TCEAL7, a putative tumor suppressor gene, negatively regulates NF-κB pathway

Abstract

We have previously shown that a frequently downregulated gene, transcription elongation factor A-like 7 (TCEAL7), promoted anchorage-independent growth and modulated Myc activity in ovarian surface epithelial cells immortalized with temperature-sensitive large T antigen and human telomerase reverse transcriptase (OSEtsT/hTERT). Analysis of protein/DNA array showed that TCEAL7 downregulation resulted in an approximately twofold increase in nuclear factor (NF)-κB binding to its target DNA sequence. In this study we showed that short hairpin RNA (shRNA)-mediated downregulation of TCEAL7 in two different immortalized OSE cells showed higher NF-κB activity, as determined using reporter and gel-shift assays. Transient transfection of TCEAL7 inhibited the activation of NF-κB in TCEAL7-downregulated clones, IOSE-523 and in other ovarian cancer cell lines (OVCAR8, SKOV3ip and DOV13), suggesting that TCEAL7 negatively regulates NF-κB pathway. Consistent with this observation, TCEAL7-downregulated clones showed higher levels of NF-κB targets, such as pro-proliferative (cyclin-D1 and cMyc), pro-angiogenic (interleukin (IL)-6, IL-8 and vascular endothelial growth factor (VEGF)), inflammatory (intercellular adhesion molecule 1 (ICAM-1) and cyclooxygenase-2 (Cox-2)) and anti-apoptotic (B-cell lymphoma-extra large (Bcl-xl)) genes when compared with vector controls. Inhibition of NF-κB by IκB kinase (IKK) inhibitor (BMS 345541) attenuated cell survival and proliferation of TCEAL-knockdown clones. Although TCEAL7 inhibited p65 transcriptional activity, it did not modulate the cytoplasmic signaling of the NF-κB pathway, by itself or by tumor necrosis factor-α (TNF-α). Chromatin immunoprecipitation (ChIP) assays revealed increased recruitment of p65 and p300 to the promoters of IL-8 and IL-6 in TCEAL7-downregulated clones. Collectively, these results indicate a novel role for TCEAL7 in the negative regulation of NF-κB signaling at the basal level by modulating transcriptional activity of NF-κB on its target gene promoters, potentially providing a novel mechanism by which NF-κB activity may be deregulated in ovarian cancer cells.

Introduction

Ovarian cancer is the fifth most common cause of death of all cancers among women in the United States and the leading cause of death from gynecological malignancies with the 5-year survival rate of only 30% (Friedlander, 1998; Kenny et al., 2008). In an effort to identify genetic alterations associated with the initiation and progression of ovarian cancer, we performed high-throughput expression-based screening and identified transcription elongation factor A-like 7 (TCEAL7) as one of the frequently downregulated genes in ovarian cancer (Shridhar et al., 2001, 2002; Chien et al., 2005).

Transcription elongation factor A-like 7 encodes TCEAL7 protein and has a sequence similarity with a small family of brain-expressed (Bex) proteins, bex1, bex2 and bex3 (Rapp et al., 1990; Mukai et al., 2000; Chien et al, 2005). It shares amino acid sequence homology with transcription elongation factor A-like 1 (TCEAL1/p21/SIIR/pp21) and pp21 homolog (WBP5/TCEAL6) (Mukai et al., 2000, 2002). Although very little is known about the role of these proteins in cancer, pp21 homolog (TCEAL1) has been shown to suppress Rous sarcoma virus long-terminal repeat promoter activity and inhibit the transformation mediated by it in chicken embryo fibroblast (Yeh and Shatkin, 1994). A closely related protein, TFIIS/TCEA is involved in transcription elongation and transcript fidelity. TFIIS/TCEA promotes 3′ endoribonuclease activity of RNA polymerase II (pol II) and allows pol II to bypass transcript pause or ‘arrest’ during elongation process (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 because of methylation of the actively expressed allele on Xq22.1. Ectopic expression of TCEAL7 in TCEAL7-non-expressing cell lines induced cell death and suppressed colony-forming abilities (Chien et al., 2005). Recent studies indicate that stable downregulation of TCEAL7 expression in ovarian surface epithelial cells immortalized with temperature-sensitive large T antigen and human telomerase reverse transcriptase (OSEtsT/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 findings suggest that loss of TCEAL7 expression may promote the survival and growth of ovarian cancer cells.

In an effort to identify transcription factors modulated by TCEAL7 loss, we previously analyzed the DNA-binding activity of 54 transcriptional factors using the protein/DNA array (TranSignal Array I, Panomics, Fremont, CA, USA) in clonal line without TCEAL7 (TCEAL7 short hairpin RNA (shRNA)) compared with the clonal line with TCEAL7 (control shRNA) expression (Chien et al., 2008). Among the 54 transcription factors tested, we identified nuclear factor (NF)-κB as one of the transcription factors whose DNA-binding activity was modulated by TCEAL7 downregulation. Because of the important role of NF-κB in cancer (Karin, 2006) and more specifically in promoting ovarian cancer progression and chemoresistance, (Mabuchi et al., 2004a, 2004b; Chen et al., 2007), we sought to determine how loss of TCEAL7 modulates the NF-κB-dependent activity in ovarian cancer.

There are five identified members of the NF-κB/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-κB. The most abundant NF-κB dimer present in most cells is composed of p50:p65 subunits. In unstimulated cells, NF-κB is usually sequestered in the cytoplasm, bound to the inhibitor IκBs (inhibitor of κB). Phosphorylation of IκB proteins by upstream kinases, IκB kinases (IKK), targets IκBs 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 NFκB-responsive elements of target genes, resulting in the activation of various genes that promote proliferation (cyclin-D1 and interleukin-6 (IL-6)), invasion (urokinase plasminogen activator, matrix metallopeptidase 9 and tumor necrosis factor-α (TNF-α)), metastasis (intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule), angiogenesis (vascular endothelial growth factor (VEGF) and IL-8) and suppression of apoptosis (B-cell lymphoma (Bcl)-2, Bcl-xl and inhibitors of apoptosis) (Aggarwal, 2004). An activated NF-κB pathway due to various aberrant upstream signaling events is characteristic of various cancer types, including ovarian cancer, and is also frequently associated with chemoresistance and tumor progression (Arlt and Schafer, 2002; Aggarwal et al., 2004; Camp et al., 2004; Karin, 2006; Chen et al., 2007). However, little is known about the molecular alterations that lead to NF-κB activation in tumor cells.

In this study, we show that loss of TCEAL7 in ovarian surface epithelial cells promotes NF-κB activation and leads to an increase in the levels of NF-κB target genes involved in proliferation, inflammation, angiogenesis and inhibition of apoptosis. These observations uncover a previously unknown pathway regulating NF-κB activity by a novel gene, TCEAL7, in ovarian surface epithelial cells.

Results

TCEAL7 negatively regulates NF-κB pathway

To examine the role of TCEAL7 in cellular signaling, we generated several stable clones expressing shRNA targeted against TCEAL7 in OSEtsT/hTERT cells and clonal lines expressing empty (control) shRNA construct. Immunoblot analysis showed that there was a complete loss of TCEAL7 expression in several clonal lines when compared with vector clones (Chien et al., 2008). shRNA-stable clones (C4-2 and C4-4), which showed significant downregulation of TCEAL7 expression compared with vector-transduced clones (V1 and V3) (Figure 1a) by real-time PCR, were chosen for further analysis.

Figure 1
figure1

Short hairpin RNA (shRNA)-mediated TCEAL7-downregulated clones show higher nuclear factor (NF)-κB activity. (a) Real-time PCR analysis of TCEAL7 in shRNA-downregulated TCEAL7 clones (C4-2 and C4-4) and vector clones (V1 and V3) in OSEtsT/hTERT cells. ***P<0.001 C4-2 and C4-4 compared with V1 and V3. (b) Nuclear extract from V1 and C4-2 clones was used to perform transcription factor (TF) array as described in Material and methods. Downregulation of endogenous TCEAL7 in OSEtsT/hTERT (TCEAL7 shRNA, C4-2) resulted in increased NF-κB transcription factors binding to target DNA compared with stable clone expressing endogenous TCEAL7 (control shRNA, V1). Densitometric tracings are shown graphically. ***P<0.001 compared with vector V1. (c) Downregulation of TCEAL7 augments the basal NF-κB promoter luciferase activity. The relative light units (RLUs) are expressed after normalizing with Renilla luciferase and expressed as mean±s.e.m. from four independent experiments. ***P<0.001, **P<0.01 compared with both vectors V1 and V3. (d) Downregulation of endogenous TCEAL7 by shRNA in IOSE-523 resulted in elevated basal NF-κB promoter luciferase activity, whereas the empty vector had no effect. Inset shows the downregulation of TCEAL7 expression by PCR compared with untransfected (Untf) and empty vector (ShV). ***P<0.001 shRNA TCEAL7 compared with shVector and untransfected. hTERT, human telomerase reverse transcriptase; OSEtsT, ovarian surface epithelial cells immortalized with temperature-sensitive large T antigen; TCEAL7, transcription elongation factor A-like 7.

Our previous analysis of protein/DNA binding assay for 54 transcription factors, using nuclear lysates from OSEtsT/hTERT clonal lines with TCEAL7 (control shRNA vector, V1) or without TCEAL7 (TCEAL shRNA clone, C4-2) (Chien et al., 2008), identified several transcription factors modulated by loss of TCEAL7 including NF-κB, which showed an approximately twofold increase in NF-κB binding to its target sequence in TCEAL7 in C4-2 clone compared with clone V1 (Figure 1b).

To test whether increased NF-κB DNA binding, which is associated with TCEAL7 knockdown, correlates with increased NF-κB promoter activities, we determined the transcriptional activity of NF-κB in these clones. TCEAL7-downregulated clones (C4-2 and C4-4) showed significantly higher NF-κB– luciferase activity when compared with vector-transduced clones (Figure 1c). To determine that this phenomenon was not unique to this one cell line, we downregulated TCEAL7 expression using shRNA in another SV40t/T immortalized IOSE-523 cell line expressing TCEAL7 (Supplementary Figure S1A). Downregulation of TCEAL7 in IOSE-523 (Inset in Figure 1d) also resulted in upregulation of basal NF-κB activity (Figure 1d). In addition, TCEAL7 shRNA-downregulated clones were found to acquire higher levels of NF-κB complex bound to its consensus site compared with vector clones, as determined using electromobility shift assay (Supplementary Figure S1B). Collectively, these results indicate that loss of TCEAL7 results in upregulation of NF-κB activity.

Re-expression of TCEAL7 inhibits NF-κB activity in ovarian cells

To determine whether TCEAL7 could attenuate NF-κB transcriptional activity, we transiently transfected TCEAL7 expression construct into OSEtsT/hTERT clones (C4-2 and C4-4) and vector clones (V1 and V3). As shown in Figure 2a, re-expression of TCEAL7 significantly abrogated the elevated NF-κB–luciferase activity in both TCEAL7 downregulated and vector clones. In addition, doxycycline-induced expression of TCEAL7 in Flp-In-293T stable cell line (Inset in Figure 2b) significantly reduced the NF-κB reporter activity (Figure 2b). Re-expression of TCEAL7 in batch IOSE-523 cells with downregulation of TCEAL7 also blocked the elevated NF-κB reporter activity (Figure 2c) compared with empty vector shRNA control cells. Transient transfection of TCEAL7 expression in DOV13, SKOV3ip and OVCAR8 (Inset in Figures 2d–f) also significantly inhibited the NF-κB activity (Figures 2d–f). Collectively, these results suggest that although loss of TCEAL7 upregulates NF-κB transcriptional activity, re-expression of TCEAL7 attenuates this increase in NF-κB transcriptional activity.

Figure 2
figure2

Re-expression of transcription elongation factor A-like 7 (TCEAL7) inhibits nuclear factor (NF)-κB activity. (a) Transient re-expression of TCEAL7 inhibits NF-κB activity. Vector (V1 and V3) and short hairpin RNA (shRNA; C4-2 and C4-4) clones were co-transfected with TCEAL7 and NF-κB 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. ***P<0.001 compared with respective pcDNA-transfected cells. (b) TCEAL7-Flip-In-293T cells were pre-treated with 1 μM doxycycline (dox) and transiently transfected with NF-κB 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. ***P<0.001 doxycycline-treated to untreated. (c) shRNA TCEAL7-downregulated IOSE-523 cells were transfected with S-tag-labeled TCEAL7 cDNA and NF-κB reporter as described above. Inset shows expression of S-tag, representing TCEAL7 expression after transfection. **P<0.001 TCEAL7 compared with pcDNA transfected. (d–f). Ovarian cell lines were transfected with TCEAL7 and NF-κB reporter and their luciferase activity was determined as described earlier. Inset shows expression of S-tag, representing TCEAL7 expression after transfection. **P<0.01, **P<0.001 TCEAL7 compared with pcDNA transfected.

Loss of TCEAL7 results in upregulation of NF-κB target genes

To determine whether upregulation of NF-κB pathway by loss of TCEAL7 results in altered levels of NF-κB target genes, we determined the levels of a subset of NF-κB target genes using enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis in TCEAL7shRNA-downregulated clones (C4-2 and C4-4) and in vector clones V1 and V3. Among the various NF-κB target genes tested, we found significant upregulation of pro-angiogenic factors, such as IL-8, IL-6 and VEGF, by ELISA (Figures 3a–c). Immunoblot analysis showed increased protein levels of pro-proliferative genes Myc and cyclin-D1, pro-inflammatory genes cyclooxygenase-2 (Cox-2) and ICAM-1 and anti-apoptotic gene Bcl-xl in OSEtsT/hTERT clones with TCEAL7 downregulation compared with the vector clones expressing endogenous TCEAL7 (Figure 3d). Expression of Cox-2, cyclin-D1, IL-6 and IL-8 were also validated using real-time PCR in both the vector and clonal pools to show regulation at the transcriptional level (Supplementary Figure S2). These data indicate that loss of TCEAL7 expression results in the upregulation of NF-κB target genes, further confirming our primary observation that activation of NF-κB pathway in the absence of TCEAL7 may be one of the mechanisms by which normal cells acquire a proliferative and survival advantage.

Figure 3
figure3

Short hairpin RNA (shRNA)-mediated downregulated clones with transcription elongation factor A-like 7 (TCEAL7) show higher expression of nuclear factor (NF)-κB targets genes. Vector control and shRNA-mediated downregulated clones were plated, and after 48 h of serum-free media, cell supernatant was used for detection of (a) interleukin (IL)-8, (b) IL-6 and (c) vascular endothelial growth factor (VEGF) using enzyme-linked immunosorbent assay (ELISA), as described under Materials and methods. Results represent mean±s.d. of three individual experiments. ***P<0.001 clones (C4-2 and C4-4) compared with vectors V1 and V3. (d) Cell lysates from TCEAL7-downregulated clones (C4-2 and C4-4) and vector clones (V1 andV3) were used for detection of NF-κB target genes: cyclin-D1, cMyc, intercellular adhesion molecule (ICAM), cyclooxygenase-2 (Cox-2), and B-cell lymphoma-extra large (Bcl-xl) proteins using immunoblot analysis. The lowest panel indicates β-tubulin levels for equal loading. Blots are representations of two independent experiments.

Inhibition of NF-κB pathway reverses the growth potential conferred by TCEAL-7 loss

We previously reported that TCEAL7 loss results in increased proliferation in OSEtsT/hTERT cells, and 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 NF-κB pathway to examine its effect on cell proliferation and survival in TCEAL7-downregulated OSEtsT/hTERT clones. Treatment of either 293T Flp-In cells (Figure 4a) or OSEtsT/hTERT clones (Figure 4b) with BMS345541 at a non-toxic concentration (5 μM), resulted in significant inhibition of the increased proliferation observed in the absence of TCEAL7 when compared with untreated cells and vector clones. To examine whether the inhibition of NF-κB pathway would affect the survival of cells in the presence or absence of TCEAL7, 293T Flp-In cells were treated with BMS345541 at cytotoxic concentrations (10–20 μM), after doxycycline induction of TCEAL7 expression. The cells expressing TCEAL7 had higher percentage of cell death with IKK inhibitor when compared with cells with no TCEAL7 induction (Figure 4c). These data suggests that inhibition of NF-κB pathway abrogates the higher proliferative state observed in the absence of TCEAL7 and that inhibition of NF-κB in the presence of TCEAL7 is more detrimental to cells. However, TCEAL7shRNA OSEtsT/hTERT clones also showed increased cell death at cytotoxic concentration of 10 and 20 μM (Figure 4d) compared with the vector-transduced controls, which could be due to oncogenic addiction to the NF-κB pathway in these clones. To validate that NF-κB oncogenic addiction is found in ovarian cancer cell lines, we selected OVCAR8 and DOV13, two cell lines showing higher basal NF-κB activity (the same level as TCEAL7-downregulated clones) and IOSE-523 and SKOV3 with low basal NF-κB activity (Figure 4e). BMS345541 treatment resulted in increased cytotoxicity in OVCAR8 and DOV13 cells when compared with IOSE-523 and SKOV3 cells (Figure 4f). These data suggests the presence of NF-κB oncogenic addiction in ovarian cancer cells lines. Interfering with this oncogene addiction by modulating NF-κB activity may lead to decreased survival of these cells.

Figure 4
figure4

IκB 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 μM doxycycline (Dox) and treated with IKK inhibitor, BMS345541 (5 μM). Cells were counted with Trypan blue over a period of 5 days. Results represents mean±s.d. from three independent experiments. **P<0.01 Dox− compared with Dox+ treated cells, ###P<0.001 Dox- BMS345541 compared with Dox− and +++P<0.001 Dox+ BMS345541 compared with Dox+. (b) Vector control and short hairpin RNA (shRNA)-mediated downregulated clones were plated and treated with BMS345541 (5 μM) and cells were counted with Trypan blue. Results represent mean±s.d. of three experiments. ***P<0.001, **P<0.01, clones C4-2 and C4-4 compared with vectors V1 and V3; +++P<0.001, 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 μM) in the presence or absence of Dox and cell survival was examined using MTT. ***P<0.001, **P<0.01, doxycycline treated (Dox+) compared with untreated (Dox−). (d) Vector control and shRNA-mediated downregulated clones were treated with IKK inhibitor (10 and 20 μM) and cell survival was examined using MTT assay. Results represent mean±s.d. of three experiments. @@P<0.01, @P<0.05 vector V1 treated with BMS345541 compared with untreated V1; ##P<0.01, #P<0.05 vector V3 treated with BMS345541 compared with untreated V3; !!!P<0.001, !!P<0.01 C4-2 treated with BMS345541 compared with untreated C4-2; &&&P<0.001 C4-4 treated with BMS345541 compared with untreated C4-4; ***P<0.001 **P<0.01 BMS345541 treated C4-2 and C4-4 compared with BMS345541 treated V1 and V3. (e) Cells were transfected with nuclear factor (NF)-κB–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. ***P<0.001 C4-2 compared with V1. **P<0.01,***P<0.001 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. *P<0.05, ***P<0.01 OVCAR 8 and DOV13 compared with IOSE and SKOV3.

Mechanism of TCEAL7-mediated regulation of NF-κB pathway

In general, NF-κB may form a hetero-dimer with any one of the five members, including p65, p50, p52, cRel and RelB, depending upon the cell type and nature of stimulation (Ahn et al., 2007). As shown previously (Supplementary Figure S1B), TCEAL7-downregulated OSEtsT/hTERT clones (C4-2 and C4-4) showed higher amounts of NF-κB complex when compared with vector clone V1. To identify the predominant component of NF-κB complex in the OSEtsT/hTERT clonal lines, we performed a supershift electromobility shift assay (Supplementary Figure S3A). The supershift of NF-κB complex was detected with p65 and p50 antibodies compared with other components (cRel, RelB or p52), indicating that the active dimer formed in OSEtsT/hTERT cells in the absence of TCEAL7 is the p65:p50 dimer (Supplementary Figure S3A, lanes 9 and 10 for C4-2 and lanes 16 and 17 for C4-4). To further delineate the mechanism underlying this observation, nuclear and cytoplasmic fractions were prepared from the vector and the TCEAL7 shRNA clones to examine whether any changes in the cytoplasmic signaling of the NF-κB pathway could be detected. The nuclear extracts of both the downregulated clones C4-2 and C4-4 showed slightly higher levels of p65 and p50 (Supplementary Figure S3B) when compared with the vector clones; however, no significant change was observed in the cytosolic extract (Supplementary Figure S3C). We did not observe any changes in the phosphorylation of IκBα and degradation of IκBα/β (Supplementary Figure S3D). These data suggest that in the absence of TCEAL7, none of the cytosolic transducers undergo any changes and possibly TCEAL7 may not be modulating the cytosolic part of NF-κB-mediated signaling.

Upstream activators of NF-κB are not modulated by TCEAL7

To further confirm this observation, we co-transfected TCEAL7 expression vector in the presence or absence of various upstream effectors of NF-κB pathway and examined the NF-κB–luciferase activity. As depicted in Figure 5a, transient transfection of TCEAL7 inhibited both the basal NF-κB activity and NIK-, IKKα-, IKKβ- and p65-mediated activation of NF-κB in 293T cells. Moreover, transient transfection of IKK-β, p65 and p50 induced higher NF-κB–luciferase activity in TCEAL7 shRNA pool compared with control vector pool (Figure 5b). Similar to 293T cells, re-expression of TCEAL7 also inhibited IKK-β and p65-mediated induction of NF-κB–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-κB activity at the nuclear level.

Figure 5
figure5

TCEAL7 expression inhibits nuclear factor (NF)-κB -dependent reporter expression. (a) 293T cells were transiently co-transfected with various NF-κB pathway signaling molecules (NIK, IκB kinase (IKK)-α, IKK-β, p65 and p50) with NF-κB 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. ***P<0.001, **P<0.01, *P<0.05, 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-β, p65 and p50 along with NF-κB 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. ***P<0.001 **P<0.01 clonal pool compared with vector pool. (c) OSEtsT/hTERT cells were transiently co-transfected with IKK-β, p65 and p50 in the presence (grey filled bar) or absence of TCEAL7 and the NF-κB reporter activity was assayed. RLUs are expressed after normalizing with Renilla and expressed as mean±s.e.m. from four independent experiments. ***P<0.001, *P<0.05, 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.

TCEAL7 inhibits p65-mediated transcriptional activity

To determine whether TCEAL7 interferes with an active complex at the NF-κB binding site of target genes, we induced the expression of TCEAL7 with doxycycline and treated the cells with TNF-α for 30 and 60 min to stimulate NF-κB signaling. As shown in Figure 6a, TNF-α treatment induced the nuclear translocation of p50 and p65 in both doxycycline-treated and -untreated cells, indicating that induction of TCEAL7 expression (by doxycycline treatment) does not affect the nuclear translocation of the NF-κB complex (Figure 6a). In addition, quantification of p50 and p65 at the binding site was determined using ELISA assay. Induction of TCEAL7 by doxycycline treatment did not affect the TNF-α-induced binding of p65 or p50 (Figures 6b and c) with NF-κB consensus sequence. This suggests that TCEAL7 does not influence TNF-α-stimulated NF-κB activity, but can inhibit the basal activity of NF-κB by an as yet unknown mechanism.

Figure 6
figure6

Transcription elongation factor A-like 7 (TCEAL7) inhibits transcriptional activity of p65. (a) Flip-In-293 T cells were plated and treated with 1 μM doxycycline (dox). After 24 h of serum-free incubation, cells were treated with tumor necrosis factor-α (TNF-α; 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 μg of the same nuclear extract at 60 m time point was used for detection of p50 or p65 bound on nuclear factor (NF)-κB consensus sequence using Active Motif enzyme-linked immunosorbent assay (ELISA) kit. Results are mean + s.d. of four values. ***P<0.001, **P<0.01 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 IκB kinase-α (IKK-α) and/or TCEAL7 expression vectors with equal content of total DNA normalized with pcDNA3. ***P<0.001 C4-2 compared with V1; ##P<0.01, ###P<0.001 TCEAL7+ p65-gal transfected compared with p65-gal transfected alone in (b). ***P<0.001 C4-2 compared with V1; ##P<0.01, ###P<0.001 p65-gal + TCEAL7+ IKK2 transfected compared with p65-gal+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 μM) for another 24 h and assayed for luciferase activity. Results are mean±s.d. of three different experiments. ***P<0.001 inhibitor-treated groups compared with untreated respective groups.

To further corroborate this observation, we used p65–DNA binding domain-gal4, a chimeric transactivator, which contains part of the transcriptional activation domain of the NF-κB-p65 protein fused to the DNA-binding domain of GAL-4 protein from yeast. Consistent with the previous results, TCEAL7-downregulated clones showed significantly higher p65-transcriptional activity compared with vector control and transient transfection of TCEAL7 reversed the p65 transcriptional activity (Figure 6d). Moreover, TCEAL7 also inhibited IKK-mediated induction of p65 transcriptional activity (Figure 6e). Figure 6f shows the specificity of the Gal-4 transfection system, in which use of the IKK inhibitor, BMS345541 abrogated p65 activation. Collectively, these results suggest that although TCEAL7 does not affect the nuclear translocation of the complex or the DNA binding when induced by TNF-α, it is still able to negatively regulate NF-κB activity through modulating p65 transcriptional activity.

TCEAL7 modulates p65 and p300 recruitment to the promoters of target genes

Cyclic adenosine monophosphate-response element-binding protein (CREB)-binding protein (CBP)/p300 interacts with p65 and is responsible for transactivation of NF-κB complex bound at the target gene promoter (Gerritsen et al., 1997). To determine whether TCEAL7 can modulate p300 interaction with NF-κB, we co-transfected p300 complementary DNA and NF-κB-reporter constructs into OSEtsT/hTERT vector and TCEAL7-downregulated clonal pools. Both the vector and clonal pool showed increase in NF-κB reporter activity in the presence of p300 (Figure 7a). However, there was a significantly higher NF-κB activity in clonal pool compared with that of vector pool (Figure 7a), indicating that loss of TCEAL7 maybe contributing to p300-mediated NF-κB activity. Co-transfection of p65 with p300 further heightened the p300-mediated NF-κB reported activity in both vector and clonal pools (Figure 7b). Co-expression of TCEAL7 significantly inhibited the p300- and the p65–p300-mediated NF-κB activity in both vector and clonal pools (Figure 7b), indicating a downstream modulation by TCEAL7.

Figure 7
figure7

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)-κB 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. ***P<0.001 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-κB 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. **P<0.01, ***P<0.001 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-κB 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.

To determine whether TCEAL7 will modulate p300–NFκB interaction at the promoter level, we performed chromatin immunoprecipitation (ChIP) assay with OSEtsT/hTERT vector 1 and TCEAL7-downregulated clone C4-2. For each, DNA/protein complexes were immunoprecipitated with anti-p65, anti-p50 and anti-p300 antibodies and the purified DNA was amplified with primers flanking NF-κB promoter site in IL-6 and IL-8 promoters. As shown in Figure 7c there is an increased presence of p300 in both IL-6 (4-fold) and IL-8 (3.5-fold) NF-κB promoter sites in the TCEAL7-downregulated clone (C4-2) when compared with TCEAL7-expressing vector clone (V1) (Figure 7c). In addition, there was a more than twofold increase in the recruitment of p65 at promoters of both the genes in the TCEAL7-downregulated clone (C4-2) compared with TCEAL7-expressing vector clone (V1) (Figure 7c). Collectively, these results suggest that TCEAL7 may interfere with the recruitment of p300 or association of p300 with p65, thus modulating the NF-κB transcriptional activity and expression of its target genes.

Discussion

Previously, we have shown that loss of TCEAL7 in immortalized, non-transformed ovarian epithelial cell line OSEtsT/hTERT resulted in increased proliferation and anchorage-independent growth, suggesting that endogenous TCEAL7 may act as a cellular repressor of transformation. Additional studies showed that TCEAL7 negatively modulated Myc activity (Chien et al., 2008). In this study, we have made several novel observations and show for the first time that TCEAL7 expression negatively regulates NF-κB at the basal level and possibly also interferes with the functioning of an active NF-κB complex at the target site, by regulating p300–p65 interactions. Our conclusion is based on the following observations: (1) loss of TCEAL7 in stable clonal lines of OSEtsT/hTERT by shRNA results in an activated NF-κB pathway, leading to increased expression of its target genes, whereas (2) transient expression of TCEAL7 in these downregulated clones and ovarian cancer cell lines inhibits NF-κB activity. However, (3) TCEAL7 neither affects the cytoplasmic signaling aspect of NF-κB pathway nor inhibits the TNF-α-stimulated nuclear translocation or DNA binding of an activated NF-κB complex at its consensus binding site, but still inhibits p65 transcriptional activity. (4) In the absence of TCEAL7, there is increased interaction of p300 and p65 at NF-κB promoter sites of its target genes. Thus, TCEAL7 may potentially be interfering with the functioning of an active NF-κB complex through p300–p65 regulation. The mechanism by which TCEAL7 modulates p300–p65 interaction is currently unknown.

The transcription factor complexes of NF-κB are one of the most widely studied in cancer because of their widespread de-regulation and transcriptional control of numerous genes that produce cytokines, chemokines, growth factors and anti-apoptotic factors that aid in the progression, maintenance and also chemoresistance of various tumors including ovarian tumors (Karin, 2006; Ahn et al., 2007). Downregulation of TCEAL7 in OSEtsT/hTERT cells promoted transcription factor activity of several transcription factors, including NF-κB (Chien et al., 2008), which translated into significantly increased NF-κB reporter activity and DNA binding in OSEtsT/hTERT clones with stably downregulated TCEAL7 expression compared with vector clones expressing TCEAL7. Reconstitution of TCEAL7 expression in these clones abrogated the increased activity. This phenomenon was not unique to this cell line, as induction of TCEAL7 in the Flp-In 293T system and its exogenous expression in IOSE-523, DOV13, SKOV3ip and OVCAR8 ovarian cancer cell lines also resulted in increased NF-κB activity. These data suggest that TCEAL7 is involved in negatively regulating NF-κB. Consistent with these observations, increased NF-κB activity resulted in the upregulation of various NF-κB target genes, such as IL-6, IL-8 and VEGF, Cox-2 and ICAM and Bcl-xl genes, which are implicated to have an important role in ovarian and other cancers (Chen et al., 2007; Collinson et al., 2008).

Ovarian tumors secrete high amounts of various pro-inflammatory cytokines, including IL-6 and IL-8. IL-6 and its downstream signaling through signal transducer and activator of transcription 3 is associated with increased proliferation, survival and secretion of matrix metalloproteinases and angiogenic mediators (Nilsson et al., 2007). IL-8 exerts an effect as a downstream signaling mediator for TNF-α and lysophosphatidic acid in ovarian cancer, augmenting proliferation, angiogenesis and invasion (Abdollahi et al., 2003). VEGF is considered as one of the causal factors for ascites formation in ovarian cancer and inhibiting VEGF by pharmacological means is currently undergoing testing as an anti-angiogenic therapy in the treatment of ovarian cancer patients. Cox-2, another target of NF-κB, is a key enzyme in prostaglandin production and is involved in tumor onset and progression and is also associated with chemotherapy resistance and poor outcome in ovarian cancer (Ferrandina et al., 2006). ICAM-1 has been reported to be upregulated in the leading edge of tumors in which it promotes recruitment of circulating macrophages and eventually neutrophils that help in breaking trans-endothelial barriers by elastase production, and support cell migration (Roland et al., 2007). Other reports have shown an association of ICAM expression with resistance in ovarian carcinoma (Giavazzi et al., 1994; Opala et al., 2003). Bcl-xl, an anti-apoptotic gene, has been shown to be overexpressed in ovarian tumors and is correlated with shorter disease-free survival after chemotherapy and recurrent disease (Williams et al., 2005; Kar et al., 2007). We have previously reported that TCEAL7 expression is lost in early-stage ovarian tumors (Chien et al., 2005). Further correlation studies involving any of the target genes and TCEAL7 expression in ovarian tumors would provide important insights into the role of these target genes in oncogenic transformation mediated by functional loss of TCEAL7.

Our effort to determine the mechanistic basis by which TCEAL7 modulated upregulation of NF-κB activity suggested that TCEAL7 regulates NF-κB activity at the nuclear rather than at the cytoplasmic level. TCEAL7 does not seem to interfere with the formation of the active NF-κB complex at its consensus site. On the other hand, loss of TCEAL7 does result in an increased NF-κB complex bound at the DNA. Studies with TNF-α induction of NF-κB activity may indicate that TCEAL7 does not affect the TNF-α–NF-κB pathway and functions at regulating the basal level of NF-κB activation in a normal cell. Under these circumstances, loss of TCEAL7 eliminates this basal regulation that results in augmentation of NF-κB activation when exposed to stimuli such as TNF-α activity. Although the mechanism/s involved in TCEAL7-mediated inhibition of NF-κB activity is currently unknown, our data suggest that TCEAL7 could interfere with the recruitment of co-activator CBP/p300 and/or modulate p65 interaction with CBP/p300. It has been reported that CBP/p300 interacts with the cyclic adenosine monophosphate-responsive element-binding protein and several other proteins in a cell signal-regulated manner. CBP/p300 with its intrinsic enzyme activity can acetylate histones, which allows the unwinding or loosening of chromatin. p300 can also acetylate p65. Phosphorylation of p65 promotes the interaction with CBP, leading to enhanced transactivation potential of NF-κB (Gerritsen et al., 1997). It acts as a bridging factor between NF-κB and DNA-binding sites. The mechanism by which TCEAL7 modulates the recruitment of p65 or the interaction of p65–p300 is currently unknown.

However, the consistent observation that TCEAL7 inhibits NF-κB activity at the basal level, in the absence of any stimuli, suggests that there is a basic mechanism by which TCEAL7 is keeping NF-κB and maybe other transcription factors (that may also require CBP/p300) in check in a normal cell. Loss of TCEAL7 eliminates this basal-level checkpoint of NF-κB, resulting in a higher activated level of NF-κB leading to vast pleiotropic effects of target genes including cytokines, growth factors, angiogenic and anti-apoptotic factors, which in conjunction with other alterations may provide a fertile ground for tumor formation or sustain the development of tumors.

Materials and methods

Reagents

TranSignal Protein/DNA array for various transcription factors, NF-κB–luciferase reporter, p65-gal and nuclear extraction kits were purchased from Panomics Inc. Antibody against pIκBα was purchased from Cell Signaling Technology (Beverly, MA, USA). [γ-32P]ATP (3000 Ci/mmol) were from PerkinElmer (Boston, MA, USA). Antibodies for p65, p50, cRel, Rel A, p52, IκB-α, IκB-β, IKK-α, IKK-β and Bcl-xl and oligonucleotides for NF-κB and NF-κB-conjugated agarose were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ChIP grade p65, p50 and p300 antibodies were from Millipore (MA, USA). TRIzol and Lipofectamine were from Invitrogen Life Technologies (Grand Island, NY, USA). The enhanced chemiluminescent-detecting reagents and nitrocellulose membrane were purchased from GE Healthcare (Piscataway, NJ, USA). Luciferase assay system was from Promega (Madison, WI, USA). IκB kinase inhibitor (BMS345541) was from Calbiochem (San Diego, CA, USA). The expression vectors of p50 and p65 were kindly provided by Dr R Pope (Northwestern University Medical School, Chicago, IL, USA). The expression vector for hemagglutinin-IKK-α and IKK-β were gift from Dr ZG Liu (National Institutes of Health, Bethesda, MD, USA). The constitutive active IKK2 was purchased from Addgene (Cambridge, MA, USA). p300 complementary DNA was a gift from Dr J Boyes (MCCSC, London, UK).

Cell culture

The ovarian epithelial cell line, OSEtsT/hTERT, was initially immortalized with temperature-sensitive SV40 T-antigen (OSEtsT) and subsequently with catalytic subunit of human telomerase (OSEtsT/hTERT) (Kalli et al., 2002). The OSEtsT/hTERT cells were grown in medium comprising Medium 1.99, MCDB105, sodium bicarbonate (1.1 g/l) and hygromycin (2 μg/ml), adjusted to pH 7.2 and supplemented with 15% fetal bovine serum (Invitrogen). SV40t/T immortalized ovarian surface epithelial cell line, IOSE-523, was kindly shared by Dr Nelly Auersperg (University of British Columbia, Canada). IOSE-523 cells were grown in Medium 1.99, MCDB105, sodium bicarbonate (1.1 g/l) with 10% fetal bovine serum. All reagents for the medium were obtained from Sigma-Aldrich (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).

Generation of shRNA downregulated TCEAL7 clones in OSEtsT/hTERT

Immortalized OSE cells transduced with a temperature-sensitive mutant of the SV40 large T antigen and catalytic subunit of human telomerase, called OSEtsT/hTERT, were used for generation of clones (Kalli et al., 2002). For downregulation of TCEAL7 expression, OSEtsT/hTERT cells were treated with retroviral supernatants to transduce pSUPER.retro constructs expressing shRNA targeting TCEAL7 mRNA (pSR-TCEAL7) or an empty pSUPER.retro vector (pSR) as described before (Chien et al., 2008).

Transcription factor array

For high-throughput analysis of transcription factor analysis, we used Panomic's TranSignal Protein/DNA array (cat no. MA1210) (Toutirais et al., 2003) containing an array membrane of 54 transcription factors as described before (Chien et al., 2008).

Semiquantitative reverse transcriptase–PCR

PCR for TCEAL7 expression was performed as described before (Chien et al., 2008).

Real-time PCR (quantitative PCR)

Real-time analysis for target genes was performed as described before (Nath et al., 2009): Cox2, IL-6, IL-8 and CyclinD1 primers were purchased from SA Biosciences (Frederick, MD, USA).

Plasmids, transfection and reporter assays

293T cells or OSEtsT/hTERT clones were seeded at 8 × 104 cells per well in 24-well plates (in triplicates) 1 day before transfection. A total of 0.15 μg TCEAL7 or empty vector as control and 0.25 μg NF-κB–luciferase constructs were co-transfected with 0.1 μg Renilla reporters. Luciferase activity was measured at 24 h after transfection with Promega's Dual-Luciferase Reporter assay system according to the manufacturer's instructions. For co-transfections, 0.15 μg NF-κB–luciferase was transfected along with 0.1 μg of TCEAL7 and or other plasmids. The relative light units are expressed after normalizing with Renilla luciferase to account for variability in transfection efficiency. Expression vector of TCEAL7 was cloned by amplifying its coding region by PCR using primers 5′-IndexTermGCAGGAAACAACAACAACATC-3′ and 3′-IndexTermTTAAATGGGATAAGGGACGGT-3′ and cloned into pcDNA3/GFP-CT TOPO cloning vector from Invitrogen following the manufacturer's recommendation.

Gel-shift

Nuclear extracts from control vector and shRNA clones were isolated and electromobility shift assay was performed, as described previously (Giri et al., 2002), with NF-κB consensus sequence, which was end labeled with [γ-32P]ATP. Nuclear extracts were normalized based on protein concentration, and equal amount of protein (5 μg) was loaded. DNA–protein complexes were resolved on 5% nondenaturing polyacrylamide gel electrophoresis in 45 mM Tris (pH 7.8), 45 mM boric acid and 1 mM EDTA (0.5 × Tris–boric–EDTA), and run at 11 V/cm. The gels were dried and then autoradiographed at −70 °C using X-ray film. For detecting supershift, the nuclear extracts were incubated with 1 μg of p65 and/or p50 antibodies for 30 min before running the gel.

Western blot

Equal amounts of protein (40 μg/lane) were separated by electrophoresis on the sodium dodecyl sulfate gel and electrophoretically transferred to polyvinylidene fluoride membrane. Blots were washed once with tris-buffered saline supplemented with 0.2% Tween-20 and then blocked with the same solution containing 5% non-fat dry milk for 1 h at room temperature. The blocking solution was replaced with a solution containing primary antibody (1:1000 dilution) in 5% milk. After overnight rocking at 4 °C, the blots were washed thrice for 5 min each in tris-buffered saline, 0.1% (w/v) Tween-20 and incubated with respective horseradish peroxidase-conjugated secondary antibody in 5% milk/tris-buffered saline supplemented with 0.2% Tween-20 at room temperature for 1 h. After washing thrice in tris-buffered saline supplemented with 0.2% Tween-20, the proteins were visualized using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). The blots were stripped and re-probed with anti-β actin (Sigma) or β-tubulin for equal protein loading control.

Enzyme-linked immunosorbent assay (ELISA)

After 48 h, serum-free supernatants of the cultured cells were taken out for detection of the levels of IL-6 and IL-8 (R&D Biosystems, Minneapolis, MN, USA) and VEGF (Immuno Biological Labs, Japan, no. 17741). The ELISA was performed according to the manufacturer's instructions. For quantitation of the levels of p65 and p50 in nuclear extract from treated and untreated cells, ELISA for p65 and p50 were performed according to the manufacturer's instructions (Active Motif, Carlsbad, CA, USA) by using equal amounts of nuclear extracts.

Cell proliferation

A total of 25–50 000 cells per well were plated according to the cell size in 24-well plates in triplicates. Next day was taken as day 0 and live cells were counted by Trypan blue exclusion dye on odd days of incubation (0, 1, 3, 5 and 7 days) in treated and untreated cells with IKK inhibitor (BMS345541) purchased from Calbiochem (Gibbstown, NJ, USA).

Cell survival

The viability of cells was evaluated using MTT assay. Viable cells take up the MTT dye and cleave tetrazolium salt to a dark 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 and 48 h of treatment.

Generation of 293T-inducible cell line

An inducible system for expression of TCEAL7 was generated using Flp-In System from Invitrogen. In brief, a Flp recombination target site was introduced in 293T cells by using vector, pFRT/lacZeo. TCEAL7 was cloned into pcDNA5/ Flp recombination target and co-transfected with pOG44 plasmid expressing Flp recombinase. The stable clones were selected using hygromycin resistance and zeocin sensitivity and expression of TCEAL7.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation was performed using Millipore Magna ChIP kit. In brief, 1 × 107 cells were crosslinked with formaldehyde and sonicated to shear the DNA to fragments of 200–1000 bp. ChIP grade antibodies from Millipore (p65, p50 or p300) were used for immunoprecipitation of the protein–DNA complex. DNA was purified according to the instructions and amplified by standard PCR and ran on agarose gels. IL-6 promoter: sense 5′-IndexTermAGTGGTGAAGAGACTCAGTG-3′ and antisense 5′-IndexTermGGCAGAATGAGCCTCAGA-3′. IL-8 promoter: sense 5′-IndexTermGGGCCATCAGTTGCAAATC-3′ and antisense 5′-IndexTermTTCCTTCCGGTGGTTTCTTC-3′.

Statistical analysis

Statistics for various parameters were analyzed with one-way multiple-range analysis of variance GraphPad Prism 3.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Significances (P-value) between groups were determined using the Newman–Keuls test. A P-value of <0.05 and above was considered significant.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This work was supported by Ovarian Cancer Research Fund to RR as a Program of Excellence grant and with funds from Mayo Foundation and Bernard and Edith Waterman Foundation to VS.

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Correspondence to V Shridhar.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Rattan, R., Narita, K., Chien, J. et al. TCEAL7, a putative tumor suppressor gene, negatively regulates NF-κB pathway. Oncogene 29, 1362–1373 (2010). https://doi.org/10.1038/onc.2009.431

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Keywords

  • TCEAL7
  • NF-κB
  • ovarian cancer
  • tumor suppressor gene

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