Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc finger RNA binding protein that regulates the stability of certain AU-rich mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared to normal cell types. Here we found that TTP expression was lower in invasive breast cancer cells (MDA-MB-231) compared to 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 act as dominant negative and increase target mRNA expression. In contrast to wt TTP, C124R TTP was able to increase certain ARE-mRNA expression in serum-stimulated breast cancer cells. Using an ARE-gene microarray, novel targets of TTP regulation were identified; urokinase plasminogen activator (uPA), uPA receptor, and matrix metallo-proteinase-1 (MMP1), all known to play prominent roles in breast cancer invasion and metastasis. Expression of these targets was upregulated in the tumorigenic types, particularly, the highly invasive MDA-MB-231. The mRNA half lives of these TTP-regulated genes were increased in TTP-knockout embryonic mouse fibroblasts as assessed by real time PCR while forced restoration of TTP by transfection led to a reduction of their mRNA levels. RNA immunoprecipitation confirmed an association of TTP, but not C124R, with these target transcripts. Moreover, TTP reduced, while 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′UTR- and ARE-dependent manner, regulates an important subset of cancer-related genes that are involved in cellular growth, invasion, and metastasis.

The activities of RNA-binding proteins are perturbed in several pathological conditions, including cancer. These proteins include tristetraprolin (TTP, ZFP36) and HuR (ELAVL1), which respectively promote the decay or stability of adenylate-uridylate-rich (AU-rich) mRNAs. Here, we demonstrated that increased stabilization and subsequent over-expression of HuR mRNA were coupled to TTP deficiency. These findings were observed in breast cancer cell lines with an invasive phenotype and were further confirmed in ZFP36-knockout mouse fibroblasts. We show that TTP–HuR imbalance correlated with increased expression of AU-rich element (ARE) mRNAs that code for cancer invasion genes. The microRNA miR-29a was abundant in invasive breast cancer cells when compared to non-tumourigenic cell types. When normal breast cells were treated with miR-29a, HuR mRNA and protein expression were up-regulated. MiR-29a recognized a seed target in the TTP 3′ UTR and a cell-permeable miR-29a inhibitor increased TTP activity towards HuR 3′ UTR. This led to HuR mRNA destabilization and restoration of the aberrant TTP–HuR axis. Subsequently, the cancer invasion factors uPA, MMP-1 and MMP-13, and cell invasiveness, were decreased. The TTP:HuR mRNA ratios were also perturbed in samples from invasive breast cancer patients when compared with normal tissues, and were associated with invasion gene expression. This study demonstrates that an aberrant ARE-mediated pathway in invasive cancer can be normalized by targeting the aberrant and functionally coupled TTP–HuR axis, indicating a potential therapeutic approach. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Defects in AU-rich elements

SummaryWe demonstrate that CXCR4 mRNA contains ARE in its 3′-UTR and regulated by the RNA-binding proteins, TTP and HuR. Normalization of the aberrant TTP-HuR axis resulted in reduced invasion and migration of breast cancer cells toward CXCL12.

Dysfunctions in post-transcriptional control are observed in cancer and chronic inflammatory diseases. Here, we employed a kinome inhibitor library (n=378) in a reporter system selective for 3-untranslated region-AU-rich elements (ARE). Fifteen inhibitors reduced the ARE reporter activity; among the targets is the polo-like kinase 1 (PLK1). RNAseq experiments demonstrated that the PLK1 inhibitor, volasertib, reduces the expression of cytokine and cell growth ARE-mRNAs. PLK1 inhibition caused accelerated mRNA decay in cancer cells and was associated with reduced phosphorylation and stability of the mRNA decay-promoting protein, tristetraprolin (ZFP36/TTP). Ectopic expression of PLK1 increased abundance and stability of high molecular weight of ZFP36/TTP likely of the phosphorylated form. PLK1 effect was associated with the MAPK-MK2 pathway, a major regulator of ARE-mRNA stability, as evident from MK2 inhibition, in vitro phosphorylation, and knockout experiments. Mutational analysis demonstrates that TTP serine 186 is a target for PLK1 effect. Treatment of mice with the PLK1 inhibitor reduced both ZFP36/TTP phosphorylation in xenograft tumor tissues, and the tumor size. In cancer patients' tissues, PLK1/ARE-regulated gene cluster was overexpressed in solid tumors and associated with poor survival. The data showed that PLK1-mediated posttranscriptional aberration could be a therapeutic target.

<p>Overall list the over-expressed ARE-mRNAs in breast cancer.</p>

<p>Supplemental methods on statistical correlation modeling, RNA-IP, and Taqman assays.</p>

<p>Reporter sequences containing the putative AREs and their AU context</p>