The RNA-binding zinc-finger protein tristetraprolin regulates AU-rich mRNAs involved in breast cancer-related processes

Abstract

Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc-finger RNA-binding protein that regulates the stability of certain AU-rich element (ARE) mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared with normal cell types. In this study we found that TTP expression was lower in invasive breast cancer cells (MDAMB231) compared with normal breast cell lines MCF12A and MCF-10. TTP targets were probed using a novel approach by expressing the C124R zinc-finger TTP mutant that functions as dominant negative and increases target mRNA expression. In contrast to wild-type TTP, C124R TTP was able to increase certain ARE-mRNA expressions in serum-stimulated breast cancer cells. Using an ARE-gene microarray, novel targets of TTP regulation were identified, namely, urokinase plasminogen activator (uPA), uPA receptor and matrix metalloproteinase-1, all known to have prominent roles in breast cancer invasion and metastasis. Expression of these targets was upregulated in tumorigenic types, particularly in highly invasive MDAMB231. The mRNA half-lives of these TTP-regulated genes were increased in TTP-knockout embryonic mouse fibroblasts, as assessed using real-time polymerase chain reaction, whereas forced restoration of TTP by transfection led to a reduction in their mRNA levels. RNA immunoprecipitation confirmed an association of TTP, but not C124R, with these target transcripts. Moreover, TTP reduced, whereas the mutant C124R TTP increased, the activity of reporter constructs fused to target ARE. As a result of TTP regulation, invasiveness of MDAMB231 cells was reduced. The data suggest that TTP, in a 3' untranslated region-and ARE-dependent manner, regulates an important subset of cancer-related genes that are involved in cellular growth, invasion and metastasis.

Figure 1

(A) TTP expression profile in normal and tumor breast cell lines. Cells were serum-starved (0.5% FBS) overnight then re-stimulated with 10% serum for 1 hour. Total RNA was then extracted and reverse transcribed for quantitative PCR. Taqman expression assays specific for human TTP were performed and quantitation was normalized to human RPLP0 as the endogenous control in the real-time PCR experiments. Values shown are means ± SEM from three independent experiments, ** P<0.01 as determined by ANOVA. ER: estrogen receptor. (B) Response of ARE-GFP reporter in HEK293 cells transfected with 25ng ARE-GFP reporter plasmid and co-transfected with various amounts of TTP, C124R (mutant TTP) or a vector plasmid alone. GFP fluorescence was measured 24 hours post transfection. (C) Level of TTP protein in untransfected and transfected cells after serum induction. 40ug of total protein was loaded onto SDS-PAGE gel and probed with an antibody against TTP or β-actin as a loading control and transfection efficiency was determined by measurement of co-transfected GFP.

Figure 2

mRNA expression profile of TTP-regulated candidate targets in normal and tumor breast cell lines. Real-time PCR experiments were performed using Taqman expression assays specific for human uPA (A), uPAR (B) and MMP1 (C) and normalized to human RPLPO as the endogenous control. Results are means ± SEM from three experiments. * P<0.05, ** P<0.005. (D) Protein expression levels of TTP, uPA, and uPAR in the ER-negative breast cell lines MCF10A (normal), and MDAMB231 (tumor). 60ug of total protein was loaded onto SDS-PAGE gel and probed with antibodies against human TTP, uPA, uPAR, or β-actin as a loading control.

Figure 3

Real-time PCR monitoring of endogenous candidate mRNAs (uPA, uPAR, and MMP1) associated with TTP or mutant TTP precipitated with anti-TTP antibody from MDAMB231 cells. Normal rabbit IgG control was used as an antibody control with both TTP and mutant TTP samples. Quantitation of associated mRNA was performed by real-time PCR and normalized to a housekeeping mRNA, RPLPO.

Figure 4

TTP regulation of candidate target mRNAs in MDAMB231 cells. MDAMB231 cells were transfected with TTP plasmid or vector control, serum-starved overnight, then serum induced for 8 hours then total RNA was extracted. Endogenous uPA, uPAR, and MMP1 mRNA expression levels were measured by real-time PCR and normalized to human RPLPO. The results are means ± SEM from five independent experiments. * P<0.05, ** P<0.005 (Student’s t test).

Figure 5

TTP regulation of TTP-regulated mRNAs in TTP^+/+ and TTP^−/− mouse fibroblasts. Cells were serum-starved then induced with 10% serum for 6 hours then total RNA was extracted. Endogenous uPA (A), uPAR (B), and MMP1 (C) mRNA levels were measured by real-time PCR and normalized to mouse β-actin. lower panel: TTP^+/+ and TTP^−/− mouse fibroblasts were serum induced for 2 hours. Results are means ± SEM of five independent experiments. * P<0.05, ** P<0.005. (D) Protein expression levels of TTP, uPA, uPAR, and MMP1 in TTP+/+ and TTP−/− mouse fibroblasts. 60ug of total protein was loaded onto SDS-PAGE gel and probed with antibodies against mouse TTP, uPA, MMP1, or β-actin as a loading control.

Figure 6

TTP-regulated mRNA decay curve in TTP^+/+ and TTP^−/− mouse fibroblasts. Cells were serum-starved overnight, induced with 10% serum for 1 hour then treated with Actinomycin D (5ug/ml) for indicated times. Total RNA was extracted and used for real-time PCR. (A) uPA; (B) uPAR; (C) MMP1. The results are means + SEM of five independent experiments. Right panels show best-fit-values of the one-phase exponential decay model as described in Materials and Methods.

Figure 7

TTP regulation of expression of EGFP reporter activity and invasion assays (A) EGFP reporter constructs were fused with uPA ARE, TNF ARE, uPAR ARE, or MMP1 3′ UTR regions (cloned in BamHI/xbaI sites in stable growth hormone 3′UTR (upper panel). (B) HEK293 cells were co-transfected with the reporters and TTP, C124R (mutant TTP), or a vector plasmid alone. GFP fluorescence was measured 24 hours post-transfection. (C) TTP modulation of invasion. MDAMB231 cells were transfected with TTP, C124R, or PCR3.1 control plasmid and co-transfected with luciferase construct. At 24 hours after transfection the cells were reseeded in invasion chambers in serum-free medium and incubated overnight. Invaded cells were lysed and luciferase was measured in illuminometer. Transfection efficiency was monitored and found comparable with the three constructs.