B16 / sh-Nodal have been constructed successfully,providing important experimental tools for the study of Nodal function in the process of melanoma EMT.