Silenced Cell Lines Research Articles

Abstract 579: Cell death induction end-points to identify new cancer indications with high likelihood of response to dual PI3K /mTOR inhibitors

Abstract Constitutive activation of the PI3K pathway is driven by a variety of genetic and epigenetic abnormalities, such as PTEN deletions, mutations, genesilencing, PIK3CA mutations and/or enhanced engagement through genetic abnormalities in upstream RTKs. Targeted therapies against specific components of the PI3K pathway are therefore expected to produce positive outcome as single agents or in combination in cancers presenting these abnormalities. Two molecular entities are in full clinical development: NVP-BEZ235, a dual PI3K/mTOR inhibitor and NVP-BKM120, a pan-class I PI3K inhibitor without mTOR activity. In order to best position these agents clinically, we investigated their efficacy in models bearing PIK3CA, PTEN, HER2, or KRAS genetic alterations. We previously demonstrated that NVP-BEZ235 in in vitro antiproferative activity was not predictive of preclinical antitumor response, in vivo. However, breast cancer models presenting either HER2 amplification or PIK3CA mutations or both were shown to be very sensitive to the compound and the use of cell death induction as an end-point was found to be predictive of strong antitumor activity in vivo. To further validate this finding, dual PI3K/mTOR targeting was phenocopied using an mTOR shRNA inducible system or an mTOR specific inhibitor (AZD8055) in combination with a pan-PI3K inhibitor, in various tumor models. Strong cell death signals were observed in 2 HER2 amplified / PIK3CA mutant breast cancer models but not in KRAS mutant cells or as PTEN silenced breast cancer cell lines, when subjected of PI3K/mTOR targeting. Moreover, in vivo, combination of a pure pan-PI3K with a pure mTOR inhibitor resulted in strong regression of HER2 amplified /PIK3CA mutant MDA-MB361 tumors.On the basis of these observations, NVP-BEZ235 was retested in a set of more then 150 tumor lines across various lineages, and cell death induction was determined. As expected, HER2 amplified /PIK3CA mutant breast cancer lines scored as sensitive. Other models from different indications and genetic background were also found to be sensitive to NVP-BEZ235. Validation studies of these new models and potential indications will be presented, and implications for further clinical trials of dual PI3K/mTOR or pure PI3K inhibitors discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 579. doi:10.1158/1538-7445.AM2011-579

Paired Box Gene 5 Is a Novel Tumor Suppressor in Hepatocellular Carcinoma Through Interaction With p53 Signaling Pathway §Δ

The paired box 5 (PAX5) is a member of PAX transcription factors family involved in the regulation of embryonic development. However, the role of PAX5 in carcinogenesis is largely unclear. We identified that PAX5 is involved in human cancer by methylation-sensitive representational difference analysis. We examined the biological functions and related molecular mechanisms of PAX5 in hepatocellular carcinoma (HCC). Promoter methylation of PAX5 was evaluated by methylation-specific polymerase chain reaction (PCR) and bisulfite genomic sequencing (BGS). The functions of ectopic PAX5 expression were determined by viability assay, colony formation, and cell cycle analyses, along with in vivo tumorigenicity assays. The PAX5 target signal pathway was identified by promoter luciferase assay, chromosome immunoprecipitation (ChIP), and pathway PCR array. PAX5 is expressed in normal human liver tissue, but silenced or down-regulated in 83% (10/12) of HCC cell lines. The mean expression level of PAX5 was significantly lower in primary HCCs as compared to their adjacent normal tissues (P < 0.0001). The promoter methylation contributes to the inactivation of PAX5. Restoring PAX5 expression in silenced HCC cell lines suppressed cell proliferation, induced apoptosis in vitro, and inhibited tumor growth in nude mice (P < 0.0001). The pathway luciferase reporter assay indicated that PAX5 activated p53 and p21 signaling. ChIP analysis demonstrated that PAX5 directly bound to the p53 promoter. The antitumorigenic function of PAX5 was at least up-regulated by p53 and its downstream targets including tumor necrosis factor, Fas ligand, leucine-rich repeats, and death domain-containing, poly(rC) binding protein 4, p21, and growth arrest and DNA-damage-inducible alpha. PAX5 is frequently inactivated by promoter methylation in HCC. PAX5 appears to be a functional tumor suppressor involved in liver carcinogenesis through direct regulation of the p53 signaling pathway.

Matrix Metalloproteinase 1: Role in Sarcoma Biology

In carcinomas stromal cells participate in cancer progression by producing proteases such as MMPs. The expression MMP1 is a prognostic factor in human chondrosarcoma, however the role in tumor progression is unknown. Laser capture microdissection and In Situ hybridization were used to determine cellular origin of MMP1 in human sarcomas. A xenogenic model of tumor progression was then used and mice were divided in two groups: each harboring either the control or a stably MMP1 silenced cell line. Animals were sacrificed; the neovascularization, primary tumor volumes, and metastatic burden were assessed. LCM and RNA-ISH analysis revealed MMP1 expression was predominantly localized to the tumor cells in all samples of sarcoma (p = 0.05). The percentage lung metastatic volume at 5 weeks (p = 0.08) and number of spontaneous deaths secondary to systemic tumor burden were lower in MMP1 silenced cell bearing mice. Interestingly, this group also demonstrated a larger primary tumor size (p<0.04) and increased angiogenesis (p<0.01). These findings were found to be consistent when experiment was repeated using a second independent MMP1 silencing sequence. Prior clinical trials employing MMP1 inhibitors failed because of a poor understanding of the role of MMPs in tumor progression. The current findings indicating tumor cell production of MMP1 by sarcoma cells is novel and highlights the fundamental differences in MMP biology between carcinomas and sarcomas. The results also emphasize the complex roles of MMP in tumor progression of sarcomas. Not only does metastasis seem to be affected by MMP1 silencing, but also local tumor growth and angiogenesis are affected inversely.

WNT5A antagonizes WNT/β-catenin signaling and is frequently silenced by promoter CpG methylation in esophageal squamous cell carcinoma

WNT5A is classified as a non-transforming WNT family member whose role in carcinogenesis is still ambiguous. It exhibits tumor suppressor activities in some cancers (thyroid, brain, breast and colorectum), but is aberrantly upregulated in cancers of lung, stomach and prostate. We investigated its epigenetic alterations and functions in esophageal squamous cell carcinoma (ESCC). With semi-quantitative reverse transcription PCR, we found that WNT5A was silenced or down-regulated in 5 of 18 ESCC cell lines, but expressed in normal esophagus tissue and immortalized normal esophageal epithelial cell lines. Promoter CpG methylation of WNT5A was detected in 4 of the 5 downregulated ESCC cell lines, while 5-aza-2'-deoxycytidine treatment induced WNT5A expression and demethylated its promoter in silenced cell lines. WNT5A promoter methylation was frequently detected in primary ESCC (24/36, 67%), but less frequently and weakly in paired surgical marginal esophageal tissues (8/36, 22%; p

Epigenetic inactivation of T-box transcription factor 5, a novel tumor suppressor gene, is associated with colon cancer

T-box transcription factor 5 (TBX5) is a member of a phylogenetically conserved family of genes involved in the regulation of developmental processes. The function of TBX5 in cancer development is largely unclear. We identified that TBX5 was preferentially methylated in cancer using methylation-sensitive arbitrarily primed PCR. We aim to clarify the epigenetic inactivation, biological function and clinical significance of TBX5 in colon cancer. Promoter methylation was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. Cell proliferation was examined by cell viability assay and colony formation assay, apoptosis by flow cytometry and cell migration by wound-healing assay. TBX5 target genes were identified by cDNA microarray analysis. Cox regression model and log-rank test were used to identify independent predictors of prognosis. TBX5 was silenced or downregulated in 88% (7/8) colon cancer cell lines, but was expressed in normal colon tissues. Loss of gene expression was associated with promoter methylation. The biological function of TBX5 in human colon cancer cells was examined. Re-expression of TBX5 in silenced colon cancer cell lines suppressed colony formation (P<0.001), proliferation (P<0.001), migration and induced apoptosis (P<0.01). Induction of apoptosis was mediated through cross-talk of extrinsic apoptosis pathway, apoptotic BCL2-associated X protein and Granzyme A signaling cascades. TBX5 suppressed tumor cell proliferation and metastasis through the upregulation of cyclin-dependent kinase inhibitor 2A, metastasis suppressor 1 and downregulation of synuclein gamma and metastasis-associated protein 1 family member 2. TBX5 methylation was detected in 68% (71/105) of primary colon tumors. Multivariate analysis showed that patients with TBX5 methylation had a significantly poor overall survival (P=0.0007). In conclusion, we identified a novel functional tumor suppressor gene TBX5 inactivated by promoter methylation in colon cancer. Detection of methylated TBX5 may serve as a potential biomarker for the prognosis of this malignancy.

The Tumor Suppressor UCHL1 Forms a Complex with p53/MDM2/ARF to Promote p53 Signaling and Is Frequently Silenced in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma is prevalent in southern China and Southeast Asia, with distinct geographic and ethnic distribution. One candidate susceptibility locus has been identified at 4p11-14, with the associated candidate gene(s) not identified yet. This study investigated the role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in nasopharyngeal carcinoma pathogenesis. UCHL1 expression and methylation were examined in nasopharyngeal carcinoma. Furthermore, the mechanism of its tumor-suppressive function was elucidated in nasopharyngeal carcinoma cells. Through genomewide expression profiling, we identified UCHL1, a 4p14 gene normally expressed in normal upper respiratory tract tissues, being silenced in all nasopharyngeal carcinoma cell lines. Its silencing is mediated by CpG methylation because UCHL1 promoter methylation was detected in all silenced cell lines, and pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. UCHL1 methylation was also frequently detected in primary tumors but only weakly detected in few normal nasopharyngeal tissues, indicating that the methylation-mediated silencing of UCHL1 is important in nasopharyngeal carcinoma pathogenesis. Ectopic UCHL1 expression dramatically inhibited the growth of nasopharyngeal carcinoma cells through promoting tumor cell apoptosis. We further found that UCHL1 formed a complex with p53/p14(ARF)/Mdm2 p53 binding protein homolog (mouse), MDM2 and activated the p53 signaling pathway. UCHL1 expression extended p53 and p14(ARF) protein half-life and shortened MDM2 protein half-life. These results indicate that UCHL1 could deubiquitinate p53 and p14(ARF) and ubiquitinate MDM2 for p53 stabilization to promote p53 signaling, thus involved in nasopharyngeal carcinoma pathogenesis, whereas it is frequently silenced in this tumor.

α‐Dystroglycan is essential for the induction of Egr3, a transcription factor important in muscle spindle formation

Muscle spindle fibers are specialized stretch receptors that allow the perception and coordination of limb movement. The differentiation of these specialized structures is initiated by signals derived from the in growing Ia sensory neurons during development. While the direct molecular signaling mechanisms between sensory neurons and developing muscle at nascent spindle fibers have been well documented in past studies the roles of muscle basal lamina components on this process have not previously been described. As such, our initial experiments addressed potential roles for agrin (AGRN) and laminin (LN) in the expression of the transcription factor Egr3. Levels of Egr3 were monitored using immunoblot analysis and both basal lamina molecules proved effective in inducing Erg3 expression. Previous work had established neuregulin (NRG) as a critical signaling component in spindle fiber development so blocking experiments with NRG and ErbB inhibitors were then used to determine if LN-induced Egr3 expression was occurring as a result of NRG-ErbB signaling and not via other, novel pathway. Inhibiting signaling through this pathway did indeed reduce the expression of Egr3. Finally, we looked at alpha-dystrogylcan, a shared receptor for AGRN and LN at neuromuscular junctions. Using a alpha-dystroglycan (alpha-DG) silenced muscle cell line and an anti-alpha-DG antibody we attempted to block basal lamina/alpha-DG interactions. Again, and in both instances, Egr3 expression was significantly decreased. Taken together, analysis of the results from these experiments revealed that indeed AGRN, LN, and alpha-DG influence Egr3 levels and therefore may play an important role in spindle fiber differentiation.

RNA interference‐mediated silencing of UBCH10 gene inhibits colorectal cancer cell growth in vitro and in vivo

Summary1. UbcH10 is the cancer‐related E2 ubiquitin‐conjugating enzyme, and its overexpression has been demonstrated in a variety of malignancies. The aim of the present study is to silence UbcH10 gene by RNA interference (RNAi) and to observe its inhibitory effect on the colorectal cancer cell growth in vitro and in vivo.2. We constructed the expression vector pGPU6/GFP/Neo/UbcH10‐RNAi (pUbcH10‐RNAi), which contained a UbcH10 short hairpin RNA expression cassette. Then the UbcH10 gene silencing cell lines LoVo/UbcH10‐RNAi and HT‐29/UbcH10‐RNAi were established. Reverse transcription‐polymerase chain reaction and western blot analysis were used to evaluate the expression of the UbcH10 gene. Cell Counting Kit‐8 was used to assess properties of tumour cell growth in vitro. Flow cytometry was used to detect the effect of pUbcH10‐RNAi on the cell cycle of colorectal cancer cells. Furthermore, the anti‐tumour effects of pUbcH10‐RNAi were evaluated in vivo in a nude mouse xenografts model.3. Results demonstrated that UbcH10 gene expression was significantly decreased in pUbcH10‐RNAi treated cells. Colorectal cancer cells growth was markedly suppressed in the pUbcH10‐RNAi group compared with control conditions and colorectal cancer cells were arrested in the G2‐M phase. In vivo, the downregulation of UbcH10 gene expression by pUbcH10‐RNAi also inhibited tumour growth in a nude mice xenograft model.4. Our study suggests that RNA interference‐mediated silencing of UbcH10 gene has anti‐tumour activity on colorectal cancer and might have therapeutic potential for the treatment of colorectal cancer.

Abstract 197: The KRAB-containing zinc finger protein ZNF545 is a functional tumor suppressor exerting proapoptotic and antiproliferation abilities with frequent epigenetic inactivation in multiple carcinomas

Abstract Epigenetic silencing of tumor suppressor gene is frequently involved in tumor development. Some zinc finger proteins (ZFPs) involved in tumorigenesis are down-regulated through CpG methylation in multiple carcinomas. Here, through genome wide-screening, we identified a novel KRAB domain-containing zinc finger protein (KRAB-ZFP), ZNF545, downregulated in multiple tumor cell lines including nasopharyngeal, esophageal, colon, stomach, liver, kidney, breast, cervix, and prostate carcinomas, in contrast to its broad expression in normal tissues. The promoter of ZNF545 contains a typical CpG island and is frequently methylated in multiple carcinoma cell lines. Transcriptional silencing of ZNF545 could be reversed by pharmacologic demethylation or genetic demethylation, indicating a direct epigenetic silencing mechanism. ZNF545 was also frequently methylated in primary tumors of NPC and ESCC, but less frequently in normal epithelial tissues and paired surgical marginal normal tissues. ZNF545 was found to be a transcriptional repressor when fused to Gal4-binding domain, as determined by the luciferase assay. Ectopic expression of ZNF545 in silenced cancer cell lines significantly inhibited their colony formation, proliferation and induced apoptosis, indicating that ZNF545 can function as a tumor suppressor. ZNF545 also can inhibit the activity of NF-KappaB and AP-1, which play an important role in tumorigenesis. We further found that ZNF545 was located in the nucleus and sequestered in the nucleolus in some cells. ZNF545 could co-localize with KAP1 and heterochromatin protein, so we proposed that ZNF545 could inhibit tumorigenesis, at least partially through downregulating the transcription of target genes, or regulating nucleolus function such as ribosome biogenesis. The tumor specific-silencing of ZNF545 could serve as an epigenetic biomarker for common cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 197.

Abstract 4386: Androgen Receptor Requires JunD as a Co-activator to Switch on an Oxidative Stress Generation Pathway in Prostate Cancer Cells

Abstract The relatively high oxidative stress in the prostate is postulated to be one of the major factors for prostate carcinogenesis and prostate cancer (CaP) progression. We focused on elucidating metabolic pathways of oxidative stress generation in CaP cells. Previously, we have shown that the transcription factor JunD is essential for the androgen-induced reactive oxygen species (ROS) production in androgen-dependent LNCaP human prostate cancer cells. We have also recently demonstrated that androgen induces the first and the regulatory enzyme spermidine/spermine N1-acetyl transferase (SSAT) in a polyamine catabolic pathway that produces copious amounts of metabolic ROS. Here, we present co-immunoprecipitation and Gaussia luciferase reconstitution assay data that show that JunD forms a complex with activated AR in situ. Our chromatin immnoprecipitation (ChIP) assay data demonstrate that JunD binds directly with a specific SSAT promoter sequence only in androgen-treated LNCaP cells. Using a vector containing a luciferase reporter gene connected to the SSAT promoter and a JunD silenced LNCaP cell line, we show that JunD is an essential co-activator of androgen-induction of SSAT gene expression. As there is no Androgen Response Element (ARE) in the SSAT promoter, we hypothesize that JunD mediates binding of androgen-activated AR to the SSAT promoter for the induction of SSAT that leads to polyamine oxidation and ROS production in prostate cells. Elucidation of this pathway may help the design of inhibitors of oxidative stress generation specifically in prostate cells to prevent prostate cancer occurrence and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4386.

Abstract 3193: EPHB2 expression parallels malignant behavior in mesothelioma

Abstract Purpose: Erythropoietin-producing hepatocellular (EPH) receptors and their Ephrin (EFN) ligands are members of the transmembrane receptor tyrosine kinase (RTK) family. EPHB2 is a RTK, promotes uncontrolled cell growth, migration and tumor progression. Preliminary gene array data indicate EPHB2 overexpression in malignant pleural mesothelioma (MPM). Here, we aim to verify EPHB2 overexpression in MPM tumors and cell lines and hypothesize that EPHB2 silencing decreases malignant behavior in MPM cell lines. Methods: EPHB2 mRNA expression was evaluated by RT-PCR in eight MPM and three benign mesothelial cell lines, and seven pairs of MPM tumors and matched normal tissue. Functional significance of EPHB2 was evaluated by transfecting EPHB2-shRNA and empty vector controls into two MPM cell lines, HP-1 and H2595. Expression of EPHB2, BCL2L1, survivin, Caspase2, Caspase8, matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) were evaluated by RT-PCR following silencing. Pooled populations of transfected cells were evaluated in scratch closure, cell proliferation by CellTitre BlueTM, MatrigelTM Invasion and colorimetric TUNELTM apoptotic assays. Comparisons were made between EPHB2 silenced and control cell lines. Results: EPHB2 was overexpressed in all MPM cell lines, but not in the benign mesothelial cell lines and in all tumors compared to matched normal tissue. Targeted knockdown of EPHB2 in MPM cell lines reduced its expression and subsequently reduced MMP-2, VEGF, BCL2L1 and survivin expression, and increased Caspase2 and Caspase8 expression. Inhibition of EPHB2 significantly decreased scratch closure, proliferation and invasion compared to the controls, and significantly increased apoptotic cells, likely related to Caspase2 and Caspase8 mediated cell death. Conclusion: Silencing of EPHB2 reduced the malignant behavior in MPM cell lines. EPHB2 appears to play an important role in MPM pathogenesis and could potentially serve as a novel therapeutic target for MPM treatment.Results of Functional AssaysAssayHP-1 ControlHP-1 EPHB2shRNAp ValueH2595 ControlH2595 EPHB2shRNAp ValueScratch Assay (% closure)69.237.6&amp;lt;0.00556.945.7&amp;lt;0.03Cell Proliferation Assay (OD value)9.56.4&amp;lt;0.058.75.7&amp;lt;0.05Matrigel Invasion Assay (cells invaded)14678&amp;lt;0.0057643&amp;lt;0.005TUNEL Apoptotic Assay (% apoptotic cells)074n/a088.5n/a Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3193.

Abstract 2353: CREB regulates AP-2α transcription to determine the malignant phenotype of melanoma

Abstract The Activator protein-2 (AP-2) family of transcription factors consists of five different proteins which are involved in the regulation of cell proliferation, differentiation, apoptosis and carcinogenesis. Among these proteins, AP-2α has been suggested to function as a tumor suppressor in many cancers including melanoma. We have previously shown that in melanoma cell lines, acquisition of the metastatic phenotype is associated with loss of AP-2α expression and gain in expression of cAMP-responsive element (CRE)-binding protein (CREB). This inverse correlation is associated with increase of melanoma invasion and metastasis resulting in overexpression of the melanoma cell adhesion molecule MCAM/MUC18 and protease-activated receptor 1 (PAR-1). However, the molecular mechanism involved in AP-2α regulation during melanoma progression remains unknown. Analyses of AP-2α promoter for transcription factor binding sites showed two distinct CRE-like sites as well as four E2F1 sites. Herein, we propose that the inhibition of AP-2α during the transition from the Radial Growth Phase (RGP) to the Vertical Growth Phase (VGP) occurs at the transcriptional level and is regulated through binding of CREB and E2F1. Hence, AP-2α protein expression was found to be increased after utilizing small hairpin RNA (shRNA) targeting CREB in two human metastatic melanoma cell lines, A375SM and C8161. Interestingly, E2F1 expression level was found to be reduced by 50% after CREB silencing in both cell lines. We also found a 20-fold increase in the luciferase activity driven by the AP-2α promoter after silencing CREB in both cell lines. In addition, point mutations at CRE-like binding sites as well as E2F1 increased the AP-2α promoter activity by more then 5-fold. To further establish the involvement of E2F1 in AP-2α regulation, we transiently overexpressed E2F1 in a low-metastatic melanoma cell line, SB-2. The AP-2α protein level as well as the promoter activity was significantly inhibited as compared to control. Finally, using Chromatin Immunoprecipitation (ChIP) assays in both CREB silenced cell lines, we were able to confirm that CREB and E2F1 bind to the AP-2α promoter thereby inhibiting its expression. Taken together, our studies demonstrate that CREB regulates AP-2α expression, thus, contributing to the metastatic phenotype of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2353.

Abstract B33: A novel isoform of the major 8p22 tumor suppressor Deleted in Liver Cancer 1 (DLC1) gene with growth suppression function is frequently silenced epigenetically in multiple tumors

Abstract Aberrant DNA methylation of Tumor Suppressor Gene (TSG) promoter regions, in addition to being a hallmark of cellular transformation, could provide potential tumor markers for early detection and monitoring of treatment effect. In a previous study to identify candidate TSGs relevant in Nasopharyngeal Carcinoma (NPC), our group identified the Deleted in Liver Cancer 1 (DLC1) gene to be epigenetically silenced not just in NPC, but in esophageal, breast and cervical carcinomas also. In this study, we report the cloning and characterization of a novel isoform of the human DLC1 gene with growth suppression function transcribing from a functional alternative promoter and silenced epigenetically in multiple tumors. Designated as DLC1 isoform 4 (DLC1-i4), this novel isoform encodes a protein of 1125 amino acids with a distinct N-terminus as compared to the other known isoforms. DLC1-i4 was expressed ubiquitously in normal tissues and immortalized normal epithelial cells, but significantly down-regulated in multiple tumor cell lines. Promoter DNA methylation was further found in 6/16 (38%) esophageal, 3/4 (75%) cervical, 2/2 (100%) colorectal, 4/4 (100%) NPC cell lines, and in 37/39 (95%) and 12/31 (39%) of primary NPC and esophageal tumors respectively. In contrast, no DNA methylation was found in normal cell lines and tissues. Pharmacological treatment with 5-aza-2′-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B could demethylate the DLC1-i4 promoter and activate transcription, indicating a direct epigenetic mechanism. Ectopic expression of DLC1-i4 had a strong inhibitory effect on the growth and colony formation ability of silenced tumor cell lines. As a whole, our results suggest that transcription of multiple DLC1 isoforms driven off alternative promoters could modulate the activity of the candidate tumor suppressor DLC1 protein in the pathogenesis of these tumors, and should be further tested as a tumor specific molecular biomarker. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B33

BMP2 is silenced by promoter hypermethylation in rhabdomyosarcoma cell lines

Aberrant DNA methylation is the most common epigenetic alteration in solid cancer and suspected to silence tumor suppressor genes. Here, we have set out to identify epigenetically silenced genes in rhabdomyosarcoma (RMS), the most common soft-tissue-sarcoma of infancy. Upon treatment with the demethylating agent 5′-aza-2′-deoxycytidine (Aza), we found increased mRNA expression levels of BMP2 (bone morphogenetic protein 2) in three RMS cell lines. BMP2 is known to play an import role not only in the differentiation and formation of many organs and tissues, but also in carcinogenesis. Detailed methylation analysis of CpG sites in the BMP2 promoter region by bisulfite sequencing revealed that a high degree of DNA methylation is causatively associated with the suppression of BMP2 in RMS cell lines. Consequently, treatment of the silenced cell lines with Aza resulted in DNA demethylation of the BMP2 promoter. Exogenous expression of BMP2 by transient and stable transfection led to a reduced growth of RMS cells, as evidenced by a MTT-proliferation assay. Altogether, these findings suggest that suppression of BMP2 by epigenetic silencing might play a critical role in the genesis of RMS.

RNA INTERFERENCE-MEDIATED SILENCING OF UBCH10 GENE INHIBITS COLORECTAL CANCER CELL GROWTH IN VITRO AND IN VIVO

1. UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme, and its overexpression has been demonstrated in a variety of malignancies. The aim of the present study is to silence UbcH10 gene by RNA interference (RNAi) and to observe its inhibitory effect on the colorectal cancer cell growth in vitro and in vivo. 2. We constructed the expression vector pGPU6/GFP/Neo/UbcH10-RNAi (pUbcH10-RNAi), which contained a UbcH10 short hairpin RNA expression cassette. Then the UbcH10 gene silencing cell lines LoVo/UbcH10-RNAi and HT-29/UbcH10-RNAi were established. Reverse transcription-polymerase chain reaction and western blot analysis were used to evaluate the expression of the UbcH10 gene. Cell Counting Kit-8 was used to assess properties of tumour cell growth in vitro. Flow cytometry was used to detect the effect of pUbcH10-RNAi on the cell cycle of colorectal cancer cells. Furthermore, the anti-tumour effects of pUbcH10-RNAi were evaluated in vivo in a nude mouse xenografts model. 3. Results demonstrated that UbcH10 gene expression was significantly decreased in pUbcH10-RNAi treated cells. Colorectal cancer cells growth was markedly suppressed in the pUbcH10-RNAi group compared with control conditions and colorectal cancer cells were arrested in the G2-M phase. In vivo, the downregulation of UbcH10 gene expression by pUbcH10-RNAi also inhibited tumour growth in a nude mice xenograft model. 4. Our study suggests that RNA interference-mediated silencing of UbcH10 gene has anti-tumour activity on colorectal cancer and might have therapeutic potential for the treatment of colorectal cancer.

The direct effect of Focal Adhesion Kinase (FAK), dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

BackgroundFocal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis.MethodsTo study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible) system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD), and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors.ResultsInduction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p < 0.05) by FAKsiRNA.ConclusionThus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal specific expression of genes affected by silencing of FAK.

Mustn1 is expressed during chondrogenesis and is necessary for chondrocyte proliferation and differentiation in vitro

Mustn1 encodes a small nuclear protein expressed specifically in the musculoskeletal system that was originally identified as a strongly up-regulated gene during bone regeneration, especially in fracture callus proliferating chondrocytes. Further experiments were undertaken to investigate its expression and role during chondrogenesis. Initially, whole mount mouse in situ hybridization was carried out and revealed Mustn1 expression in areas of active chondrogenesis that included limb buds, branchial arches and tail bud. To elucidate its function, experiments were carried out to perturb Mustn1 by overexpression and silencing in the pre-chondrocytic RCJ3.1C5.18 (RCJ) cell line. In these cells, Mustn1 is normally differentially regulated, with a spike in expression 2 days after induction of differentiation. Further, Mustn1 was successfully overexpressed in multiple RCJ cell lines by ∼2–6 fold, and reduced to ∼32–52% in silenced cell lines as compared to parental Mustn1 levels. Overexpressing, silenced, control, and parental RCJ cell lines were assayed for proliferation and differentiation. No statistically significant changes were observed in either proliferation or proteoglycan production when Mustn1 overexpressing lines were compared to parental and control. By contrast, both proliferation rate and differentiation were significantly reduced in Mustn1 silenced cell lines. Specifically, RNAi silenced cell lines showed reductions in populations of ∼55–75%, and also ∼34–40% less matrix (proteoglycan) production as compared to parental and random control lines. Further, this reduction in matrix production was accompanied by significant downregulation of chondrogenic marker genes, such as Sox9, Collagen type II (Col II), and Collagen type X (Col X). Lastly, reintroduction of Mustn1 into a silenced cell line rescued this phenotype, returning proliferation rate, matrix production, and chondrogenic marker gene expression back to parental levels. Taken together these data suggest that Mustn1 is a necessary regulator of chondrocyte function.

Decreased expression of EZH2 is associated with ESR1 upregulation and response to anti-estrogens.

Abstract Abstract #6127 Background: In prostate and breast cancer high levels of Enhancer of Zeste Homolog 2 (EZH2) are associated with tumor progression. EZH2, a histone H3 methyl transferase, is part of the polycomb complex. Together with histone deacetylases (HDACs) EZH2 regulates “genome wide” gene transcription silencing. The aims of this study are a) to correlate EZH2 expression with endocrine therapy response in patients with recurrent disease treated with first-line tamoxifen monotherapy and b) to determine the role of EZH2 in endocrine therapy response using in vitro cell line models.&amp;#x2028; Material and Methods: EZH2 mRNA levels were measured with quantitative real-time PCR (qRT-PCR) in 297 retrospectively collected hormone receptor positive (HR+) primary breast tumor specimens of patients with recurrent disease who did respond (N=110) or were resistant (N=187) to first-line tamoxifen monotherapy. In vitro, EZH2 and estrogen receptor (ESR1) expression was downregulated with siRNAs in the human breast cancer cell line MCF7 and assessed for their sensitivity to the selective estrogen receptor degrader ICI164.384 after 96hrs treatment (N=3). To establish therapy response in vitro, cell number counts were determined. All p-values are two-sided and significant if P&amp;lt;0.05.&amp;#x2028; Results: In 297 HR+ breast tumors, EZH2 as continuous variable, associated significantly with poor response (OR= 0.67 [0.49-0.91]; P=0.001) and a shorter progression-free survival (PFS) (HR=1.28 [1.11-1.47], P&amp;lt;0.001). In the multivariate model with traditional predictive factors, the tertile with highest EZH2 levels was independently related with response (OR= 0.50 [0.26-0.98]; P=0.045) and PFS (HR=1.82 [1.33-2.49], P&amp;lt;0.001).&amp;#x2028; In vitro, EZH2 downregulation in MCF7 with siRNAs showed a significant decrease in cell number compared to the mock silenced cell line (40%). Moreover, ICI treatment of EZH2 silenced MCF7 cells resulted in a 70% cell number decrease versus 23% decrease in the controls (P&amp;lt;0.001). In addition, EZH2 downregulation is associated with a twofold upregulation with ESR1 (P&amp;lt;0.001). Conversely, silencing of ESR1 in MCF7 resulted did not alter EZH2 levels but, as expected, increases HER2 protein levels.&amp;#x2028; Conclusion: In primary breast tumors, high EZH2 mRNA levels are associated with poor outcomes after tamoxifen therapy. These results suggest that EZH2 may identify patients at risk for tamoxifen therapy failure. In vitro studies show that downregulation of EZH2 inhibits cell proliferation and on the other hand results in upregulation of ESR1 levels. The latter may explain the increased sensitivity to ICI164.384 of EZH2 silenced cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6127.

Epigenetic Inactivation of Notch Signaling Target Genes HES in B Cell Acute Lymphoblastic Leukemia.

The Notch signaling pathway has been implicated in multiple functions during normal hemato-lymphoid development. It also plays critical roles in T-cell leukemogenesis through influencing T-cell proliferation, differentiation and survival. In contrast, we have previously reported a tumor suppressor role in B-cell leukemias, where Notch signaling leads to growth inhibition and apoptosis. The Notch target genes Hairy/Enhancer of Split (HES1-7) encode transcriptional repressors with basic helix-loop-helix (bHLH) domains. Functional and phenotypic analyses of some of the HES family members have been reported, however, expression and epigenetic regulation of the HES family in leukemia is largely unknown. Using Methylated CpG Island Amplification (MCA) / DNA promoter microarray, we identified several HES family genes as hypermethylated in B cell acute lymphoblastic leukemia (B ALL). We further investigated the comprehensive methylation profiles of HES family genes in a panel of leukemia cell lines and ALL patient samples by bisulfite pyrosequencing. Aberrant DNA methylation of HES2, HES4, HES5 and HES6 was detected in most B ALL cell lines including B-JAB, RS4:11, REH, Raji and Ramos but not in normal B cell controls. In contrast, in T cell leukemia cell lines such as Molt4, PEER, T-ALL1 and J-TAG, these genes were generally unmethylated. In B ALL patient samples, the frequencies of DNA methylation in the promoter regions of these genes were 25% for HES2, 50% for HES4, 76% for HES5 and 71% for HES6. Expression analysis of HES4, HES5 and HES6 in leukemia cell lines by real-time PCR further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with DNA methyltransferase inhibitor 5‘-aza-2‘-deoxycytidine resulted in HES gene re-expression. Finally, forced re-expression of HES5 and HES6 in methylation silenced Rs4 and REH cell lines inhibited cell growth. These results suggest that the Notch/HES signaling pathway is epigenetically-inactivated in B ALL. These data support the role of the HES family as tumor suppressors in pre-B ALL and establish epigenetic modulation as a novel mechanism of Notch pathway regulation. We anticipate that therapies capable of activating Notch/HES signaling may have therapeutic potential in B cell leukemias.

Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines

Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines in vitro. However, the replacement of the knocked down GHRH expression by exogenous GHRH (1–29)NH2 re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1–29)NH2 and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor.