The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase

Introduction

Cervical cancer is the second most common cancer among women worldwide [1]. A necessary factor in the development of nearly all cases of cervical cancer is infection with the high-risk human papillomavirus (HPV) types 16 and 18 [2]. These subtypes of HPV promote cellular transformation through expression of the early viral genes E6 and E7. The HPV E7 protein neutralizes the retinoblastoma (Rb) tumor suppressor pathway by sequestering Rb from E2F and promoting its destabilization [3,4], while the E6 protein promotes degradation of the p53 tumor suppressor and activates transcription of the human telomerase reverse transcriptase gene (hTERT) [5,6]. The oncogenic functions of E6 occur through its interaction with a number of cellular regulatory proteins and one of the best characterized E6-binding partners is the E6-associated protein (E6-AP) [7,8]. E6-AP belongs to a class of HECT ubiquitin-protein ligases [9] and its interaction with E6 facilitates cell transformation through enhanced p53 protein degradation and activation of hTERT gene expression [10]. Deregulation of these critical factors through the combined action of E6 and E7 oncoproteins allows for continued cell proliferation and genomic instability ultimately leading to HPV-mediated cellular transformation.

Messenger RNA turnover is a tightly regulated process that plays a central role in controlling mammalian gene expression. The significance of this is evident in disease and tumorigenesis where loss of post-transcriptional gene regulation accounts for the aberrant overexpression of a variety of genes encoding growth factors, inflammatory cytokines and proto-oncogenes [11,12]. A majority of cancer-associated immediate-early response genes that control growth and inflammation display conserved cis-acting adenylate- and uridylate (AU)-rich elements (ARE) in the mRNA 3' untranslated region (3'UTR). A primary function of the ARE is to target mRNAs for rapid decay through interaction with trans-acting RNA-binding proteins that have high affinity for AREs. Among the best characterized ARE-binding proteins involved in promoting ARE-mediated mRNA decay is tristetraprolin (TTP, ZFP36, TIS11). TTP is a member of a small family of tandem Cys3His zinc finger proteins originally identified as an inducible immediate-early response gene [13]. Initially thought to be a transcription factor, various studies have established the role of TTP as an mRNA decay protein that binds to AREs in the mRNA of various inflammatory mediators (e.g. TNF-α, GM-CSF, COX-2) [14-16]. The binding of TTP to ARE-mRNAs targets them for rapid degradation through association with various decay enzymes [14,17-21]. The physiological role of TTP is significant as TTP deficient mice develop a number of inflammatory syndromes. These abnormalities have been shown to be due to excessive levels of pro-inflammatory factors resulting from defects in ARE-mediated decay in these mice [22,23].

In this study, we examined the role of TTP in HPV-mediated cervical carcinogenesis. Expression of TTP in HPV18-positive HeLa cells dramatically inhibited cell growth by inducing cellular senescence through a mechanism involving p53 protein stabilization and inhibition of telomerase expression. It was found that TTP induced cellular senescence through rapid decay of E6-AP ubiquitin ligase mRNA that was mediated through the ARE-containing 3'UTR of E6-AP. Furthermore, we demonstrate that TTP expression is lost in cervical cancer compared to normal tissue, implying a tumor suppressor function for TTP in cervical tissue. These novel findings not only add another attribute to the already established anti-inflammatory role of this ARE-binding protein but also bring new insights into the mechanism of HPV-mediated cervical carcinogenesis.

Results

Discussion

Normal cellular growth is associated with rapid decay of ARE-containing mRNAs and targeted mRNA decay is an essential way of controlling their pathogenic overexpression. However, a number of observations have implicated loss of ARE-mediated post-transcriptional regulation in the neoplastic transformation of cells [11]. Based upon the inherent genetic instability of tumor cells, it might be expected that mutations in AREs are a frequent event. However, few naturally occurring mutations in AREs have been described [37]. This implies that loss of ARE function in tumor cells is primarily due to altered recognition of AU-rich sequences by trans-acting RNA-binding regulatory factors.

Through their ability to selectively bind and control expression of many cancer-associated transcripts [12], ARE RNA-binding proteins are being acknowledged as central regulators influencing various aspects of tumorigenesis. Along with its recognized ability to target rapid decay of an array of inflammatory mediators, the ARE-binding protein TTP has been shown to inhibit expression of a wide range of cancer-associated factors [25,38-44]. Consistent with this, expression of TTP was shown to inhibit cell growth and tumorigenesis in a mast cell tumor model [45] and attenuate colon cancer cell growth and proliferation [44]. These aspects, taken together with the results presented here, indicate that TTP can serve in a tumor suppressor capacity by controlling ARE-containing gene expression.

Through its ability to promote rapid mRNA decay, the tumor suppressor ability of TTP should reflect the ARE-containing mRNAs needed for enhanced tumor cell growth and survival. Previous findings have indicated that TTP overexpression can promote apoptosis in various cells lines [46]. The results presented here using HPV-positive cervical cancer cells demonstrate the ability of TTP to inhibit cell growth. However, in this TTP-inducible system evidence of apoptosis was not observed as indication of caspase-3 activation, nuclear condensation, and DNA fragmentation was not apparent in HeLa cells expressing TTP over a 7 day time course (data not shown) indicating that TTP-mediated growth inhibition was occurring through an alternate mechanism in HeLa cells. In our findings, HeLa cells expressing TTP exhibited a flattened morphology and elevated levels of β-galactosidase activity indicating they have undergone replicative senescence. These findings are in agreement with recent results demonstrating TTP-mediated growth inhibition using a similar TTP-inducible HeLa cell model [25]. These differences in phenotypic outcome resulting from TTP expression in cells may reflect a specific variation in the ARE-containing mRNAs targeted for TTP-mediated decay in the differing cell types.

In HeLa cells, repression of viral E6 and E7 oncogene expression can trigger endogenous senescence pathways [47-50]. Although our results could be explained through the ability of TTP to inhibit E6 or E7 expression, we did not observe any TTP-dependent changes in E6/E7 transcript level (Figure 5A). Interestingly, an AU-rich region has been identified within the 3'UTR of HPV16 E6/E7 RNA that can mediate rapid decay [51]. The results presented here (Figure 8) and that of others [36] demonstrate TTP to be abundantly expressed in normal uterine cervix. Based on these observations, it is plausible that that TTP may play a protective role in the early stages of HPV infection by targeting E6/E7 RNA for rapid degradation. However, this viral ARE is lost in cells containing integrated HPV16 genomes through the process of viral DNA linearization and host genome integration [51] and the consequences of this would make E6/E7 RNA resistant to TTP-mediated decay. This loss of post-transcriptional control, coupled with disruption of the viral E2 transcriptional repressor [52], would potentiate persistent E6/E7 oncogene expression needed for cell transformation.

Actively growing HeLa cells maintain a dormant p53 pathway and elevated telomerase activities [49]. The results presented here demonstrate the ability of TTP to promote p53 protein expression, which is consistent with senescent growth arrest that is often associated with an active p53 pathway [53]. In normal cells, p53 levels are under negative regulation of Mdm2 ubiquitin ligase and p53 pathway activation primarily involves signal-dependent escape from degradation [54,55]. Whereas in high-risk HPV-transformed cervical cancer cells, the viral oncoprotein E6 binds to p53 and with the help of the cellular ubiquitin ligase E6-AP, p53 is targeted for constitutive degradation through the ubiquitin proteasomal pathway [6,32,56].

Replicative senescence in somatic cells is in part attributed to gradual loss of telomeres, while high telomerase activity is observed in a majority of cancer cells [30]. Another characteristic of cervical cancer cell transformation is reactivation of hTERT gene expression, which is the catalytic component of telomerase. Although the mechanism of E6-dependent activation of hTERT is not entirely defined in HPV-transformed cells, current observations indicate the involvement of E6-AP in targeting a regulator of hTERT expression [5,33,57]. Furthermore, p53 can serve as a negative regulator of hTERT expression [58], suggesting that E6/E6-AP-dependent degradation of p53 may also play a causal role in hTERT promoter activation.

Central to the deregulation of these factors in cervical cancer is E6-AP and the results presented here are readily explained with E6-AP being a novel target of TTP-mediated post-transcriptional regulation (Figure 9). Within the 3'UTR of E6-AP, the presence of AU-rich elements provide a binding site for TTP and this functional interaction targets E6-AP mRNA for rapid ARE-mediated decay. These results are supported by the observations that the presence of E6-AP 3'UTR to a luciferase reporter renders it susceptible to TTP-mediated downregulation and deletion of the 3'UTR from E6-AP makes it resistant to TTP-mediated mRNA decay (Figure 7). The functional consequences of TTP-mediated suppression of E6-AP leads to p53 stabilization, hTERT inhibition, and cellular senescence. These results are consistent with those using RNA interference to downregulate E6-AP expression indicating the central role E6-AP has in promoting HPV-associated cervical cancer [59].

Recent findings have demonstrated that loss of TTP expression is observed in a variety of tumor types [25,36,44,60]. Consistent with this, we also observe a similar loss of TTP in cervical cancer cells and tumors. This loss of TTP expression appears to be a critical factor in the progression of high-risk HPV-associated cervical cancer, since the presence of TTP in cervical tumor cells impedes their tumorigenic potential through rapid decay of E6-AP mRNA. Also observed with the loss of TTP expression is increased expression of the ARE-mRNA stabilization factor HuR in cervical cancers [61]. Through these combined defects of TTP loss-of-function and HuR gain-of-function, aberrant mRNA stabilization can occur leading to over-expression of cancer-associated factors in cervical cancer similar to what is seen in colon cancer [44]. Moreover, the potential of TTP to promote senescence may be through its ability to antagonize HuR-mediated stabilization of proliferative ARE-mRNAs [44,62] similar to observations showing that reduction in HuR levels in fibroblasts promoted a senescent phenotype [63].

The mechanisms underlying the loss of TTP expression in cervical cancer cells and tumors are largely undefined. The TTP gene (ZFP36) is located on 19q13.1 and does not appear to be a target of genomic loss or rearrangement in cervical cancer [64]. One explanation for the lack of TTP expression observed in tumor tissue may reside in epigenetic silencing of the TTP promoter. Within the proximal 3' region of the human TTP promoter lies a putative CpG island and the presence of hypermethylation of this region was observed in HeLa cells (unpublished observations). Based on this we hypothesize that epigenetic alterations occurring through changes in DNA methylation and altered chromatin structure promote TTP gene silencing in cervical tumors. This is consistent with observations demonstrating that various tumor suppressor genes have been silenced or display decreased expression resulting from abnormal promoter hypermethylation in HPV-associated cervical carcinoma [65].

The results presented here provide a novel link between post-transcriptional gene regulation and HPV-associated cervical tumorigenesis. Based on these findings we conclude that TTP promotes cellular senescence in cervical cancer cells through rapid decay of E6-AP mRNA leading to p53 protein stabilization and inhibition of hTERT transcription. Moreover, absence of TTP expression in cervical cancer strongly implicates that loss of TTP expression is a critical step that occurs early in HPV-mediated carcinogenesis. These findings demonstrate the novel ability of TTP to servein a tumor suppressor capacity by regulating ARE-mRNA gene expression and identify how defects in post-transcriptional regulation can contribute to tumorigenesis.

Methods

Cell culture, DNA transfection, and adenoviral infection. Human cervical cancer cell lines HeLa (HPV18+), SiHa (HPV16+) and CaSki (HPV16+) cells were obtained from ATCC; HeLa Tet-Off cells were purchased from BD Clontech. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Hyclone); HeLa Tet-Off cell media was supplemented with 100 μg/ml G418 (Cellgro). The Tet-responsive pTRE2hyg/TTP-Flag vector was created by cloning an N-terminal Flag epitope-tagged TTP cDNA from pcDNA3-Flag-TTP (kindly provided by N. Kedersha, Brigham and Women's Hospital, Boston, MA) into pTRE2hyg (Clontech). HeLa Tet-Off cells were stably transfected with pTRE2hyg/TTP-Flag using Lipofectamine Plus (Invitrogen) according to the vendor's protocol. Stably transfected cells were selected in normal growth medium containing 100 μg/ml G418, 200 μg/ml hygromycin B (Invitrogen), and 2 μg/ml doxycycline (Dox) (Clontech) for 2-3 weeks. Individual clones were isolated by limiting dilution in 96-well plates. Positive HeLa-Tet-Off/TTP-Flag clones were screened by growing cells in the presence or absence of Dox (2 μg/ml) to induce expression of TTP-Flag, respectively; the absence of Dox allows for TTP-Flag expression. For stable cell maintenance the hygromycin B concentration was reduced to 100 μg/ml. Unless otherwise indicated, HeLa Tet-Off/TTP-Flag cells were grown in the absence of Dox for 48 hr to induce TTP-Flag expression.

HeLa Tet-Off/TTP-Flag cells were transiently transfected with p53-responsive promoter luciferase reporter vector pp53-Luc or control vector pTA-Luc (Clontech) along with control pRL-TK renilla vector (Promega) using Lipofectamine Plus. The E6-AP coding region or 3'UTR were PCR amplified from HeLa cDNA as described [66]. E6-AP coding region was cloned into the expression vector pcDNA3.1/Zeo (Invitrogen) to generate pcDNA3.1/E6-APΔ3'UTR. Luciferase reporter construct containing the E6-AP 3'UTR was prepared by cloning E6-AP 3'UTR into pcDNA3.1/Zeo containing the luciferase cDNA [66]. Cells were transfected in DMEM for 3 hr after which cells were grown in complete medium in the presence or absence of 2 μg/ml Dox for 48 hr. Transfected cells were lysed in reporter lysis buffer (Promega) and assayed for luciferase and renilla activities using the Dual-Luciferase Assay System (Promega). Luciferase reporter gene activities were normalized to renilla activity and all results represent the average of triplicate experiments.

TTP-Flag expressing adenovirus was created by cloning TTP-Flag cDNA into the shuttle vector Dual-CCM-CMV-EGFP (Vector Biolabs) that contains dual CMV promoters to drive expression of TTP-Flag and GFP. Construction of TTP-expressing adenoviral vector (AdGFP/TTP) and production of viral stocks were conducted by Vector Biolabs. Control GFP-expressing adenovirus (AdGFP) was purchased from Vector Biolabs. HeLa, SiHa and CaSki cells were infected with AdGFP or AdGFP/TTP using a MOI of 100 in serum-free DMEM for 2 hr after which FBS was added to a final concentration of 10%. 48 hr after infection, cells were harvested in SDS-PAGE lysis buffer for western blot analysis.

Immunoblot analysis. Cells were lysed in SDS-PAGE lysis buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and protein content was determined using a BCA protein assay with BSA as standard (Pierce Biotechnology). Where indicated, nuclear lysates were prepared by resuspending cells in lysis buffer (10 mM HEPES pH 7.9, 2 mM MgCl₂, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% NP-40) containing 0.5 mM PMSF and protease inhibitor cocktail (Sigma) and incubated on ice for 10 min. Cells were centrifuged at 13000 rpm for 10 min and the nuclear pellet was washed 3 times with lysis buffer. Nuclei were lysed in RIPA buffer (50 mM Tris-Cl pH 8.0, 156 mM NaCl, 4 mM EDTA, 1% SDS, 1 % Triton X-100, 1% Na-deoxycholate). Lysates (50 μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes (Bio-Rad), and probed with antibodies against Flag epitope (M2; Sigma), TTP (Ab-36558, Abcam), p53 (DO-1, Calbiochem), hTERT (Ab-1, Calbiochem), and E6-AP (BD Biosciences) at dilutions specified by the vendor. Blots were stripped and then probed with antibodies against β-actin (Clone C4, MP Biomedicals) or nucleoporin (BD Biosciences). Detection and quantitation of blots were performed as described [66].

mRNA analysis. Total RNA was extracted from cells using Trizol reagent (Invitrogen). Northern blotting was performed as previously described [67] and probed with P³²-labeled DNA probes synthesized for TTP, E6-AP and actin (Promega). cDNA synthesis and RT-PCR analysis of mRNA was accomplished as described [66]. The sequences for PCR primers used were: TTP sense, 5'-TCCACAACCCTAGCGAAGAC-3' and TTP anti-sense, 5'-GAGAAGGCAGAGGGTGACAG-3'; p53 sense, 5'-CAGCCAAGTCTGTGACTTGCACGTAC-3' and p53 antisense, 5'-CTATGTCGAAAAGTGTTT CTGTCATC-3'; hTERT sense, 5'-GTGACCGTGGTT TCTGTGTG-3' and hTERT antisense, 5'-TCGCCTGA GGAGTAGAGGAA-3'; HPV18 E6 sense, 5'-CGCGC TTTGAGGATCCAA-3' and HPV18 E6 antisense, 5'-TATGGCATGCAGCATGCG-3'; HPV18 E7 sense, 5'-TATGCATGGACCTAAGGCAAC-3' and HPV18 E7 antisense, 5'-TTACTGCTGGGATGCACACC-3'; E6-AP sense, 5'-GCTTGAGGTTGAGCCTTTTG-3' and E6-AP antisense, 5'-CCAATTTCTCCCTTCCTTCC-3'; GAPDH sense, 5'-CCACCCATGGCAAATTCCAT GGCA-3' and GAPDH antisense, 5'-TCTAGACGGCA GGTCAGGTCCACC-3'. Real-time PCR (qPCR) was performed using the 7300 Real-Time PCR Assay System (Applied Biosystems) with SYBR green PCR master mix (Applied Biosystems) and primers for E6-AP and GAPDH according to the vendor's protocol.

Cell growth and senescence. Cell growth was assayed using the MTT-based cell growth determination kit (Sigma) as previously described [68]. For cellular senescence studies, 1 x 10⁴ HeLa Tet-Off/TTP-Flag cells were grown in 35mm diameter dishes in the presence or absence of 2 μg/ml Dox. Twelve days later, the cells were stained at pH 6.0 with X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; Cell Signaling Technology) to visualize senescence associated-β-galactosidase (SA-β-gal) activity.

Fluorescence microscopy. HeLa Tet-Off/TTP-Flag cells were plated on coverslips in a 24-well plate and grown in the presence or absence of 2 μg/ml Dox. After 48 hrs, the cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were blocked with 10% normal goat serum and 3% BSA diluted in PBST (PBS + 0.1% Tween-20) for 1 hr. Cells were incubated for 1 hr at RT with anti-p53 antibody (DO-1, Calbiochem; 1:100) diluted in blocking buffer. After washing, the cells were incubated with fluorescein-conjugated goat anti-mouse secondary antibody (MP Biomedicals; 1:150) for 1 hr at RT. DAPI (Invitrogen) was used for nuclear counter-staining. Coverslips were mounted on glass slides and visualized using an Axiovert 200 inverted microscope (Zeiss). Cell morphology was examined by staining fixed and permeabilized cells with DAPI and rhodamine phalloidin (Invitrogen) according to the vendor's instructions.

Telomerase activity. Telomerase activity was determined in HeLa Tet-Off/TTP-Flag lysates 48 hr after TTP induction using the PCR-based TRAP assay as previously described [69]. PCR products were resolved on a 10% non-denaturing polyacrylamide gel and visualized by silver staining [70].

Immunoprecipitations. HeLa Tet-Off/TTP-Flag (1.25 x 10⁵ cells) were grown in 100 mm diameter dishes in the presence or absence of 2 μg/ml Dox for 48 hr. Cells were lysed in polysome lysis buffer (100 mM KCl, 5mM MgCl₂, 10mM HEPES pH 7.0, 0.5% NP-40, and 1 mM DTT) containing 100 U/ml RNase inhibitor (Ambion) and protease inhibitor cocktail (Sigma). Cytoplasmic extracts were separated from nuclei by centrifugation at 20,000g for 30 min. 700 μl of IP buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% NP-40) was added to 500 μg of lysate and immunoprecipitation of TTP-bound RNA was accomplished by incubating lysates with equal amounts (30 μg) of anti-Flag mAb or mouse IgG pre-coated to protein A/G PLUS agarose (Santa Cruz Biotechnology) overnight at 4°C. Immunoprecipitates were collected by brief centrifugation and washed 4 times with IP buffer. Total RNA was isolated using 1 ml Trizol per IP reaction and then used for cDNA synthesis [66]. Real-time PCR reactions were performed using 1 μl of cDNA. Data was plotted as 1/C_t to represent the abundance of E6-AP or GAPDH mRNA in each IP sample.

Immunohistochemical analysis. Immunohistochemical analysis of TTP expression was performed using cervical cancer tissue array CXC96101 (Pantomics) that contained 12 cases of normal and inflammatory tissues of cervix and 36 cases of cervical cancer graded by histology. TTP immunostaining was performed using TTP polyclonal antibody (Ab-36558, Abcam) at 8 μg/ml (1:400). Standard staining protocol was performed and stained tissue sections were evaluated for intensity of staining as described [44] using two blinded investigators (S.S and V.K.). For each tissue section, the percentage of positive cells was scored on a scale of 0 to 4 : 0 (0% positive cells), 1 (< 25%), 2 (25-50%), 3 (50-75%) or 4 (> 75%). Staining intensity was scored on a scale of 0 to 3; 0-negative, 1-weak, 2-moderate, or 3-strong. The two scores were multiplied to give an immunoreactivity score (IRS) ranging from 0 to 12, with scores in the range of 0-6 grouped in the category of Low IRS and those in the range of 7-12 representing High IRS.

Statistical analysis. The data are expressed as the mean +/- SD. Student's t-test was used to determine significant differences. P-values less than 0.05 were considered significant.