Post-transcriptional regulation of BIRC5/survivin expression and induction of apoptosis in breast cancer cells by tristetraprolin.

Inhibition of apoptosis is one of the hallmarks of cancer and is a target of various therapeutic interventions. BIRC5 is an inhibitor of apoptosis that is aberrantly expressed in cancer leading to sustained growth of tumours. Post-transcriptional control mechanisms involving RNA-binding proteins and AU-rich elements (AREs) are fundamental to many cellular processes and changes in the expression or function of these proteins can promote an aberrant and pathological phenotype. BIRC5 mRNA has an ARE in its 3' UTR making it a candidate for regulation by the RNA binding proteins tristetraprolin (TTP) and HuR (ELAVL1). In this study, we investigated the binding of TTP and HuR by RNA-immunoprecipitation assays and found that these proteins were associated with BIRC5 mRNA to varying extents. Consequently, BIRC5 expression decreased when TTP was overexpressed and apoptosis was induced. In the absence of TTP, BIRC5 mRNA was stabilized, protein expression increased and the number of apoptotic cells declined. As an ARE-mRNA stabilizing protein, recombinant HuR led to upregulation of BIRC5 expression, whereas HuR silencing was concomitant with downregulation of BIRC5 mRNA and protein and increased cell death. Survival analyses demonstrated that increased TTP and low BIRC5 expression predicted an overall better prognosis compared to dysregulated TTP and high BIRC5. Thus, the results present a novel target of ARE-mediated post-transcriptional regulation.

Kinome inhibition reveals a role for polo-like kinase 1 in targeting post-transcriptional control in cancer.

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). RNA-seq 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 post-transcriptional aberration could be a therapeutic target.

Systematic Analysis of AU-Rich Element Expression in Cancer Reveals Common Functional Clusters Regulated by Key RNA-Binding Proteins.

Defects in AU-rich elements (ARE)-mediated posttranscriptional control can lead to several abnormal processes that underlie carcinogenesis. Here, we performed a systematic analysis of ARE-mRNA expression across multiple cancer types. First, the ARE database (ARED) was intersected with The Cancer Genome Atlas databases and others. A large set of ARE-mRNAs was over-represented in cancer and, unlike non-ARE-mRNAs, correlated with the reversed balance in the expression of the RNA-binding proteins tristetraprolin (TTP, ZFP36) and HuR (ELAVL1). Serial statistical and functional enrichment clustering identified a cluster of 11 overexpressed ARE-mRNAs (CDC6, KIF11, PRC1, NEK2, NCAPG, CENPA, NUF2, KIF18A, CENPE, PBK, TOP2A) that negatively correlated with TTP/HuR mRNA ratios and was involved in the mitotic cell cycle. This cluster was upregulated in a number of solid cancers. Experimentally, we demonstrated that the ARE-mRNA cluster is upregulated in a number of tumor breast cell lines when compared with noninvasive and normal-like breast cancer cells. RNA-IP demonstrated the association of the ARE-mRNAs with TTP and HuR. Experimental modulation of TTP or HuR expression led to changes in the mitosis ARE-mRNAs. Posttranscriptional reporter assays confirmed the functionality of AREs. Moreover, TTP augmented mitotic cell-cycle arrest as demonstrated by flow cytometry and histone H3 phosphorylation. We found that poor breast cancer patient survival was significantly associated with low TTP/HuR mRNA ratios and correlated with high levels of the mitotic ARE-mRNA signature. These results significantly broaden the role of AREs and their binding proteins in cancer, and demonstrate that TTP induces an antimitotic pathway that is diminished in cancer. Cancer Res; 76(14); 4068-80. ©2016 AACR.

Posttranscriptional control of the chemokine receptor CXCR4 expression in cancer cells.

CXCR4 is a chemokine receptor that is overexpressed in certain cancer types and involved in migration toward distant organs. The molecular mechanisms underlying CXCR4 expression in invasive cancer, particularly posttranscriptional regulation, are poorly understood. Here, we find that CXCR4 harbors AU-rich elements (AREs) in the 3'-untranslated region (3'-UTR) that bind and respond to the RNA-binding proteins, tristetraprolin (TTP/ZFP36) and HuR (ELAVL1). Different experimental approaches, including RNA immunoprecipitation, 3'-UTR reporter, RNA shift and messenger RNA (mRNA) half-life studies confirmed functionality of the CXCR4 ARE. Wild-type TTP, but not the zinc finger mutant, C124R, was able to bind CXCR4 mRNA and ARE. In the invasive breast cancer phenotype, aberrant expression of CXCR4 is linked to both TTP deficiency and HuR overexpression. HuR silencing led to decreased CXCR4 mRNA stability and expression, and significant reduction in migration of the cells toward the CXCR4 ligand, CXCL12. Derepression of TTP using miR-29a inhibitor led to significant reduction in CXCR4 mRNA stability, expression and migration capability of the cells. The study shows that CXCR4 is regulated by ARE-dependent posttranscriptional mechanisms that involve TTP and HuR, and that aberration in this pathway helps cancer cells migrate toward the CXCR4 ligand. Targeting posttranscriptional control of CXCR4 expression may constitute an alternative approach in cancer therapy.

miR-29a inhibition normalizes HuR over-expression and aberrant AU-rich mRNA stability in invasive cancer.

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.