Inhibition of IRES-dependent translation of caspase-2 by HuR confers chemotherapeutic drug resistance in colon carcinoma cells.

HuR plays an important role in tumor cell survival mainly through posttranscriptional upregulation of prominent anti-apoptotic genes. In addition, HuR can inhibit the translation of pro-apoptotic factors as we could previously report for caspase-2. Here, we investigated the mechanisms of caspase-2 suppression by HuR and its contribution to chemotherapeutic drug resistance of colon carcinoma cells. In accordance with the significant drug-induced increase in cytoplasmic HuR abundance, doxorubicin and paclitaxel increased the interaction of cytoplasmic HuR with the 5'untranslated region (5'UTR) of caspase-2 as shown by RNA pull down assay. Experiments with bicistronic reporter genes furthermore indicate the presence of an internal ribosome entry site (IRES) within the caspase-2-5'UTR. Luciferase activity was suppressed either by chemotherapeutic drugs or ectopic expression of HuR. IRES-driven luciferase activity was significantly increased upon siRNA-mediated knockdown of HuR implicating an inhibitory effect of HuR on caspase-2 translation which is further reinforced by chemotherapeutic drugs. Comparison of RNA-binding affinities of recombinant HuR to two fragments of the caspase-2-5'UTR by EMSA revealed a critical HuR-binding site residing between nucleotides 111 and 241 of caspase-2-5'UTR. Mapping of critical RNA binding domains within HuR revealed that a fusion of RNA recognition motif 2 (RRM2) plus the hinge region confers a full caspase-2-5'UTR-binding. Functionally, knockdown of HuR significantly increased the sensitivity of colon cancer cells to drug-induced apoptosis. Importantly, the apoptosis sensitizing effects by HuR knockdown were rescued after silencing of caspase-2. The negative caspase-2 regulation by HuR offers a novel therapeutic target for sensitizing colon carcinoma cells to drug-induced apoptosis.

Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2.

Increased abundance of the mRNA-binding protein human antigen R (HuR) is a characteristic feature of many cancers and frequently associated with a high grade malignancy and therapy resistance. HuR elicits a broad cell survival program mainly by stabilizing or increasing the translation of mRNAs coding for anti-apoptotic effector proteins. Conversally, we previously identified the pro-apoptotic caspase-2 as a novel HuR target which is mainly regulated at the level of translation. In this study, we investigated whether siRNA-mediated HuR knockdown interferes with cell survival and radiation sensitivity by monitoring apoptosis, DNA repair and three-dimensional (3D) clonogenic survival. We observed a significant elevation in caspase-2 upon HuR depletion and in turn, a sensitization of colorectal DLD-1 and HCT-15 cells to radiation-induced apoptosis as implicated by the dose-dependent elevation of sub-G1 phase cell entry and increased caspase-2, -3 and poly ADP-ribose polymerase (PARP)-cleavage, respectively. Coincidentally, HuR deficiency significantly elevated the number of radiation-induced γH2AX/53BP1-positive foci indicating an increase in DNA damage. Accordingly, the irradiation-dependent reduction in clonogenic cell survival was further impaired after knockdown of HuR. Importantly, HuR knockdown remained ineffective to radiation-induced cell responses after additional knockdown of caspase-2. Furthermore, by using RNA-pull down assay we demonstrate that irradiation (6 Gy) robustly increased HuR binding to caspase-2 mRNA. Collectively, sensitization of colon carcinoma cells to radiation-induced cell death and DNA-damage by HuR knockdown critically depends on caspase-2 and may represent a valuable approach to intervene with therapy resistance of colorectal cancer (CRC).

Cytoskeleton-Dependent Transport as a Potential Target for Interfering with Post-transcriptional HuR mRNA Regulons.

The ubiquitous mRNA binding protein human antigen R (HuR), a member of the embryonal lethal abnormal vision protein family has a critical impact on the post-transcriptional control of AU-rich element bearing mRNA regulons implied in inflammation, senescence, and carcinogenesis. HuR in addition to mRNA stability can affect many other aspects of mRNA processing including splicing, polyadenylation, translation, modulation of miRNA repression, and intracellular mRNA trafficking. Since many of the identified HuR mRNA targets ("HuR mRNA regulons") encode tumor-related proteins, HuR is not only discussed as an useful biomarker but also as promising therapeutic target for treatment of various human cancers. HuR which is most abundantly localized in the nucleus is translocated to the cytoplasm which is fundamental for most of the described HuR functions on target mRNAs. Accordingly, an elevation in cytoplasmic HuR was found in many tumors and correlated with a high grade of malignancy and a poor prognosis of patients. Therefore, direct interference with the intracellular trafficking of HuR offers an attractive approach to intervene with pathologically deregulated HuR functions. Data from several laboratories implicate that the integrity of the cytoskeleton is critical for HuR-mediated intracellular mRNA localization and translation. This review will particularly focus on drugs which have proven a direct inhibitory effect on HuR translocation. Based on the results from those studies, we will also discuss on the principle value of targeting cytoskeleton-dependent transport of HuR by natural or synthetic inhibitors as a potential therapeutic avenue for interfering with dysregulated post-transcriptional HuR mRNA regulons and related tumor cell functions. In spite of that, interfering with cytoplasmic HuR transport could highlight a so far underestimated action of microtubule inhibitors clinically used for cancer chemotherapy.

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking.

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC.