Oncogene (2010) 29, 1362–1373 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE TCEAL7, a putative tumor suppressor , negatively regulates NF-jB pathway

R Rattan1, K Narita2, J Chien1, JL Maguire1, R Shridhar3, S Giri1 and V Shridhar1

1Department of Experimental Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA; 2Department of Anatomy and Cell Biology, University of Yamanashi, Faculty of Medicine, Chuo, Yamanashi, Japan and 3Gershenson Radiation Oncology Center, Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA

We have previously shown that a frequently down- Oncogene (2010) 29, 1362–1373; doi:10.1038/onc.2009.431; regulated gene, transcription elongation factor A-like 7 published online 7 December 2009 (TCEAL7), promoted anchorage-independent growth and modulated Myc activity in ovarian surface epithelial cells Keywords: TCEAL7; NF-kB; ovarian cancer; tumor immortalized with temperature-sensitive large T antigen suppressor gene and human telomerase reverse transcriptase (OSEtsT/ hTERT). Analysis of /DNA array showed that TCEAL7 downregulation resulted in an approximately twofold increase in nuclear factor (NF)-jB binding to its Introduction target DNA sequence. In this study we showed that short hairpin RNA (shRNA)-mediated downregulation of Ovarian cancer is the fifth most common cause of TCEAL7 in two different immortalized OSE cells showed death of all cancers among women in the United States higher NF-jB activity, as determined using reporter and and the leading cause of death from gynecological gel-shift assays. Transient transfection of TCEAL7 malignancies with the 5-year survival rate of only 30% inhibited the activation of NF-jB in TCEAL7-down- (Friedlander, 1998; Kenny et al., 2008). In an effort to regulated clones, IOSE-523 and in other ovarian cancer identify genetic alterations associated with the initiation cell lines (OVCAR8, SKOV3ip and DOV13), suggesting and progression of ovarian cancer, we performed high- that TCEAL7 negatively regulates NF-jB pathway. throughput expression-based screening and identified Consistent with this observation, TCEAL7-downregulated transcription elongation factor A-like 7 (TCEAL7) as clones showed higher levels of NF-jB targets, such as pro- one of the frequently downregulated in ovarian proliferative (cyclin-D1 and cMyc), pro-angiogenic (inter- cancer (Shridhar et al., 2001, 2002; Chien et al., 2005). leukin (IL)-6, IL-8 and vascular endothelial growth factor Transcription elongation factor A-like 7 encodes (VEGF)), inflammatory (intercellular adhesion molecule 1 TCEAL7 protein and has a sequence similarity with a (ICAM-1) and cyclooxygenase-2 (Cox-2)) and anti- small family of brain-expressed (Bex) , bex1, apoptotic (B-cell lymphoma-extra large (Bcl-xl)) genes bex2 and bex3 (Rapp et al., 1990; Mukai et al., 2000; when compared with vector controls. Inhibition of NF-jB Chien et al, 2005). It shares amino acid sequence by IjB kinase (IKK) inhibitor (BMS 345541) attenuated homology with transcription elongation factor A-like 1 cell survival and proliferation of TCEAL-knockdown (TCEAL1/p21/SIIR/pp21) and pp21 homolog (WBP5/ clones. Although TCEAL7 inhibited p65 transcriptional TCEAL6) (Mukai et al., 2000, 2002). Although very activity, it did not modulate the cytoplasmic signaling of little is known about the role of these proteins in cancer, the NF-jB pathway, by itself or by tumor necrosis factor- pp21 homolog (TCEAL1) has been shown to suppress a (TNF-a). Chromatin immunoprecipitation (ChIP) Rous sarcoma virus long-terminal repeat promoter assays revealed increased recruitment of p65 and p300 activity and inhibit the transformation mediated by it to the promoters of IL-8 and IL-6 in TCEAL7-down- in chicken embryo fibroblast (Yeh and Shatkin, 1994). regulated clones. Collectively, these results indicate a A closely related protein, TFIIS/TCEA is involved in novel role for TCEAL7 in the negative regulation of NF-jB transcription elongation and transcript fidelity. TFIIS/ signaling at the basal level by modulating transcriptional TCEA promotes 30 endoribonuclease activity of RNA activity of NF-jB on its target gene promoters, potentially polymerase II (pol II) and allows pol II to bypass providing a novel mechanism by which NF-jB activity transcript pause or ‘arrest’ during elongation process may be deregulated in ovarian cancer cells. (Jeon and Agarwal, 1996; Thomas et al., 1998). We previously reported that TCEAL7 expression is lost in >90% of primary ovarian tumors and cell lines Correspondence: Dr V Shridhar, Department of Laboratory Medicine because of methylation of the actively expressed allele and Experimental Pathology, Mayo Clinic College of Medicine, on Xq22.1. Ectopic expression of TCEAL7 in TCEAL7- Rochester, MN 55905, USA. E-mail: [email protected] non-expressing cell lines induced cell death and sup- Received 9 March 2009; revised 16 July 2009; accepted 2 October 2009; pressed colony-forming abilities (Chien et al., 2005). published online 7 December 2009 Recent studies indicate that stable downregulation of TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1363 TCEAL7 expression in ovarian surface epithelial cells observations uncover a previously unknown pathway immortalized with temperature-sensitive large T antigen regulating NF-kB activity by a novel gene, TCEAL7, in and human telomerase reverse transcriptase (OSEtsT/ ovarian surface epithelial cells. hTERT) cells confers increased proliferation potential and an ability to form soft-agar colonies and results in upregulation of Myc activity (Chien et al., 2008). These Results findings suggest that loss of TCEAL7 expression may promote the survival and growth of ovarian cancer cells. TCEAL7 negatively regulates NF-kB pathway In an effort to identify transcription factors modu- To examine the role of TCEAL7 in cellular signaling, we lated by TCEAL7 loss, we previously analyzed the generated several stable clones expressing shRNA targeted DNA-binding activity of 54 transcriptional factors using against TCEAL7 in OSEtsT/hTERT cells and clonal lines the protein/DNA array (TranSignal Array I, Panomics, expressing empty (control) shRNA construct. Immunoblot Fremont, CA, USA) in clonal line without TCEAL7 analysis showed that there was a complete loss of (TCEAL7 short hairpin RNA (shRNA)) compared with TCEAL7 expression in several clonal lines when compared the clonal line with TCEAL7 (control shRNA) expres- with vector clones (Chien et al., 2008). shRNA-stable sion (Chien et al., 2008). Among the 54 transcription clones (C4-2 and C4-4), which showed significant down- factors tested, we identified nuclear factor (NF)-kBas regulation of TCEAL7 expression compared with vector- one of the transcription factors whose DNA-binding transduced clones (V1 and V3) (Figure 1a) by real-time activity was modulated by TCEAL7 downregulation. PCR, were chosen for further analysis. Because of the important role of NF-kB in cancer (Karin, 2006) and more specifically in promoting ovarian cancer progression and chemoresistance, (Mabuchi et al., 2004a, b; Chen et al., 2007), we sought to determine how loss of TCEAL7 modulates the NF-kB-dependent activity in ovarian cancer. There are five identified members of the NF-kB/Rel family, including p50, p65 (RelA), p52, Rel B and cRel; of which p65, Rel B and cRel contain the transactivation domain required for positive regulation of NF-kB. The most abundant NF-kB dimer present in most cells is composed of p50:p65 subunits. In unstimulated cells, NF-kB is usually sequestered in the cytoplasm, bound to the inhibitor IkBs (inhibitor of kB). Phosphorylation of IkB proteins by upstream kinases, IkB kinases (IKK), targets IkBs to ubiquitin-mediated degradation, resulting in the translocation of the p50:p65 dimer into the nucleus (Aggarwal, 2004; Karin, 2006). Once in the nucleus, the p50:p65 dimer binds to the NFkB- responsive elements of target genes, resulting in the activation of various genes that promote proliferation Figure 1 Short hairpin RNA (shRNA)-mediated TCEAL7-down- regulated clones show higher nuclear factor (NF)-kB activity. (cyclin-D1 and interleukin-6 (IL-6)), invasion (urokinase (a) Real-time PCR analysis of TCEAL7 in shRNA-downregulated plasminogen activator, matrix metallopeptidase 9 and TCEAL7 clones (C4-2 and C4-4) and vector clones (V1 and V3) in tumor necrosis factor-a (TNF-a)), metastasis (intercel- OSEtsT/hTERT cells. ***Po0.001 C4-2 and C4-4 compared with lular adhesion molecule 1 (ICAM-1) and vascular cell V1 and V3. (b) Nuclear extract from V1 and C4-2 clones was used adhesion molecule), angiogenesis (vascular endothelial to perform (TF) array as described in Material and methods. Downregulation of endogenous TCEAL7 in growth factor (VEGF) and IL-8) and suppression of OSEtsT/hTERT (TCEAL7 shRNA, C4-2) resulted in increased apoptosis (B-cell lymphoma (Bcl)-2, Bcl-xl and inhibi- NF-kB transcription factors binding to target DNA compared with tors of apoptosis) (Aggarwal, 2004). An activated stable clone expressing endogenous TCEAL7 (control shRNA, NF-kB pathway due to various aberrant upstream V1). Densitometric tracings are shown graphically. ***Po0.001 compared with vector V1. (c) Downregulation of TCEAL7 signaling events is characteristic of various cancer types, augments the basal NF-kB promoter luciferase activity. The including ovarian cancer, and is also frequently asso- relative light units (RLUs) are expressed after normalizing with ciated with chemoresistance and tumor progression Renilla luciferase and expressed as mean±s.e.m. from four (Arlt and Schafer, 2002; Aggarwal et al., 2004; Camp independent experiments. ***Po0.001, **Po0.01 compared with et al., 2004; Karin, 2006; Chen et al., 2007). However, both vectors V1 and V3. (d) Downregulation of endogenous TCEAL7 by shRNA in IOSE-523 resulted in elevated basal NF-kB little is known about the molecular alterations that lead promoter luciferase activity, whereas the empty vector had no to NF-kB activation in tumor cells. effect. Inset shows the downregulation of TCEAL7 expression by In this study, we show that loss of TCEAL7 in PCR compared with untransfected (Untf) and empty vector (ShV). ovarian surface epithelial cells promotes NF-kB acti- ***Po0.001 shRNA TCEAL7 compared with shVector and untransfected. hTERT, human telomerase reverse transcriptase; vation and leads to an increase in the levels of OSEtsT, ovarian surface epithelial cells immortalized with NF-kB target genes involved in proliferation, inflam- temperature-sensitive large T antigen; TCEAL7, transcription mation, angiogenesis and inhibition of apoptosis. These elongation factor A-like 7.

Oncogene TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1364 Our previous analysis of protein/DNA binding assay target genes, we determined the levels of a subset of NF- for 54 transcription factors, using nuclear lysates from kB target genes using enzyme-linked immunosorbent OSEtsT/hTERT clonal lines with TCEAL7 (control assay (ELISA) and/or immunoblot analysis in shRNA vector, V1) or without TCEAL7 (TCEAL TCEAL7shRNA-downregulated clones (C4-2 and C4-4) shRNA clone, C4-2) (Chien et al., 2008), identified and in vector clones V1 and V3. Among the various several transcription factors modulated by loss of NF-kB target genes tested, we found significant TCEAL7 including NF-kB, which showed an approxi- upregulation of pro-angiogenic factors, such as IL-8, mately twofold increase in NF-kB binding to its target IL-6 and VEGF, by ELISA (Figures 3a–c). Immunoblot sequence in TCEAL7 in C4-2 clone compared with analysis showed increased protein levels of pro-prolif- clone V1 (Figure 1b). erative genes Myc and cyclin-D1, pro-inflammatory To test whether increased NF-kB DNA binding, genes cyclooxygenase-2 (Cox-2) and ICAM-1 and anti- which is associated with TCEAL7 knockdown, corre- apoptotic gene Bcl-xl in OSEtsT/hTERT clones with lates with increased NF-kB promoter activities, we TCEAL7 downregulation compared with the vector determined the transcriptional activity of NF-kBin clones expressing endogenous TCEAL7 (Figure 3d). these clones. TCEAL7-downregulated clones (C4-2 and Expression of Cox-2, cyclin-D1, IL-6 and IL-8 were also C4-4) showed significantly higher NF-kB– luciferase validated using real-time PCR in both the vector and activity when compared with vector-transduced clones clonal pools to show regulation at the transcriptional (Figure 1c). To determine that this phenomenon was level (Supplementary Figure S2). These data indicate not unique to this one cell line, we downregulated that loss of TCEAL7 expression results in the upregula- TCEAL7 expression using shRNA in another SV40t/T tion of NF-kB target genes, further confirming our immortalized IOSE-523 cell line expressing TCEAL7 primary observation that activation of NF-kB pathway (Supplementary Figure S1A). Downregulation of in the absence of TCEAL7 may be one of the TCEAL7 in IOSE-523 (Inset in Figure 1d) also resulted mechanisms by which normal cells acquire a prolifera- in upregulation of basal NF-kB activity (Figure 1d). In tive and survival advantage. addition, TCEAL7 shRNA-downregulated clones were found to acquire higher levels of NF-kB complex bound to its consensus site compared with vector clones, as Inhibition of NF-kB pathway reverses the growth determined using electromobility shift assay (Supple- potential conferred by TCEAL-7 loss mentary Figure S1B). Collectively, these results indicate We previously reported that TCEAL7 loss results in that loss of TCEAL7 results in upregulation of NF-kB increased proliferation in OSEtsT/hTERT cells, and activity. forced expression of TCEAL7 promoted apoptosis in ovarian and cervical cancer cell lines (Chien et al., 2008). We used an IKK inhibitor (BMS345541) to inhibit Re-expression of TCEAL7 inhibits NF-kB activity NF-kB pathway to examine its effect on cell prolifera- in ovarian cells tion and survival in TCEAL7-downregulated OSEtsT/ To determine whether TCEAL7 could attenuate NF-kB hTERT clones. Treatment of either 293T Flp-In cells transcriptional activity, we transiently transfected (Figure 4a) or OSEtsT/hTERT clones (Figure 4b) with TCEAL7 expression construct into OSEtsT/hTERT BMS345541 at a non-toxic concentration (5 mM), re- clones (C4-2 and C4-4) and vector clones (V1 and V3). sulted in significant inhibition of the increased prolifera- As shown in Figure 2a, re-expression of TCEAL7 tion observed in the absence of TCEAL7 when significantly abrogated the elevated NF-kB–luciferase compared with untreated cells and vector clones. To activity in both TCEAL7 downregulated and vector examine whether the inhibition of NF-kB pathway clones. In addition, doxycycline-induced expression of would affect the survival of cells in the presence or TCEAL7 in Flp-In-293T stable cell line (Inset in absence of TCEAL7, 293T Flp-In cells were treated with Figure 2b) significantly reduced the NF-kB reporter BMS345541 at cytotoxic concentrations (10–20 mM), activity (Figure 2b). Re-expression of TCEAL7 in batch after doxycycline induction of TCEAL7 expression. IOSE-523 cells with downregulation of TCEAL7 also The cells expressing TCEAL7 had higher percentage blocked the elevated NF-kB reporter activity (Figure 2c) of cell death with IKK inhibitor when compared with cells compared with empty vector shRNA control cells. with no TCEAL7 induction (Figure 4c). These data Transient transfection of TCEAL7 expression in suggests that inhibition of NF-kB pathway abrogates DOV13, SKOV3ip and OVCAR8 (Inset in Figures 2d–f) the higher proliferative state observed in the absence also significantly inhibited the NF-kBactivity(Figures of TCEAL7 and that inhibition of NF-kBinthepresence 2d–f). Collectively, these results suggest that although loss of TCEAL7 is more detrimental to cells. However, of TCEAL7 upregulates NF-kB transcriptional activity, TCEAL7shRNA OSEtsT/hTERT clones also showed re-expression of TCEAL7 attenuates this increase in increased cell death at cytotoxic concentration of 10 and NF-kB transcriptional activity. 20 mM (Figure 4d) compared with the vector-transduced controls, which could be due to oncogenic addiction to Loss of TCEAL7 results in upregulation of NF-kB the NF-kB pathway in these clones. To validate that target genes NF-kB oncogenic addiction is found in ovarian cancer To determine whether upregulation of NF-kB pathway cell lines, we selected OVCAR8 and DOV13, two cell by loss of TCEAL7 results in altered levels of NF-kB lines showing higher basal NF-kB activity (the same

Oncogene TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1365

Figure 2 Re-expression of transcription elongation factor A-like 7 (TCEAL7) inhibits nuclear factor (NF)-kB activity. (a) Transient re-expression of TCEAL7 inhibits NF-kB activity. Vector (V1 and V3) and short hairpin RNA (shRNA; C4-2 and C4-4) clones were co-transfected with TCEAL7 and NF-kB promoter and luciferase activity was determined. The relative light units (RLUs) are expressed after normalizing with Renilla and expressed as mean ± s.e.m. from four independent experiments. ***Po0.001 compared with respective pcDNA-transfected cells. (b) TCEAL7-Flip-In-293T cells were pre-treated with 1 mM doxycycline (dox) and transiently transfected with NF-kB reporter and thymidine kinase promoter-Renilla luciferase reporter plasmid (PRL-TK) constructs. After 24 h, cells were processed for luciferase activity and normalized with Renilla. Results are mean±s.d. of four values. The inset shows doxycycline-mediated induction of TCEAL7 using immunoblot analysis. ***Po0.001 doxycycline-treated to untreated. (c) shRNA TCEAL7-downregulated IOSE-523 cells were transfected with S-tag-labeled TCEAL7 cDNA and NF-kB reporter as described above. Inset shows expression of S-tag, representing TCEAL7 expression after transfection. **Po0.001 TCEAL7 compared with pcDNA transfected. (d–f). Ovarian cell lines were transfected with TCEAL7 and NF-kB reporter and their luciferase activity was determined as described earlier. Inset shows expression of S-tag, representing TCEAL7 expression after transfection. **Po0.01, **Po0.001 TCEAL7 compared with pcDNA transfected. level as TCEAL7-downregulated clones) and IOSE-523 and RelB, depending upon the cell type and nature and SKOV3 with low basal NF-kB activity (Figure 4e). of stimulation (Ahn et al., 2007). As shown previously BMS345541 treatment resulted in increased cyto- (Supplementary Figure S1B), TCEAL7-downregulated toxicity in OVCAR8 and DOV13 cells when compared OSEtsT/hTERT clones (C4-2 and C4-4) showed higher with IOSE-523 and SKOV3 cells (Figure 4f). These data amounts of NF-kB complex when compared with vector suggests the presence of NF-kB oncogenic addiction in clone V1. To identify the predominant component of ovarian cancer cells lines. Interfering with this oncogene NF-kB complex in the OSEtsT/hTERT clonal lines, we addiction by modulating NF-kB activity may lead to performed a supershift electromobility shift assay decreased survival of these cells. (Supplementary Figure S3A). The supershift of NF-kB complex was detected with p65 and p50 antibodies compared with other components (cRel, RelB or p52), Mechanism of TCEAL7-mediated regulation of NF-kB indicating that the active dimer formed in OSEtsT/ pathway hTERT cells in the absence of TCEAL7 is the p65:p50 In general, NF-kB may form a hetero-dimer with any dimer (Supplementary Figure S3A, lanes 9 and 10 for one of the five members, including p65, p50, p52, cRel C4-2 and lanes 16 and 17 for C4-4). To further delineate

Oncogene TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1366 Figure 5a, transient transfection of TCEAL7 inhibited both the basal NF-kB activity and NIK-, IKKa-, IKKb- and p65-mediated activation of NF-kB in 293T cells. Moreover, transient transfection of IKK-b, p65 and p50 induced higher NF-kB–luciferase activity in TCEAL7 shRNA pool compared with control vector pool (Figure 5b). Similar to 293T cells, re-expression of TCEAL7 also inhibited IKK-b and p65-mediated induction of NF-kB–luciferase activity in OSEtsT/ hTERT cell line (Figure 5c). These data indicate that TCEAL7 functions downstream of all these signaling mediators and maybe modulates NF-kB activity at the nuclear level.

TCEAL7 inhibits p65-mediated transcriptional activity To determine whether TCEAL7 interferes with an active complex at the NF-kB binding site of target genes, we induced the expression of TCEAL7 with doxycycline and treated the cells with TNF-a for 30 and 60 min to Figure 3 Short hairpin RNA (shRNA)-mediated downregulated stimulate NF-kB signaling. As shown in Figure 6a, clones with transcription elongation factor A-like 7 (TCEAL7) TNF-a treatment induced the nuclear translocation of show higher expression of nuclear factor (NF)-kB targets genes. p50 and p65 in both doxycycline-treated and -untreated Vector control and shRNA-mediated downregulated clones were plated, and after 48 h of serum-free media, cell supernatant was cells, indicating that induction of TCEAL7 expression used for detection of (a) interleukin (IL)-8, (b) IL-6 and (c) vascular (by doxycycline treatment) does not affect the nuclear endothelial growth factor (VEGF) using enzyme-linked immuno- translocation of the NF-kB complex (Figure 6a). In sorbent assay (ELISA), as described under Materials and methods. addition, quantification of p50 and p65 at the binding Results represent mean±s.d. of three individual experiments. site was determined using ELISA assay. Induction of ***Po0.001 clones (C4-2 and C4-4) compared with vectors V1 and V3. (d) Cell lysates from TCEAL7-downregulated clones (C4-2 TCEAL7 by doxycycline treatment did not affect the and C4-4) and vector clones (V1 andV3) were used for detection of TNF-a-induced binding of p65 or p50 (Figures 6b and c) NF-kB target genes: cyclin-D1, cMyc, intercellular adhesion with NF-kB consensus sequence. This suggests that molecule (ICAM), cyclooxygenase-2 (Cox-2), and B-cell lymphoma- TCEAL7 does not influence TNF-a-stimulated NF-kB extra large (Bcl-xl) proteins using immunoblot analysis. The lowest panel indicates b-tubulin levels for equal loading. Blots are activity, but can inhibit the basal activity of NF-kB representations of two independent experiments. by an as yet unknown mechanism. To further corroborate this observation, we used p65– DNA binding domain-gal4, a chimeric transactivator, which contains part of the transcriptional activation the mechanism underlying this observation, nuclear and domain of the NF-kB-p65 protein fused to the cytoplasmic fractions were prepared from the vector and DNA-binding domain of GAL-4 protein from yeast. the TCEAL7 shRNA clones to examine whether any Consistent with the previous results, TCEAL7-down- changes in the cytoplasmic signaling of the NF-kB regulated clones showed significantly higher p65-tran- pathway could be detected. The nuclear extracts of both scriptional activity compared with vector control and the downregulated clones C4-2 and C4-4 showed slightly transient transfection of TCEAL7 reversed the p65 higher levels of p65 and p50 (Supplementary Figure transcriptional activity (Figure 6d). Moreover, TCEAL7 S3B) when compared with the vector clones; however, also inhibited IKK-mediated induction of p65 transcrip- no significant change was observed in the cytosolic tional activity (Figure 6e). Figure 6f shows the specificity extract (Supplementary Figure S3C). We did not of the Gal-4 transfection system, in which use of the observe any changes in the phosphorylation of IkBa IKK inhibitor, BMS345541 abrogated p65 activation. and degradation of IkBa/b (Supplementary Figure Collectively, these results suggest that although S3D). These data suggest that in the absence of TCEAL7 does not affect the nuclear translocation of TCEAL7, none of the cytosolic transducers undergo the complex or the DNA binding when induced by any changes and possibly TCEAL7 may not be TNF-a, it is still able to negatively regulate NF-kB modulating the cytosolic part of NF-kB-mediated activity through modulating p65 transcriptional activity. signaling.

TCEAL7 modulates p65 and p300 recruitment to the Upstream activators of NF-kB are not modulated promoters of target genes by TCEAL7 Cyclic adenosine monophosphate-response element- To further confirm this observation, we co-transfected binding protein (CREB)-binding protein (CBP)/p300 TCEAL7 expression vector in the presence or absence of interacts with p65 and is responsible for transactivation various upstream effectors of NF-kB pathway and of NF-kB complex bound at the target gene promoter examined the NF-kB–luciferase activity. As depicted in (Gerritsen et al., 1997). To determine whether TCEAL7

Oncogene TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1367

Figure 4 IkB kinase (IKK) inhibitor attenuates the cell proliferation and survival in the absence of transcription elongation factor A-like (TCEAL) expression. (a) Flp-In-293T cells were plated in the presence or absence of 1 mM doxycycline (Dox) and treated with IKK inhibitor, BMS345541 (5 mM). Cells were counted with Trypan blue over a period of 5 days. Results represents mean±s.d. from three independent experiments. **Po0.01 DoxÀ compared with Dox þ treated cells, ###Po0.001 Dox- BMS345541 compared with DoxÀ and þþþPo0.001 Dox þ BMS345541 compared with Dox þ .(b) Vector control and short hairpin RNA (shRNA)-mediated downregulated clones were plated and treated with BMS345541 (5 mM) and cells were counted with Trypan blue. Results represent mean±s.d. of three experiments. ***Po0.001, **Po0.01, clones C4-2 and C4-4 compared with vectors V1 and V3; þþþPo0.001, C4- 2 and C4-4 treated with BMS345541 compared with C4-2 and C4-4 untreated. (c) Flip-In-293T cells were treated with cytotoxic concentrations of BMS345541 (10 and 20 mM) in the presence or absence of Dox and cell survival was examined using MTT. ***Po0.001, **Po0.01, doxycycline treated (Dox þ ) compared with untreated (DoxÀ). (d) Vector control and shRNA-mediated downregulated clones were treated with IKK inhibitor (10 and 20 mM) and cell survival was examined using MTT assay. Results represent mean±s.d. of three experiments. @@Po0.01, @Po0.05 vector V1 treated with BMS345541 compared with untreated V1; ##Po0.01, #Po0.05 vector V3 treated with BMS345541 compared with untreated V3; !!!Po0.001, !!Po0.01 C4-2 treated with BMS345541 compared with untreated C4-2; &&&Po0.001 C4-4 treated with BMS345541 compared with untreated C4-4; ***Po0.001 **Po0.01 BMS345541 treated C4-2 and C4-4 compared with BMS345541 treated V1 and V3. (e) Cells were transfected with nuclear factor (NF)-kB–luciferase and luciferase activity determined as before. The relative light units (RLUs) are expressed after normalizing with Renilla and represent mean±s.d. of three experiments. ***Po0.001 C4-2 compared with V1. **Po0.01,***Po0.001 OVCAR 8 and DOV13 compared with IOSE and SKOV3. (f) Cells were treated with IKK inhibitor (BMS345541) and MTT was performed after 48 h. The data are representation of two individual experiments. *Po0.05, ***Po0.01 OVCAR 8 and DOV13 compared with IOSE and SKOV3. can modulate p300 interaction with NF-kB, we co- activity in clonal pool compared with that of vector transfected p300 complementary DNA and pool (Figure 7a), indicating that loss of TCEAL7 maybe NF-kB-reporter constructs into OSEtsT/hTERT vector contributing to p300-mediated NF-kB activity. Co- and TCEAL7-downregulated clonal pools. Both the transfection of p65 with p300 further heightened vector and clonal pool showed increase in NF-kB the p300-mediated NF-kB reported activity in both reporter activity in the presence of p300 (Figure 7a). vector and clonal pools (Figure 7b). Co-expression However, there was a significantly higher NF-kB of TCEAL7 significantly inhibited the p300- and the

Oncogene TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1368

Figure 5 TCEAL7 expression inhibits nuclear factor (NF)-kB -dependent reporter expression. (a) 293T cells were transiently co- transfected with various NF-kB pathway signaling molecules (NIK, IkB kinase (IKK)-a, IKK-b, p65 and p50) with NF-kB reporter along with thymidine kinase promoter-Renilla luciferase reporter plasmid (PRL-TK). After 24 h, cells were processed for luciferase activity. pcDNA3 was used to normalize total DNA content. The relative light units (RLUs) are expressed after normalizing with Renilla and expressed as mean±s.e.m. from four independent experiments. ***Po0.001, **Po0.01, *Po0.05, NS, nonsignificant, TCEAL transfected compared with pcDNA transfected. (b) Batch vector (black filled bar) and clonal pool (grey filled bar) were transiently co-transfected with IKK-b, p65 and p50 along with NF-kB reporter, and luciferase activity was examined at 24 h after transfection as described before. RLUs are expressed after normalizing with Renilla and expressed as mean±s.e.m. from four independent experiments. ***Po0.001 **Po0.01 clonal pool compared with vector pool. (c) OSEtsT/hTERT cells were transiently co-transfected with IKK-b, p65 and p50 in the presence (grey filled bar) or absence of TCEAL7 and the NF-kB reporter activity was assayed. RLUs are expressed after normalizing with Renilla and expressed as mean±s.e.m. from four independent experiments. ***Po0.001, *Po0.05, NS, nonsignificant, TCEAL transfected compared with pcDNA transfected. hTERT, human telomerase reverse transcriptase; OSEtsT, ovarian surface epithelial cells immortalized with temperature-sensitive large T antigen; TCEAL7, transcription elongation factor A-like 7.

p65–p300-mediated NF-kB activity in both vector and Discussion clonal pools (Figure 7b), indicating a downstream modulation by TCEAL7. Previously, we have shown that loss of TCEAL7 in To determine whether TCEAL7 will modulate immortalized, non-transformed ovarian epithelial cell p300–NFkB interaction at the promoter level, we line OSEtsT/hTERT resulted in increased proliferation performed chromatin immunoprecipitation (ChIP) and anchorage-independent growth, suggesting that assay with OSEtsT/hTERT vector 1 and TCEAL7- endogenous TCEAL7 may act as a cellular repressor downregulated clone C4-2. For each, DNA/protein of transformation. Additional studies showed that complexes were immunoprecipitated with anti-p65, TCEAL7 negatively modulated Myc activity (Chien anti-p50 and anti-p300 antibodies and the purified et al., 2008). In this study, we have made several novel DNA was amplified with primers flanking NF-kB observations and show for the first time that TCEAL7 promoter site in IL-6 and IL-8 promoters. As shown expression negatively regulates NF-kB at the basal level in Figure 7c there is an increased presence of p300 in and possibly also interferes with the functioning of an both IL-6 (4-fold) and IL-8 (3.5-fold) NF-kB promoter active NF-kB complex at the target site, by regulating sites in the TCEAL7-downregulated clone (C4-2) when p300–p65 interactions. Our conclusion is based on the compared with TCEAL7-expressing vector clone (V1) following observations: (1) loss of TCEAL7 in stable (Figure 7c). In addition, there was a more than twofold clonal lines of OSEtsT/hTERT by shRNA results in an increase in the recruitment of p65 at promoters of activated NF-kB pathway, leading to increased expres- both the genes in the TCEAL7-downregulated clone sion of its target genes, whereas (2) transient expression (C4-2) compared with TCEAL7-expressing vector clone of TCEAL7 in these downregulated clones and ovarian (V1) (Figure 7c). Collectively, these results suggest that cancer cell lines inhibits NF-kB activity. However, (3) TCEAL7 may interfere with the recruitment of p300 TCEAL7 neither affects the cytoplasmic signaling aspect or association of p300 with p65, thus modulating the of NF-kB pathway nor inhibits the TNF-a-stimulated NF-kB transcriptional activity and expression of its nuclear translocation or DNA binding of an activated target genes. NF-kB complex at its consensus binding site, but still

Oncogene TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1369

Figure 6 Transcription elongation factor A-like 7 (TCEAL7) inhibits transcriptional activity of p65. (a) Flip-In-293 T cells were plated and treated with 1 mM doxycycline (dox). After 24 h of serum-free incubation, cells were treated with tumor necrosis factor-a (TNF-a; 30 and 60 m) and nuclear extract was prepared. Immunoblot analysis of p50 and p65 was performed in nuclear extract of treated samples. Lamin B was used as control for loading and nuclear fraction. Last panel represents the expression of TCEAL7 induced by dox treatment. Blot is representation of two individually performed experiments. (b, c) A total of 100 mg of the same nuclear extract at 60 m time point was used for detection of p50 or p65 bound on nuclear factor (NF)-kB consensus sequence using Active Motif enzyme-linked immunosorbent assay (ELISA) kit. Results are mean þ s.d. of four values. ***Po0.001, **Po0.01 TNF-treated cells compared with untreated; NS, nonsignificant, doxycycline treated compared with untreated. (d) Vector (V1) and TCEAL7- downregulated clones (C4-2) were transiently transfected with Gal-p65 or DNA binding domain-gal4 (Gal-DBD) along with PTL- luciferase in the absence or (e) presence of IkB kinase-a (IKK-a) and/or TCEAL7 expression vectors with equal content of total DNA normalized with pcDNA3. ***Po0.001 C4-2 compared with V1; ##Po0.01, ###Po0.001 TCEAL7 þ p65-gal transfected compared with p65-gal transfected alone in (b). ***Po0.001 C4-2 compared with V1; ##Po0.01, ###Po0.001 p65-gal þ TCEAL7 þ IKK2 transfected compared with p65-gal þ IKK2 transfected alone; NS, nonsignificant C4-2 transfected compared with vector transfected (last two lanes) in (c). (f) At 24 h after transfection, cells were treated with IKK inhibitor, BMS345541 (5 mM) for another 24 h and assayed for luciferase activity. Results are mean±s.d. of three different experiments. ***Po0.001 inhibitor-treated groups compared with untreated respective groups. inhibits p65 transcriptional activity. (4) In the absence of activity. This phenomenon was not unique to this cell TCEAL7, there is increased interaction of p300 and p65 line, as induction of TCEAL7 in the Flp-In 293T system at NF-kB promoter sites of its target genes. Thus, and its exogenous expression in IOSE-523, DOV13, TCEAL7 may potentially be interfering with the SKOV3ip and OVCAR8 ovarian cancer cell lines also functioning of an active NF-kB complex through resulted in increased NF-kB activity. These data suggest p300–p65 regulation. The mechanism by which that TCEAL7 is involved in negatively regulating NF- TCEAL7 modulates p300–p65 interaction is currently kB. Consistent with these observations, increased unknown. NF-kB activity resulted in the upregulation of various The transcription factor complexes of NF-kB are one NF-kB target genes, such as IL-6, IL-8 and VEGF, Cox-2 of the most widely studied in cancer because of their and ICAM and Bcl-xl genes, which are implicated to widespread de-regulation and transcriptional control of have an important role in ovarian and other cancers numerous genes that produce cytokines, chemokines, (Chen et al., 2007; Collinson et al., 2008). growth factors and anti-apoptotic factors that aid in the Ovarian tumors secrete high amounts of various pro- progression, maintenance and also chemoresistance of inflammatory cytokines, including IL-6 and IL-8. IL-6 various tumors including ovarian tumors (Karin, 2006; and its downstream signaling through signal transducer Ahn et al., 2007). Downregulation of TCEAL7 in and activator of transcription 3 is associated with OSEtsT/hTERT cells promoted transcription factor increased proliferation, survival and secretion of matrix activity of several transcription factors, including NF-kB metalloproteinases and angiogenic mediators (Nilsson (Chien et al., 2008), which translated into signifi- et al., 2007). IL-8 exerts an effect as a downstream cantly increased NF-kB reporter activity and DNA signaling mediator for TNF-a and lysophosphatidic acid binding in OSEtsT/hTERT clones with stably down- in ovarian cancer, augmenting proliferation, angio- regulated TCEAL7 expression compared with vector genesis and invasion (Abdollahi et al., 2003). VEGF is clones expressing TCEAL7. Reconstitution of TCEAL7 considered as one of the causal factors for ascites expression in these clones abrogated the increased formation in ovarian cancer and inhibiting VEGF by

Oncogene TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1370

Figure 7 Transcription elongation factor A-like 7 (TCEAL7) regulates recruitment of p300 and p65. (a) Batch vector and clonal pool were transiently co-transfected with p300 along with nuclear factor (NF)-kB reporter, and luciferase activity was examined at 24 h after transfection as described before. The relative light units (RLUs) are expressed after normalizing with Renilla and expressed as mean±s.e.m. from three independent experiments. ***Po0.001 p300 transfected (black bars) compared with pcDNA3 transfected (white bars). (b) Batch vector and clonal pool were transiently co-transfected with p300 and or p65 and TCEAL7 along with NF-kB reporter, and luciferase activity was examined at 24 h after transfection as described before. RLUs are expressed after normalizing with Renilla and expressed as mean±s.e.m. from four independent experiments. **Po0.01, ***Po0.001 TCEAL7 transfected (black bars) compared with others. (c) Chip assay was performed to show levels of in vivo binding of p65, p50 and p300 on interleukin (IL)-8 and IL-6 promoter at the NF-kB site both vector (V1) and TCELA7-downregulated clone (C4-2). Immunoglobulin G was used to precipitate as a negative control. Input control DNA was diluted fivefold before PCR amplification. The band intensity is expressed as fold-change underneath the panels.

pharmacological means is currently undergoing testing formation of the active NF-kB complex at its consensus as an anti-angiogenic therapy in the treatment of site. On the other hand, loss of TCEAL7 does result in ovarian cancer patients. Cox-2, another target of NF- an increased NF-kB complex bound at the DNA. kB, is a key enzyme in prostaglandin production and is Studies with TNF-a induction of NF-kB activity may involved in tumor onset and progression and is also indicate that TCEAL7 does not affect the TNF-a–NF-kB associated with chemotherapy resistance and poor pathway and functions at regulating the basal level of outcome in ovarian cancer (Ferrandina et al., 2006). NF-kB activation in a normal cell. Under these ICAM-1 has been reported to be upregulated in the circumstances, loss of TCEAL7 eliminates this basal leading edge of tumors in which it promotes recruitment regulation that results in augmentation of NF-kB of circulating macrophages and eventually neutrophils activation when exposed to stimuli such as TNF-a that help in breaking trans-endothelial barriers by activity. Although the mechanism/s involved in elastase production, and support cell migration (Roland TCEAL7-mediated inhibition of NF-kB activity is et al., 2007). Other reports have shown an association of currently unknown, our data suggest that TCEAL7 ICAM expression with resistance in ovarian carcinoma could interfere with the recruitment of co-activator (Giavazzi et al., 1994; Opala et al., 2003). Bcl-xl, an anti- CBP/p300 and/or modulate p65 interaction with CBP/ apoptotic gene, has been shown to be overexpressed in p300. It has been reported that CBP/p300 interacts with ovarian tumors and is correlated with shorter disease- the cyclic adenosine monophosphate-responsive free survival after chemotherapy and recurrent disease element-binding protein and several other proteins in a (Williams et al., 2005; Kar et al., 2007). We have cell signal-regulated manner. CBP/p300 with its intrinsic previously reported that TCEAL7 expression is lost in enzyme activity can acetylate histones, which allows the early-stage ovarian tumors (Chien et al., 2005). Further unwinding or loosening of chromatin. p300 can also correlation studies involving any of the target genes and acetylate p65. Phosphorylation of p65 promotes the TCEAL7 expression in ovarian tumors would provide interaction with CBP, leading to enhanced transactiva- important insights into the role of these target genes in tion potential of NF-kB (Gerritsen et al., 1997). It acts oncogenic transformation mediated by functional loss of as a bridging factor between NF-kB and DNA-binding TCEAL7. sites. The mechanism by which TCEAL7 modulates the Our effort to determine the mechanistic basis by recruitment of p65 or the interaction of p65–p300 is which TCEAL7 modulated upregulation of NF-kB currently unknown. activity suggested that TCEAL7 regulates NF-kB However, the consistent observation that TCEAL7 activity at the nuclear rather than at the cytoplasmic inhibits NF-kB activity at the basal level, in the absence level. TCEAL7 does not seem to interfere with the of any stimuli, suggests that there is a basic mechanism

Oncogene TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1371 by which TCEAL7 is keeping NF-kB and maybe other For downregulation of TCEAL7 expression, OSEtsT/hTERT transcription factors (that may also require CBP/p300) cells were treated with retroviral supernatants to transduce in check in a normal cell. Loss of TCEAL7 eliminates pSUPER.retro constructs expressing shRNA targeting TCEAL7 this basal-level checkpoint of NF-kB, resulting in a mRNA (pSR-TCEAL7) or an empty pSUPER.retro vector higher activated level of NF-kB leading to vast (pSR) as described before (Chien et al., 2008). pleiotropic effects of target genes including cytokines, growth factors, angiogenic and anti-apoptotic factors, Transcription factor array which in conjunction with other alterations may provide For high-throughput analysis of transcription factor analysis, a fertile ground for tumor formation or sustain the we used Panomic’s TranSignal Protein/DNA array (cat development of tumors. no. MA1210) (Toutirais et al., 2003) containing an array membrane of 54 transcription factors as described before (Chien et al., 2008).

Materials and methods Semiquantitative reverse transcriptase–PCR PCR for TCEAL7 expression was performed as described Reagents before (Chien et al., 2008). TranSignal Protein/DNA array for various transcription factors, NF-kB–luciferase reporter, p65-gal and nuclear Real-time PCR (quantitative PCR) extraction kits were purchased from Panomics Inc. Antibody Real-time analysis for target genes was performed as described against pIkBa was purchased from Cell Signaling Technology before (Nath et al.,2009):Cox2,IL-6,IL-8andCyclinD1primers 32 (Beverly, MA, USA). [g- P]ATP (3000 Ci/mmol) were from were purchased from SA Biosciences (Frederick, MD, USA). PerkinElmer (Boston, MA, USA). Antibodies for p65, p50, cRel, Rel A, p52, IkB-a,IkB-b, IKK-a, IKK-b and Bcl-xl and oligonucleotides for NF-kB and NF-kB-conjugated agarose Plasmids, transfection and reporter assays 4 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 293T cells or OSEtsT/hTERT clones were seeded at 8 Â 10 ChIP grade p65, p50 and p300 antibodies were from Millipore cells per well in 24-well plates (in triplicates) 1 day before (MA, USA). TRIzol and Lipofectamine were from Invitrogen transfection. A total of 0.15 mg TCEAL7 or empty vector as Life Technologies (Grand Island, NY, USA). The enhanced control and 0.25 mg NF-kB–luciferase constructs were co- chemiluminescent-detecting reagents and nitrocellulose mem- transfected with 0.1 mg Renilla reporters. Luciferase activity brane were purchased from GE Healthcare (Piscataway, NJ, was measured at 24 h after transfection with Promega’s USA). Luciferase assay system was from Promega (Madison, Dual-Luciferase Reporter assay system according to the WI, USA). IkB kinase inhibitor (BMS345541) was from manufacturer’s instructions. For co-transfections, 0.15 mg Calbiochem (San Diego, CA, USA). The expression vectors of NF-kB–luciferase was transfected along with 0.1 mgof p50 and p65 were kindly provided by Dr R Pope (North- TCEAL7 and or other plasmids. The relative light units are western University Medical School, Chicago, IL, USA). The expressed after normalizing with Renilla luciferase to account expression vector for hemagglutinin-IKK-a and IKK-b were for variability in transfection efficiency. Expression vector of gift from Dr ZG Liu (National Institutes of Health, Bethesda, TCEAL7 was cloned by amplifying its coding region by PCR 0 0 MD, USA). The constitutive active IKK2 was purchased using primers 5 -GCAGGAAACAACAACAACATC-3 and 0 0 from Addgene (Cambridge, MA, USA). p300 complementary 3 -TTAAATGGGATAAGGGACGGT-3 and cloned into DNA was a gift from Dr J Boyes (MCCSC, London, UK). pcDNA3/GFP-CT TOPO cloning vector from Invitrogen following the manufacturer’s recommendation.

Cell culture Gel-shift The ovarian epithelial cell line, OSEtsT/hTERT, was initially Nuclear extracts from control vector and shRNA clones were immortalized with temperature-sensitive SV40 T-antigen isolated and electromobility shift assay was performed, as (OSEtsT) and subsequently with catalytic subunit of human described previously (Giri et al., 2002), with NF-kB consensus telomerase (OSEtsT/hTERT) (Kalli et al., 2002). The OSEtsT/ sequence, which was end labeled with [g-32P]ATP. Nuclear hTERT cells were grown in medium comprising Medium 1.99, extracts were normalized based on protein concentration, and MCDB105, sodium bicarbonate (1.1 g/l) and hygromycin equal amount of protein (5 mg) was loaded. DNA–protein (2 mg/ml), adjusted to pH 7.2 and supplemented with 15% complexes were resolved on 5% nondenaturing polyacryl- fetal bovine serum (Invitrogen). SV40t/T immortalized ovarian amide gel electrophoresis in 45 mM Tris (pH 7.8), 45 mM boric surface epithelial cell line, IOSE-523, was kindly shared by Dr acid and 1 mM EDTA (0.5 Tris–boric–EDTA), and run at Nelly Auersperg (University of British Columbia, Canada). Â 11 V/cm. The gels were dried and then autoradiographed at IOSE-523 cells were grown in Medium 1.99, MCDB105, 70 1C using X-ray film. For detecting supershift, the nuclear sodium bicarbonate (1.1 g/l) with 10% fetal bovine serum. À extracts were incubated with 1 mg of p65 and/or p50 antibodies All reagents for the medium were obtained from Sigma-Aldrich for 30 min before running the gel. (St Louis, MO, USA). 293T cells were obtained from American Type Culture Collection center and grown in Dulbecco’s modified Eagle’s medium (Mediatech, Inc., Herndon, VA, USA). Western blot Equal amounts of protein (40 mg/lane) were separated by electrophoresis on the sodium dodecyl sulfate gel and Generation of shRNA downregulated TCEAL7 clones in electrophoretically transferred to polyvinylidene fluoride OSEtsT/hTERT membrane. Blots were washed once with tris-buffered saline Immortalized OSE cells transduced with a temperature- supplemented with 0.2% Tween-20 and then blocked with the sensitive mutant of the SV40 large T antigen and catalytic same solution containing 5% non-fat dry milk for 1 h at room subunit of human telomerase, called OSEtsT/hTERT, temperature. The blocking solution was replaced with a were used for generation of clones (Kalli et al., 2002). solution containing primary antibody (1:1000 dilution) in 5%

Oncogene TCEAL7 negatively regulates NF-jB pathway R Rattan et al 1372 milk. After overnight rocking at 4 1C, the blots were washed recombination target site was introduced in 293T cells thrice for 5 min each in tris-buffered saline, 0.1% (w/v) Tween-20 by using vector, pFRT/lacZeo. TCEAL7 was cloned into and incubated with respective horseradish peroxidase-conjugated pcDNA5/ Flp recombination target and co-transfected with secondary antibody in 5% milk/tris-buffered saline supplemented pOG44 plasmid expressing Flp recombinase. The stable clones with 0.2% Tween-20 at room temperature for 1 h. After washing were selected using hygromycin resistance and zeocin sensitivity thrice in tris-buffered saline supplemented with 0.2% Tween-20, and expression of TCEAL7. the proteins were visualized using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). The blots were Chromatin immunoprecipitation assay stripped and re-probed with anti-b actin (Sigma) or b-tubulin for Chromatin immunoprecipitation was performed using Millipore equal protein loading control. Magna ChIP kit. In brief, 1 Â 107 cells were crosslinked with formaldehyde and sonicated to shear the DNA to Enzyme-linked immunosorbent assay (ELISA) fragments of 200–1000 bp. ChIP grade antibodies from After 48 h, serum-free supernatants of the cultured cells were Millipore (p65, p50 or p300) were used for immunoprecipita- taken out for detection of the levels of IL-6 and tion of the protein–DNA complex. DNA was purified IL-8 (R&D Biosystems, Minneapolis, MN, USA) and VEGF according to the instructions and amplified by standard PCR (Immuno Biological Labs, Japan, no. 17741). The ELISA was and ran on agarose gels. IL-6 promoter: sense 50-AGTGGT performed according to the manufacturer’s instructions. For GAAGAGACTCAGTG-30 and antisense 50-GGCAGAAT quantitation of the levels of p65 and p50 in nuclear extract GAGCCTCAGA-30. IL-8 promoter: sense 50-GGGCCATCA from treated and untreated cells, ELISA for p65 and p50 were GTTGCAAATC-30 and antisense 50-TTCCTTCCGGTGGT performed according to the manufacturer’s instructions TTCTTC-30. (Active Motif, Carlsbad, CA, USA) by using equal amounts of nuclear extracts. Statistical analysis Cell proliferation Statistics for various parameters were analyzed with one-way A total of 25–50 000 cells per well were plated according to the multiple-range analysis of variance GraphPad Prism 3.0 cell size in 24-well plates in triplicates. Next day was taken as software (GraphPad Software, Inc., La Jolla, CA, USA). day 0 and live cells were counted by Trypan blue exclusion dye Significances (P-value) between groups were determined using on odd days of incubation (0, 1, 3, 5 and 7 days) in treated and the Newman–Keuls test. A P-value of o0.05 and above was untreated cells with IKK inhibitor (BMS345541) purchased considered significant. from Calbiochem (Gibbstown, NJ, USA).

Cell survival Conflict of interest The viability of cells was evaluated using MTT assay. Viable cells take up the MTT dye and cleave tetrazolium salt to a dark The authors declare no conflict of interest. blue formazon product by mitochondrial dehydrogenase. The absorbance of the product was measured at 570 nm and a reference wavelength of 630 nm. MTT was performed after 24 Acknowledgements and 48 h of treatment. This work was supported by Ovarian Cancer Research Fund Generation of 293T-inducible cell line to RR as a Program of Excellence grant and with funds from An inducible system for expression of TCEAL7 was generated Mayo Foundation and Bernard and Edith Waterman Foun- using Flp-In System from Invitrogen. In brief, a Flp dation to VS.

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Oncogene