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

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Cancer Letters xxx (2017) 1e10

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Original Articles

Q6 Q5

Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2 € del b, Amel Badawi a, 1, Stephanie Hehlgans b, 1, Josef Pfeilschifter a, Franz Ro a, * Wolfgang Eberhardt a b

pharmazentrum frankfurt/ZAFES, University of Frankfurt, Medical School, Frankfurt/Main, Germany Department of Radiotherapy and Oncology, University of Frankfurt, Medical School, Frankfurt/Main, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 November 2016 Received in revised form 2 February 2017 Accepted 10 February 2017

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 gH2AX/ 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 CRC. © 2017 Published by Elsevier Ireland Ltd.

Keywords: Apoptosis Caspase-2 Colorectal carcinoma cells HuR Radiotherapy resistance

Introduction Colorectal cancer (CRC) is one of the most common cancers in the western world. Despite current treatment options including surgery, chemo- and radiotherapy, these therapies are still associated with numerous side effects and with the occurrence of therapy resistance. For this reason, an implementation of molecular

Abbreviations: CRC, colorectal cancer; DDR, DNA-damage response; HuR, human antigen R; PARP, poly ADP-ribose polymerase; 50 UTR, 50 -untranslated region. * Corresponding author. pharmazentrum frankfurt/ZAFES, Klinikum der Johann €t, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Wolfgang Goethe-Universita Germany. E-mail address: [email protected] (W. Eberhardt). 1 These authors contributed equally to this work.

approaches aiming on a sensitization of CRC cells is a strong need to improve the efficacy of current tumor therapies. Experimental evidence of the last decade implicates that besides variations in the transcriptome of tumor cells, post-transcriptional mechanisms play an important role for an increased cell survival of tumor cells [for review see: Refs. [1,2]]. In line with that, the RNA-binding protein HuR which regulates AU- and U-rich element (ARE) bearing mRNAs, among many of them encoding cancer-related proteins, has emerged as a promising target of potential cancer therapeutics. Upon binding to these RNA signatures which in most cases reside within the 30 untranslated region (30 UTR) of the mRNA, HuR can affect many fates of an mRNA including splicing, stabilization, intracellular transport and translation [for a review see: Refs. [3,4]]. The critical impact of HuR-dependent posttranscriptional gene regulation in carcinogenesis is highlighted in many tumors wherein

http://dx.doi.org/10.1016/j.canlet.2017.02.010 0304-3835/© 2017 Published by Elsevier Ireland Ltd.

Please cite this article in press as: A. Badawi, et al., Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.02.010

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the increased HuR abundance tightly correlates with high-grade malignancy and with a poor outcome as has been extensively documented in human colon cancer [5e7]. Prominent target genes of HuR implement key regulators of cell cycle control, angiogenesis, cancer cell invasion or metastasis and evasion of immune recognition [3,4]. In addition, HuR elicits a broad cell survival program mainly by stabilizing and/or enhancing the translation of prominent anti-apoptotic effector proteins [1,4]. Since some prominent HuR target genes including the Xlinked inhibitor of apoptosis (XIAP) and Survivin are also involved in radiation resistance [8,9], the anti-apoptotic program by HuR becomes particularly relevant under conditions when tumor cells are exposed to ionizing radiation. Mechanistically, radiation of cells initiates the DNA-damage response (DDR), a network of signaling pathways which coordinate cell cycle checkpoints and arrest, DNA repair or apoptosis [10e12]. Increasing evidence from many laboratories indicates that evasion from DDR-induced cell death predisposes cells to malignancy and furthermore induces an important step towards therapy resistance of tumor cells [13,14]. Importantly, signaling pathways of the DDR can jointly influence HuR activity either by the alteration of cytoplasmic HuR abundance and/or by affecting HuR's binding affinity to target mRNAs [15e17]. Searching for genes functionally relevant for apoptosis resistance in colorectal carcinoma cells, we previously have identified the pro-apoptotic Caspase-2 (synonym: Caspase2L, ICH-1L) as a novel target of HuR [18]. In contrast to most prototypical target genes, HuR inhibits caspase-2 translation via a constitutive binding to the 50 untranslated region (UTR) thereby conferring a reduced sensitivity towards drug-induced apoptosis [18]. Unlike other caspases, the defined role of caspase-2, the evolutionally most conserved caspase in apoptotic pathways is not fully understood. However, caspase-2 by acting as an apical caspase is critically involved in the intrinsic, mitochondrial apoptotic pathway induced by genotoxic stress, chemotherapeutic drugs and ionizing radiation [19e21]. Functionally, a deficiency of caspase-2 promotes an aberrant DDR and genetic instability [22,23]. Given the functional impact of HuR for tumor cell survival, we investigated whether siRNA-mediated depletion of HuR would sensitize colorectal cancer cells to ionizing radiation-induced cell death and consequently may impair clonogenic survival in threedimensional (3D) grown colorectal cancer cells. The functional contribution of caspase-2 in the HuR depletion-dependent sensitization to irradiation-induced apoptosis and clonogenic survival was tested by double knockdown of HuR and caspase-2. Materials and methods Reagents All cell culture media, supplements and modifying enzymes were purchased from Invitrogen (Karlsruhe, Germany). Laminin-rich extracellular matrix (lrECM; BME Growth Factor Reduced PathClear) was obtained from Biozol (Eching, Germany). Cell culture The human colorectal carcinoma cell lines DLD-1 and HCT-15 were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and from the American Type Culture Collection (LGC-Promochem, Wiesbaden, Germany), respectively. Both cell lines are heterozygous for p53 mutation [24]. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37  C, 5% CO2 and 95% humidity. RNA interference Transfection of subconfluent cells with siRNAs was performed by using the Oligofectamine reagent (Invitrogen) according to the manufacturer's instructions. Gene silencing was performed by transfecting 50 nM of a mixture of small interfering RNA (siRNA)-duplexes from Santa Cruz (Heidelberg, Germany; siRNA-

HuR, sc-35619) and FlexiTube siRNAs for human HuR (SI00300139, SI03166436, SI103246551 and SI03246887) or the same amount of siRNAs for caspase-2 (SI00299551) from Qiagen (Hilden, Germany). For double siRNA transfections, each siRNA was applied at 25 nM. 48 h after transfection, the cells were treated for specific applications before lyzed for Western blot anaylsis. Irradiation procedure Irradiation with single doses of 2, 4 or 6 Gy was performed using a linear accelerator (SL-15, Elekta, Crawley, UK) with 6 MeV/100 cm focus-surface distance and a dose rate of 4 Gy/min. To guarantee equal conditions mock-treated cells were kept under equivalent surroundings in the irradiation control room. Western blot analysis Whole-cell lysates were prepared as described previously (Winkler et al., 2014). Total cell lysates containing 30 mg of protein were prepared in SDS sample buffer and resolved by 12%e15% SDS-PAGE, and transferred for immunodetection with the following specific primary antibodies: anti-HuR (sc-5261) and anti-BID (sc-373939), both from Santa Cruz, anti-caspase-2 (#611022, BD Biosciences, Heidelberg, Germany), anti-caspase-3 (#9662) and anti-PARP (#9542) both from Cell Signaling, (Frankfurt, Germany). For detection, blots were incubated with goat anti-rabbit (sc2054) or goat anti-mouse (sc-20559) HRP-linked antibodies (Santa Cruz Biotechnology) and finally visualized with chemiluminescence using an ECL system from Amersham Biosciences (Freiburg, Germany). To confirm equal loading of protein amounts, blots were re-probed with a b-actin antibody (#A2228, SigmaeAldrich Deisenhofen, Germany). Biotin pull-down assay Biotin pull-down assay was performed as described previously [18]. Briefly, a biotinylated RNA sense probe was generated by using 10 mg of linearized plasmid pCR2.1-50 -UTR-caspase-2 using the “RiboMax Large scale RNA production system T7” (Promega, Mannheim, Germany) and T7 RNA polymerase and biotin-CTP (Invitrogen). 15 mg of the biotinylated RNAs were conjugated to streptavidinconjugated agarose beads in incubation buffer (10 mM TriseHCl, pH 7.5; 150 mM KCl; 1.5 mM MgCl2; 0.5 mM DTT; 40 U/ml RNasin) at 4  C for 2 h with continuous rotation. Subsequently, 300 mg of total cell lysates were added to the beads and incubated for 45 min at 4  C. After intensive washing with incubation buffer, RNA-bound proteins were collected by addition of 35 ml of 1 Laemmli buffer and the pull-down material was subsequently analyzed by Western blot analysis by probing the membranes successively with a HuR-specific antibody. Equal input material was confirmed by Western blotting using the same antibody. After incubation with the secondary antibody, the immunopositive signals were visualized by ECL. Cell cycle and apoptosis analysis The analysis of cell cycle distribution and the sub-G1 population of different colorectal carcinoma cells was performed with a FACSCanto II flow cytometer (Becton Dickinson, Heidelberg, Germany). Briefly, cells were seeded in 60 mm dishes and transfected with the relevant siRNAs as described before. At 24 h after siRNA transfection, the cells were trypsinized, centrifuged (300  g for 5 min), resuspended in growth medium supplemented with 10% heatinactivated fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin before 4.0e6.0  105 cells were seeded on 60 mm petri dishes. 24 h later the cells were exposed to irradiation and a subsequent 24 h later trypsinized, washed in PBS and fixed overnight in absolute ethanol at 20  C. After centrifugation (300  g for 2 min), cell pellets were resuspended in 0.3 ml hypotonic buffer containing 50 mg/ml propidium iodide (SigmaeAldrich); 0.1% sodium citrate; 0.1% Triton X-100 and 10 mg/ml RNase A and incubated for 30 min at 37  C before measurement. Finally, cells were gated to exclude cell debris and analyzed by flow cytometry in linear mode by using the FACSDiva Software (Becton Dickinson). Immunofluorescence staining and quantification of gH2AX/53BP1 foci formation Analysis of residual DNA double-strand breaks (DSBs) was performed by counting of gH2AX/53BP1-positive nuclear foci. Colorectal carcinoma cells were subjected to siRNA transfection and 24 h later plated on microscope cover glasses in 12-well plates (neoLab Migge, Heidelberg, Germany). Cells were irradiated (0, 2, 6 Gy) after 24 h, fixed and permeabilized with 3.7% paraformaldehyde and 0.25% Triton X-100 (AppliChem, Darmstadt, Germany) in PBS for 10 min at 24 h after irradiation. After blocking in 5% bovine serum albumin (Applichem), staining was accomplished with anti-gH2AX (clone JBW301, #05-636, Millipore, Schwalbach, Germany) and anti-53BP1 (#100-304, Novus Biologicals, Cambridge, UK) primary and Alexa-labeled secondary antibodies (Alexa Fluor 594 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, Life Technologies, Darmstadt, Germany). Nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) solution (Life Technologies). Cover slips were mounted with Vectashield mounting medium (Alexis, Grünberg, Germany). gH2AX/53BP1-positive foci were microscopically counted

Please cite this article in press as: A. Badawi, et al., Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.02.010

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using an AxioImager Z1 microscope (Carl Zeiss, Jena, Germany) and 50 nuclei were evaluated for each data point from four independent experiments. Fluorescence images were obtained with the AxioImager Z1 microscope and AxioVision 4.6 software (Carl Zeiss). 3D colony formation assay Analysis of three-dimensional (3D) clonogenic survival was accomplished as previously described [25]. Briefly, knockdown cell cultures were plated in 0.5 mg/ ml lrECM, supplemented with RPMI (HCT-15) or DMEM (DLD-1) medium, 10% FCS and 1% Penicillin/Streptomycin. Cells were irradiated 24 h thereafter (0e6 Gy, single doses) and colony formation was assessed microscopically 6 days (DLD-1) or 7 days (HCT-15) after plating by counting of colonies > 50 cells (inverted microscope Axio Vert.A1, 2.5 objective, Carl Zeiss). Images of typical 3D colonies were obtained by phase contrast microscopy using an Axio Observer inverted microscope (Carl Zeiss) equipped with a 10 objective. Calculation of plating efficiencies was performed according to: numbers of colonies formed/ numbers of cells plated. Surviving fractions (SF) were evaluated as follows: numbers of colonies formed/(numbers of cells plated (irradiated)  plating efficiency (non-irradiated)). Each point on survival curves represents the mean surviving fraction from four independent experiments, each performed in triplicate. Survival variables a, b were fitted according to the linear quadratic equation SF ¼ exp [a  D  b  D2] with D ¼ dose (EXCEL software, Microsoft, Redmond, USA). Statistical analysis Data are given as means ± SD. For statistical analysis, the unpaired two-tailed ttest was applied (GraphPad Prism, GraphPad Software, Inc., La Jolla, CA, USA). A p value  0.05 was considered significant.

Results Kinetics of siRNA-mediated HuR knockdown in DLD-1 cells Previously, we reported on caspase-2 as a novel HuR target gene which is regulated by HuR-mediated repression of translation [18]. Given the possible causal role of caspase-2 in DNA damaged-induced apoptosis [22,26], we investigated whether an increased level of caspase-2 after knockdown of HuR would sensitize DLD-1 cells to radiation-induced apoptosis. For estimation of time-dependent effectiveness of HuR knockdown, we monitored the time-course of HuR levels in response to HuR knockdown (siHuR) in DLD-1 cells. Western blotting revealed a robust and stable decrease in HuR protein expression compared to cells transfected with a scrambled siRNA (siCtrl.) (Fig. 1). By contrast, the increase in caspase-2 levels by HuR knockdown was only transient and significant at 24 and 48 h after transfection (Fig. 1). For the following experiments, colon carcinoma cells were routinely transfected for 48 h before they were exposed to ionizing radiation. HuR-silencing sensitizes DLD-1 cells to radiation-induced apoptosis To test the functional impact of transient HuR knockdown on radiation-induced cell-death, we compared apoptosis in control-siRNA with siHuR-transfected DLD-1 cells by monitoring cleavage of caspase-2 and -3 at 24 h after irradiation, respectively. In addition to the pro-caspase, a longer exposure of the Western blots revealed a weak increase in the constitutively generated caspase-2 cleavage product running at 32 kDa (Casp2*) after HuR depletion (Fig. 2A). Addtionally, generation of a second caspase-2 cleavage product at 18/19 kDa (Casp2**) appeared if combining HuR knockdown with radiation (2 and

Fig. 1. Time-dependent changes in caspase-2 protein levels after HuR knockdown. Subconfluent DLD-1 cells were transfected with control siRNA duplexes (siCtrl.) or

with siRNA duplexes of HuR (siHuR) for the indicated time periods before the content of total HuR or caspase-2 (Casp2) was monitored by Western blot analysis and b-actin was used as loading control. Data in the lower panels represent means ± SD (n ¼ 5) *P  0.05, **P  0.01, ***P  0.005, siCtrl. vs. siHuR cells.

Please cite this article in press as: A. Badawi, et al., Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.02.010

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Fig. 2. Transient knockdown of HuR sensitizes colon carcinoma cells to irradiation-induced apoptosis. (A). Subconfluent DLD-1 cells were transfected with control siRNA duplexes (siCtrl.) or with siRNA duplexes of HuR (siHuR) for 48 h before cells were irradiated with the indicated single doses of X-ray and harvested 24 h later. Cleavage of caspases and PARP (indicated by asterisks) and the knockdown efficiency of HuR was determined by Western blot analysis. (B). Densitometrical analysis of cleaved caspase-3 (upper panel) and PARP (lower panel) levels in relationship to the contents of corresponding mature proteins. Data represent means ± SD (n ¼ 3) *P  0.05, **P  0.01 compared with control-siRNAtransfected (siCtrl.) cells (C). Cells were treated similar as described in panel (A) before sub-G1 arrest was analyzed by flow cytometry (FACS) by propidium iodide (PI) staining. Values represent means ± SD (n ¼ 3) and are depicted as percentage of cells in the sub-G1-phase *P  0.05, versus the indicated transfectants and are depicted as percentage of cells in the sub-G1 phase. The graph in (D) shows cell-cycle kinetics from the same experiments. Error bars, SD from three independent experiments.

6 Gy). The increase of this smaller caspase-2 18/19 kDa fragment in HuR knockdown cells after irradiation implicate an increase in the second cleavage step of caspase-2 which fully corresponded with the enhanced cleavage of caspase-3 (Fig. 2A). Similarly, the basal and radiation-induced processing of the effector caspase-3 was significantly increased after HuR knockdwon and most prominent after 6 Gy irradiation (Fig. 2A and B upper panel). Also, the presence of cleaved PARP after irradiation was markedly increased upon HuR knockdown supporting an increased activation of the caspase cascade after

HuR depletion (Fig. 2A and B, lower panel). Next, the impact of HuR silencing and irradiation on apotosis was confirmed by determination of sub-G1 fractions. The knockdown of HuR resulted in a significant increase in sub-G1 phase cells after siCtrl. treatment and was dose-dependently increased after irradiation with the highest cell counts in the sub-G1 phase measured at 6 Gy (Fig. 2C and Supplementary Fig. 1). Concomitantly, silencing of HuR had a moderate negative effect on cell counts in the G2/M phase following irradiation at 6 Gy (Fig. 2D).

Please cite this article in press as: A. Badawi, et al., Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.02.010

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Fig. 3. Caspases-2 is indispensable for irradiation-induced apoptosis in HuR depleted DLD-1 cells. (A). DLD-1 cells were transfected with control siRNA duplexes (siCtrl.) or with siRNA duplexes of caspase-2 (siCasp2) or HuR (siHuR) or alternatively, double transfected with HuR plus caspase-2-specific siRNA duplexes (siHuR þ Casp2) for 48 h before cells were irradiated with the indicated doses (single doses, X-ray). 24 h after irradiation cells were harvested for total protein extraction and cleavage (asterisk) of caspase-3 (upper panel) and PARP (lower panel) determined by Western blot using b-actin as a loading control. To achieve a better resolution of PARP processing, samples from the same experiments were separately loaded on a 12% PAGE gel. (B). Densitometrical analysis of cleaved caspase-3 (upper panel) or PARP (lower panel) levels in a relationship to the contents of corresponding uncleaved proteins in knockdown cell cultures Data represent means ± SD (n ¼ 3) *P  0.05, **P  0.01 siCtrl. vs. siHuR cells and #P  0.05, ##P  0.01 siHuR/Casp2 vs. siHuR.

Fig. 4. Caspase-2 is indispensable for irradiation-induced apoptosis in HuR depleted HCT-15 cells. (A). HCT-15 cells were transfected with control siRNA duplexes (siCtrl.) or with siRNA duplexes of caspase-2 (siCasp2) or HuR (siHuR) or alternatively, double transfected with HuR plus caspase-2-specific siRNA duplexes (siHuR þ Casp2) for 48 h before cells were irradiated with the indicated ionizing doses (single doses, X-ray). 24 h after irradiation the processing of caspase-3 (upper panel) and PARP (lower panel) was determined by Western blot analysis. To achieve a better resolution of uncleaved and cleaved PARP, samples from the same experiments were additionally loaded on a 12% PAGE gel. (B). Densitometrical analysis of cleaved caspase-3 (upper panel) or PARP (lower panel) levels in relationship to the contents of uncleaved caspase-3 and PARP in knockdown cell cultures Data represent means ± SD (n ¼ 3) *P  0.05, **P  0.01 siCtrl. vs. siHuR cells and #P  0.05, ##P  0.01 siHuR/Casp2 vs. siHuR.

Please cite this article in press as: A. Badawi, et al., Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.02.010

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Caspase-2 is indispensable for irradiation-induced apoptosis in colorectal carcinoma cells Next, the functional impact of caspase-2 on apoptosis sensitization by HuR knockdown was assessed by additional silencing of caspase-2. To confirm our findings in a second colorectal cancer cell line, experiments were additionally performed in HCT-15 cells. First, the knockdown efficacy of both proteins in both cell lines was confirmed by Western blotting (Supplementary Fig. 2). Interestingly, in both cell lines the HuR depletion-dependent increase in caspase-3 cleavage was strongly reduced after the additional silencing of caspase-2 (Figs. 3A and 4A). Accordingly, cells receiving radiation treatment did not show an increase in caspase-3 cleavage after the single knockdown of caspase-2 or HuR/caspase-2 double knockdown (Figs. 3 and 4). Similarly, the increased PARP processing after HuR knockdown in both cell lines was impaired after additonal knockdown of caspase-2 (Figs. 3 and 4). Furthermore, the HuR depletion-dependent increase in sub-G1 phase fractions in DLD-1 and HCT-15 was dose-dependently enhanced by irradiation but ablated after the additional knockdown of caspase-2 (Fig. 5A and B and Supplmentary Fig. 3A and B). Together, these results demonstrate that silencing of HuR expression sensitizes colorectal cancer cells to basal as well as radiation-induced apoptosis by a mechanism which critically depends on caspase-2.

knockdown nor HuR/caspase-2 double knockdown had an impact on the basal plating efficiency of colorectal carcinoma cells (Fig. 8A and B). In contrast, HuR knockdown significantly radiosensitized DLD-1 and HCT-15 cells (Fig. 8A and C). Again, the sensitizing effect by HuR knockdown was rescued by simultaneous caspase-2 depletion, to levels which were obtained after control siRNA transfection (siCtrl.), whereas a single knockdown of caspase-2 even increased the clonogenic radiation survival of DLD-1 cells (Fig. 8C). Together, these data demonstrate that HuR depletion-dependent sensitization of colon carcinoma cells towards irradiation-induced apoptosis is partially due to an increase in DNA damage by a mechanism which critically depends on caspase-2.

Constitutive HuR binding to the 50 UTR of caspase-2 is enhanced by irradiation Given the previously reported constitutive HuR binding to the 50 UTR of caspase-2 which is relevant for a reduction in caspase-2 translation [18], we next investigated whether ionizing radiation would affect the constitutive binding of endogeneous HuR to caspase-2. For this purpose, we employed a pull-down assay with an in-vitro transcribed biotinylated mRNA encompassing the complete 50 UTR of caspase-2 (Fig. 6). Exposure of DLD-1 cells with irradiation (6 Gy) caused a significant and transient increase in HuR affinity to the 50 UTR of caspase-2 6 h after radiation (Fig. 6). Caspase2 knockdown protects from HuR depletion-induced DNA damage To test whether the caspase-2 dependent increase in apoptosis after HuR knockdown may coincide with a hampered DNA repair capacity, DSBs were determined by counting gH2AXand 53BP1-positive nuclear foci as described in “Materials and Methods”. Thereby, measurement of repair of radiation-induced DNA DSB in HuR and/or Caspase-2 knockdown cells revealed a significant increase of gH2AX/53BP1 foci after HuR depletion in 2 Gy and 6 Gy irradiated colorectal cancer cells whereas no significant changes were observed after single knockdown of Caspase-2 (Fig. 7A and B). In contrast, the additional knockdown of caspase 2 in both cell lines rescued from radiation- and HuR depletion-induced DNA damage (Fig. 7B) indicating a critical role of caspase 2 in the increased DNA damage upon HuR knockdown. The radiosensitization of colorectal carcinoma upon HuR knockdown critically depends on caspase-2 Finally, we evaluated the long-term effect of HuR and/or caspase-2 depletion in combination with irradiation in a more physiological cell culture model by applying a 3D clonogenic survival assay (Fig. 8A). Neither HuR or caspase-2 single

Fig. 5. Depletion of caspase-2 abrogates the irradiation-induced accumulation of colorectal carcinoma cells in sub-G1 phase upon HuR knockdown. DLD-1 (A) or HCT-15 (B) cells were transfected with control siRNA duplexes (siCtrl.) or with siRNA duplexes of caspase-2 (siCasp2) or HuR (siHuR) or alternatively, double transfected with HuR plus caspase-2-specific siRNA duplexes (siHuR þ Casp2) for 48 h before cells were irradiated with the indicated doses (single doses, X-ray). 24 h after irradiation, sub-G1 arrest was analyzed by flow cytometry (FACS) by propidium iodide (PI) staining. Values represent means ± SD (n ¼ 3) *P  0.05, **P  0.01, ***P  0.005 siHuR vs. siCtrl. and # P  0.05, ##P  0.01, ###P  0.005 siHuR/Casp2 vs. siHuR.

Please cite this article in press as: A. Badawi, et al., Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.02.010

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Fig. 6. Irradiation enhances the constitutive HuR binding to the 50 UTR of Caspase-2. (Upper panel). Schematic representation of the human caspase-2 mRNA coding for the proapoptotic caspase-2 (Casp2). The relative position of exons is depicted by boxes and triangles show the position of start (grey) and stop codons (black) of the coding sequence (CDS). The doubleheaded arrow depicts the length of the RNA probe used for biotin pull-down assay. (Lower panel). A representative biotin pull-down assay showing irradiationinduced changes in HuR binding to the 50 UTR of caspase-2. For pull-down assay, the biotinylated transcript encompassing 241 nucleotides of the the 50 UTR of human caspase-2 was incubated with 300 mg of total cell lysates from DLD-1 cells which were left untreated () or irradiated (þ) for the indicated time with a single dose (6 Gy). HuR binding to the pulldown material was assessed by Western blot analysis and equal amount of input material was confirmed by Western bloting (HuRinput). Graphs show means ± SD (n ¼ 3) and depict the relative HuR mRNA-binding in relation to input HuR levels in untreated (open bars) or irradiated (filled bars) DLD-1 cells. **P  0.01 versus untreated conditions.

Discussion The evasion of apoptosis most likely constitutes a major reason for therapy resistance of tumor cells towards anticancer treatment including chemo/radiotherapy and substantially limits the efficacy of those therapies. Several lines of evidence from tumor patient specimens and animal models implicate that elevations in the RNA-binding protein HuR in various cancers tightly correlate with cancer progression and chemotherapeutic drug resistance [6,27e32]. Given the broad anti-apoptotic capacity of HuR, an inhibition of this ubiquitous RNA-binding protein reflects a promising therapeutic avenue to improve the efficacy of current therapy regimes. In this study we demonstrate a significantly increased sensitivity towards irradiation-induced DNA damage and clonogenic survival after knockdown of HuR. In addition to an increased DNA-damage, we found a significant elevation in radiation-induced apoptosis. Consistently with the HuR-depletion dependent increase in apoptosis, the transient knockdown of HuR significantly increased the number of gH2AX/53BP1 foci in colon carcinoma cells implicating an increased number of DNA-DSBs is concomitant with an enhanced radiation response. In contrast, HuR depletion had only a minor impact on the radiation-induced distribution in other cell cycle phases (Fig. 2D). This indicates that the increase in radiation sensitivity does not rely on HuR mediated alterations in cell cycle phase distribution. In contrast, using RNApulldown assay we show, that irradiation transiently increased the constitutive HuR binding to the 50 UTR of caspase-2 suggesting a modulation of RNA-binding affinity underlies radiotherapyinduced cell survival mechanisms by HuR (Fig. 6). Importantly, we have previously demonstrated that HuR exerts a direct repressive effect on caspase-2 translation through its constitutive binding to the 50 UTR of caspase-2 mRNA a mechansims which critically contributes to the anti-apoptotic program of HuR [18]. Consequently, the irradiation-induced HuR binding to the 50 UTR of

caspase-2 via downregulation of caspase-2 may indicate an activation of the HuR-mediated pro-survival mechanisms under genotoxic stress conditions. In contrast to other caspases, the defined role of caspase-2, the evolutionally most conserved caspase in apoptotic pathways is still on debate [19e21]. According to a current model, caspase-2 by acting as an apical caspase can be critically involved in the intrinsic, mitochondrial apoptotic pathway induced by DNA-damage by a mechanism wich depends on a trimolecular complex of p53induced protein with death domain (PIDD), receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD) and caspase-2 [22]. However, studies by Manzl et al. could demonstrate an alternative and PIDDosomeindependent mechanism of caspase-2 activation [33] or reported on conditions under which DNA damage-induced cell death is not uniquely dependent on caspase-2 or the PIDDoseome [34]. Therefore, the role of caspase-2 in DNA-damage induced apoptosis seems highly cell-type and stimulus specific. A mechanism which could explain caspase-3 induction by caspase-2 includes the activation of the proapoptotic protein BH3 interacting domain death agonist (BID) leading to an increased mitochondrial release of cytochrome C [35,36]. Importantly, when monitoring for BID activation of DLD1 cells by Western-blot analysis, we found a weak increase in BID cleavage in HuR knockdown cells mainly after high doses of radiation (Supplementary Fig. 4) indicating that BID cleavage is probably involved in the activation of intrinsic apoptosis by caspase-2. In addition to apoptosis, caspase-2 is implied in several nonapoptotic responses including tumor suppression [37], genetic stability [22,23] and modulation of autophagy [38]. Interestingly, the increase in caspase-2 upon HuR knockdown was only transient although the knockdown efficacy of HuR remained constant (Fig. 1). We did observe this phenomenon also in other colon carcinoma cells (data not shown), suggesting that these tumor cells have evolved different mechanisms to keep caspase-2 in check.

Please cite this article in press as: A. Badawi, et al., Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.02.010

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Fig. 7. HuR depletion increases radiation-induced residual DNA-DSB, an effect reversed by concomitant caspase-2 knockdown. (A). Representative images of residual gH2AX/53BP1 nuclear foci 24 h after irradiation of indicated colorectal cancer knockdown cell cultures (siCtrl., non-specific control siRNA; siHuR, HuR siRNA; siCasp2, caspase-2 siRNA; siHuR/ Casp2, HuR þ Caspase-2 siRNA) with doses of 0e6 Gy (single doses, X-ray). Nuclei were counterstained for DAPI. Bar, 5 mm. (B). Simultaneous caspase-2 depletion rescues HuRmediated hampered DNA repair. Quantification of residual gH2AX/53BP1-positive nuclear foci of DLD-1 (left) and HCT-15 (right) non-specific control (siCtrl.), HuR and/or caspase-2 (siHuR, siCasp2, siHuR/Casp2) knockdown cells 24 h after irradiation with 0, 2 or 6 Gy. Columns represent means þ SD (n ¼ 4). *P  0.05, **P  0.01, ***P  0.005 siHuR vs. siCtrl. and #P  0.05, ##P  0.01, ###P  0.005 siHuR/Casp2 vs. siHuR.

Noteworthy, in accordance to our results, a recently published study could demonstrate that siRNA-mediated HuR silencing sensitizes human breast cancer cells to radiotherapy by a mechanism including increased oxidative stress [39]. The authors of the study did not investigate the effects on apoptosis, but importantly, reactive oxygen species (ROS) account as potent inducers of caspase-2 [40]. Therefore, it is tempting to speculate that in addition to a direct translational suppression of caspase-2, HuR through an inhibition of ROS may additionally interfere with the activation of caspase-2. In keeping with the known function of caspase-2 to act as a “damage sensor” the irradiation-induced binding of HuR to the 50 UTR of caspase-2 mRNA may reflect an additional mechanism contributing to the complex regulation of the DNA damage response in tumor cells [17]. In line with that DNA damage pathways in response to ionizing irradiation can jointly influence HuR functions mainly by affecting its binding affinity to target mRNA. In particular, the checkpoint kinases Chk2 the main effector of the ataxia telangiectasia mutated (ATM)-related DDR-kinases, can affect HuR's binding affinity to target mRNA via direct phosphorylation [41]. Similar to HuR, caspase-2 can be activated by the ATR/ ATM kinases as an evolutionary ancient DNA-damage response pathway independent of p53 [38]. Prior work from other laboratories revealed that ionizing radiation has a strong impact on the

dynamic association of HuR with target mRNAs by a mechanism which depends on the DNA-damage kinase ATM [43]. Future experiments are needed to define the potential role of HuR specific posttranslational modifications especially by different DNAdamage kinases for suppression of caspase-2 translation in response to ionizing radiation and genotoxic drugs. In summary, our study implicates that HuR-dependent inhibition of caspase-2 represents a so far undiscovered survival mechanism by colorectal carcinoma cells. Targeting the molecular interaction between HuR and caspase-2 may consequently represent a valuable approach to enhance the efficacy of current preoperative therapies for treatrment of CRC.

Conflict of interest The authors declare that they have no conflicts of interest.

Uncited reference [42].

Please cite this article in press as: A. Badawi, et al., Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.02.010

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Fig. 8. Radiosensitization of colorectal cancer cells by HuR depletion is rescued by caspase-2 knockdown. (A). Phase contrast images of representative 3D DLD-1 and HCT-15 control (siCtrl.) or HuR siRNA (siHuR)-treated and irradiated (0, 6 Gy) colonies 6e7 days after plating of single cells in laminin-rich extracellular matrix (lrECM, 0.5 mg/ml). bar, 100 mm. (B). HuR and/or caspase-2 knockdown does not modulate plating efficiencies of non-irradiated colorectal cell cultures. DLD-1 or HCT-15 knockdown cell cultures (siCtrl., non-specific control siRNA; siHuR, HuR siRNA; siCasp2, caspase-2 siRNA; siHuR/Casp2, HuR þ caspase-2 siRNA) were plated in 3D lrECM. Colonies >50 cells were counted microscopically and plating efficiencies (numbers of colonies formed/numbers of cells plated) relative to siCtrl. were calculated. Results represent means of n ¼ 4 independent experiments þ SD. (C). HuR-dependent radiosensitization of colorectal cancer cells is mediated by caspase-2 induction. Indicated knockdown cells were plated in 3D lrECM and irradiated 24 h thereafter (0, 2, 4, 6 Gy, single doses, X-ray). At 6 or 7 days after plating, colonies >50 cells were microscopically quantified, surviving fractions were calculated and survival variables a, b were fitted according to the linear quadratic equation SF ¼ exp [a  D  b  D2]; D ¼ dose. Data represent means ± SD (n ¼ 4) *P  0.05, **P  0.01, ***P  0.005 siHuR vs. siCtrl. #P  0.05, ##P  0.01, ###P  0.005 siHuR/Casp2 vs. siHuR.

Please cite this article in press as: A. Badawi, et al., Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.02.010

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Acknowledgements We gratefully acknowledge Roswitha Müller and Julius Oppermann for their excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft [EB 257/6-1, Excellence Cluster “Cardiopulmonary System (ECCPS)” EXC 147/1] and the German Federal Ministry of Education and Research (BMBF) [GREWIS: 02NUK017F]. A.B. was financially supported by a scholarship from the DAAD and by the University of Khartoum (Sudan). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2017.02.010. References [1] K. Abdelmohsen, A. Lal, H.H. Kim, M. Gorospe, Posttranscriptional orchestration of an anti-apoptotic program by HuR, Cell Cycle 6 (2007) 1288e1292. [2] K. Mazan-Mamczarz, P.R. Hagner, S. Corl, S. Srikantan, W.H. Wood, K.G. Becker, et al., Post-transcriptional gene regulation by HuR promotes a more tumorigenic phenotype, Oncogene 27 (2008) 6151e6163.  pez de Silanes, A. Lal, M. Gorospe, HuR: post-transcriptional paths to [3] I. Lo malignancy, RNA Biol. 2 (2005) 11e13. [4] K. Abdelmohsen, M. Gorospe, Posttranscriptional regulation of cancer traits by HuR, Wiley Interdiscip. Rev. RNA 1 (2010) 214e229. [5] D.A. Dixon, N.D. Tolley, P.H. King, L.B. Nabors, T.M. McIntyre, G.A. Zimmerman, et al., Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells, J. Clin. Invest. 108 (2001) 1657e1665. [6] C. Denkert, I. Koch, N. von Keyserlingk, A. Noske, S. Niesporek, M. Dietel, et al., Expression of the ELAV-like protein HuR in human colon cancer: association with tumor stage and cyclooxygenase-2, Mod. Pathol. 19 (2006) 1261e1269.  pez de Silanes, J. Fan, X. Yang, A.B. Zonderman, O. Potapova, E.S. Pizer, et [7] I. Lo al., Role of the RNA-binding protein HuR in colon carcinogenesis, Oncogene 22 (2003) 7146e7154. [8] D. Moussata, S. Amara, B. Siddeek, M. Decaussin, S. Hehlgans, R. Paul-Bellon, et al., XIAP as a radioresistance factor and prognostic marker for radiotherapy in human rectal adenocarcinoma, Am. J. Pathol. 181 (2012) 1271e1278. € del, T. Sprenger, B. Kaina, T. Liersch, C. Ro €del, S. Fulda, et al., Survivin as a [9] F. Ro prognostic/predictive marker and molecular target in cancer therapy, Curr. Med. Chem. 19 (2012) 3679e3688. [10] J.H. Petrini, T.H. Stracker, The cellular response to DNA double-strand breaks: defining the sensors and mediators, Trends Cell. Biol. 13 (2003) 458e462. [11] Y. Liang, S.Y. Lin, F.C. Brunicardi, J. Goss, K. Li, DNA damage response pathways in tumor suppression and cancer treatment, World J. Surg. 33 (2009) 661e666. [12] H. Wang, X. Zhang, L. Teng, R.J. Legerski, DNA damage checkpoint recovery and cancer development, Exp. Cell Res. 334 (2015) 350e358. [13] J. Bartkova, Z. Horejsí, K. Koed, A. Kr€ amer, F. Tort, K. Zieger, et al., DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis, Nature 434 (2005) 864e870. [14] F.A. Mallette, M.F. Gaumont-Leclerc, G. Ferbeyre, The DNA damage signaling pathway is a critical mediator of oncogene-induced senescence, Genes Dev. 21 (2007) 43e48. [15] M. Gorospe, R. de Cabo, AsSIRTing the DNA damage response, Trends Cell Biol. 18 (2008) 77e83. [16] H.H. Kim, K. Abdelmohsen, M. Gorospe, Regulation of HuR by DNA damage response kinases, J. Nucleic Acids 25 (2010) 2010. €pker, et al., [17] J. Boucas, A. Riabinska, M. Jokic, G.S. Herter-Sprie, S. Chen, K. Ho Posttranscriptional regulation of gene expression-adding another layer of complexity to the DNA damage response, Front. Genet. 3 (2012) 159. [18] C. Winkler, A. Doller, G. Imre, A. Badawi, T. Schmid, S. Schulz, et al., Attenuation of the ELAV1-like protein HuR sensitizes adenocarcinoma cells to the intrinsic apoptotic pathway by increasing the translation of caspase-2L, Cell Death Dis. 5 (2014) e1321. [19] L. Bouchier-Hayes, The role of caspase-2 in stress-induced apoptosis, J. Cell. Mol. Med. 14 (2010) 1212e1224.

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