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

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

Sandhya Sanduja et al. Aging (Albany NY). 2009.

Abstract

The RNA-binding protein tristetraprolin (TTP) regulates expression of many cancer-associated and proinflammatory factors through binding AU-rich elements (ARE) in the 3'-untranslated region (3'UTR) and facilitating rapid mRNA decay. Here we report on the ability of TTP to act in an anti-proliferative capacity in HPV18-positive HeLa cells by inducing senescence. HeLa cells maintain a dormant p53 pathway and elevated telomerase activity resulting from HPV-mediated transformation, whereas TTP expression counteracted this effect by stabilizing p53 protein and inhibiting hTERT expression. Presence of TTP did not alter E6 and E7 viral mRNA levels indicating that these are not TTP targets. It was found that TTP promoted rapid mRNA decay of the cellular ubiquitin ligase E6-associated protein (E6-AP). RNA-binding studies demonstrated TTP and E6-AP mRNA interaction and deletion of the E6-AP mRNA ARE-containing 3'UTR imparts resistance to TTP-mediated downregulation. Similar results were obtained with high-risk HPV16-positive cells that employ the E6-AP pathway to control p53 and hTERT levels. Furthermore, loss of TTP expression was consistently observed in cervical cancer tissue compared to normal tissue. These findings demonstrate the ability of TTP to act as a tumor suppressor by inhibiting the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated carcinogenesis.

Keywords: AU-rich element; E6-AP; HPV; senescence; tristetraprolin.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1.

Figure 1.. TTP inhibits HeLa cell proliferation through induction of senescence.

(A) HeLa Tet-Off/TTP-Flag cells grown in the presence or absence of 2 μg/ml Dox for 48 hr. The expression of TTP-Flag was detected by western blot (WB) using antibodies against the Flag epitope (left panel) or TTP (right panel). Actin was used as a loading control. (B) Growth curves of HeLa Tet-Off/TTP-Flag (circles) and parental HeLa Tet-Off (triangles) cells in the presence (open symbols) or absence (filled symbols) of 2 μg/ml Dox. On day 4 of growth, Dox was added to HeLa Tet-Off/TTP-Flag cells to repress TTP expression. Each point represents the mean of 4 replicates. (C) HeLa Tet-Off/TTP-Flag cells were grown in the presence or absence of Dox to repress (- TTP) or induce (+ TTP) TTP, respectively. Phase contrast (top panels) and fluorescence (middle panels) microscopy of cells after 48 hr of TTP expression; original magnification 200X and 400X, respectively. Nuclei (blue) and cytoskeleton (red) are shown in fluorescent micrographs. HeLa-Tet-Off/TTP-Flag cells were stained for SA-β-gal activity (bottom panels) after 12 days of TTP expression.

Figure 2.

Figure 2.. TTP promotes p53 expression through protein stabilization.

(A) HeLa Tet-Off/TTP-Flag cells grown in presence or absence of Dox for 48 hr (left panel) and HeLa cells infected with AdGFP or AdGFP/TTP virus for 48 hr (right panel) were examined for TTP and p53 expression by western blotting. Actin was used as a loading control. (B) RT-PCR analysis of p53 mRNA expression in HeLa Tet-Off/TTP-Flag cells grown in presence or absence of Dox for 48 hr. Induction TTP-Flag mRNA is shown along with loading control GAPDH. (C) TTP promotes increased stability of p53 protein. HeLa Tet-Off/TTP-Flag cells grown in presence (- TTP) or absence (+ TTP) of Dox for 48 hr were incubated with 20 μg/ml cycloheximide (CHX) to inhibit protein synthesis for the indicated times. Decay of p53 protein was examined by western blot (left panels) using actin as a loading control. Decay curves of p53 protein (right panel) in the presence (open circles) and absence (filled circles) of TTP was obtained by western blot analysis and normalized to the internal control actin.

Figure 3.

Figure 3.. Enhanced p53 activity in HeLa cells expressing TTP.

(A) Immunofluorescent detection of p53, shown in green, in HeLa Tet-Off/TTP-Flag cellsin the absence or presence of TTP for 48 hr. DAPI nuclear staining and merged images are shown. (B) Expression of TTP induces p53 transcriptional activity. Luciferase reporter constructs containing either a p53-dependent promoter (pp53-Luciferase) or control vector (pTA-Luciferase) were transfected into HeLa Tet-Off/TTP-Flag cells and allowed to grow without (grey bars) or with (black bars) TTP induction for 48 hr. Relative activity was assessed as luciferase activity normalized to renilla activity and are the averages of 3 experiments. (*) P < 0.01

Figure 4.

Figure 4.. TTP-mediated inhibition of hTERT expression.

(A) hTERT expression in HeLa Tet-Off/TTP-Flag cells growing in absence or presence of TTP for 48 hr was examined by RT-PCR analysis (top panel) and western blot using nuclear lysates (bottom panel). GAPDH and nucleoporin were detected as loading controls, respectively. (B) TRAP assay showing inhibition of telomerase activity in TTP-expressing cells. 0.5 and 1 μg of lysate from cells grown in the absence or presence of TTP was used for TRAP assay as described in Methods. Control reaction lacks Taq polymerase.

Figure 5.

Figure 5.. TTP downregulates E6-AP mRNA and protein expression.

(A) Northern blot (left panel) of TTP and E6-AP mRNA in HeLa Tet-Off/TTP-Flag cells 48 hr after TTP induction. RT-PCR assay (right panel) of HPV18-E6 and -E7 RNA levels in TTP-expressing cells. Actin and GAPDH were used as loading controls. (B) Western blot of E6-AP protein in HeLa Tet-Off/TTP-Flag cells (left panel) and adenovirus-infected HeLa cells (right panel) expressing TTP for 48 hr. Actin was used as a loading control. (C) TTP-dependent downregulation of E6-AP mRNA. HeLa Tet-Off/TTP-Flag cells were initially grown without Dox for 48 hr to induce TTP. At time zero, Dox was added to the culture medium and E6-AP mRNA levels were evaluated by qPCR over the indicated time course. E6-AP mRNA levels were normalized to control GAPDH mRNA. Cells grown in presence of Dox were used as control. All values shown are normalized to E6-AP expression of control-treated cells and are the averages of 3 experiments. (*) P < 0.01 (D) HPV 16-positive cells, SiHa (left panel) and CaSki (right panel) were infected with control AdGFP or AdGFP/TTP virus at an MOI of 100 or left untreated (NT). 48 hr after infection, cell lysates were examined for TTP, p53, and E6-AP expression by western blot. Actin was used as a loading control.

Figure 6.

Figure 6.. TTP binds E6-AP mRNA and targets it for rapid decay.

(A) Schematic representation of E6-AP mRNA. The grey bar corresponds to E6-AP coding region and number-labeled black ovals represent putative 3' UTR AU-rich elements (AREs). m7G, 7-methyl-guanosine cap; AAAAn, polyadenylated tail. (B) E6-AP mRNA half-life was assayed in HeLa Tet-Off/TTP-Flag cells grown in the presence (triangles; labeled -TTP) or absence (circles; labeled +TTP) of Dox to induce TTP expression. After 48 hr, 5 μg/ml of actinomycin D was added to the cells and E6-AP mRNA decay was analyzed by qPCR using GAPDH mRNA as a normalization control. The data shown is the average of triplicate experiments. (C, D) Binding of TTP and E6-AP mRNA. Control and TTP-expressing (48 hr) HeLa Tet-Off/TTP-Flag cells were lysed and immunoprecipitation was performed on equal amounts of cytoplasmic lysates using control IgG or anti-Flag mAb. RNA purified from immuno-precipitates was subjected to RT-PCR (C) or qPCR (D) to detect E6-AP and GAPDH mRNA. The ethidium bromide-stained agarose gel depicting the 292bp E6-AP PCR product is shown in reverse image. The relative amounts of immuno-precipitated E6-AP mRNA is reported as the average 1/Ct value of triplicate experiments. (*) P < 0.01

Figure 7.

Figure 7.. E6-AP 3' UTR is necessary for TTP-mediated decay.

(A) HeLa Tet-Off/TTP-Flag cells were transfected with an expression vector containing the coding region of E6-AP (E6-APΔ3'UTR). Cells were grown in the absence or presence of TTP for 48 hr and lysates were analyzed for E6-AP and TTP protein expression by western blot. Actin was detected as a loading control. (B) HeLa cells were transfected with a luciferase-reporter construct containing the 1.6 kb E6-AP 3'UTR (Luc+E6-AP 3'UTR) or the control luciferase vector (no 3'UTR) along with a TTP expression construct (pcDNA3-TTP-Flag) or empty vector. Relative luciferase reporter activity in the absence (black bars) or presence (grey bars) of TTP is shown. Relative activity was assessed as luciferase activity normalized to its respective protein concentration for each transfection in the absence or presence of TTP. The data shown is the average of duplicate experiments. (*) P < 0.01

Figure 8.

Figure 8.. TTP protein expression is lost in human cervical cancer.

(A) Immunohistochemical detection of TTP expression in normal cervix and squamous cell carcinoma. Representative tissue sections were examined for TTP expression and counterstained with hematoxylin. Original magnification x 200. (B) Immunoreactivity scores (IRS) for TTP expression in tissue sections of normal cervix and squamous cell carcinoma. The line indicates the division of samples with high IRS from 7-12 and low IRS from 0-6.

Figure 9.

Figure 9.. TTP-mediated regulation of E6-AP in cervical cancer cells.

The binding of TTP to the ARE-containing E6-AP mRNA targets it for rapid degradation. Black ovals represent putative 3' UTR AU-rich elements (AREs). The subsequent loss of E6-AP expression allows for concurrent p53 protein stabilization and inhibition of hTERT transcription leading to cellular senescence.

Comment in

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