Thymidine phosphorylase mRNA stability and protein levels ... - Nature

Oncogene (2009) 28, 1904–1915

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ORIGINAL ARTICLE

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells L-C Chen1, H-P Liu1, H-P Li2, C Hsueh1,3, J-S Yu1,2, C-L Liang4 and Y-S Chang1,2 1

Chang Gung Molecular Medicine Research Center, Chang Gung University, Kwei-Shan, Taoyuan, Taiwan; 2Graduate Institute of Basic Medical Sciences, Chang Gung University, Kwei-Shan, Taoyuan, Taiwan; 3Department of Pathology, Chang Gung Memorial Hospital at Lin-Kou, Kwei-Shan, Taoyuan, Taiwan and 4Department of Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan

The cytoplasmic level of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is significantly correlated with the elevated expression of thymidine phosphorylase (TP), and high levels of both proteins are predictive of a poor prognosis in nasopharyngeal carcinoma (NPC). We herein show that TP is highly induced by serum deprivation in NPC cells, and that this is due to an increase in the halflife of the TP mRNA, as shown by nuclear run-on and actinomycin D assays. We further show that the CU-rich element of the TP mRNA directly interacts with hnRNP K, as demonstrated by immunoprecipitation RT–PCR assays, and the nucleus-to-cytoplasm translocation of hnRNP K. Blockade of hnRNP K expression reduces TP expression, suggesting that hnRNP K acts in the upregulation of TP. Mechanistically, both MEK inhibitor and the hnRNP K ERK-phosphoacceptor-site mutant decrease cytoplasmic accumulation of hnRNP K, suggesting that ERK-dependent phosphorylation is critical for TP induction. Furthermore, we found that hnRNP Kmediated TP induction allows NPC cells to resist hypoxia-induced apoptosis. Our results collectively establish the regulation and role of ERK-mediated cytoplasmic accumulation of hnRNP K as an upstream modulator of TP, suggesting that hnRNP K may be an attractive candidate as a future therapeutic target for cancer. Oncogene (2009) 28, 1904–1915; doi:10.1038/onc.2009.55; published online 30 March 2009 Keywords: hnRNP K; thymidine phosphorylase; ERK signaling; nasopharyngeal carcinoma

Introduction Heterogeneous nuclear ribonucleoprotein K (hnRNP K) belongs to the hnRNP family of proteins, which directly interact with DNA and RNA through their K homology Correspondence: Professor Y-S Chang, Chang Gung Molecular Medicine Research Center and Graduate Institute of Basic Medical Sciences, Chang Gung University, 259, Wen-Hwa 1st Rd., Kwei-Shan, Taoyuan 333, Taiwan. E-mail: [email protected] Received 10 September 2008; revised 4 February 2009; accepted 23 February 2009; published online 30 March 2009

domains and regulate gene expression at multiple levels, including transcription, RNA splicing, RNA stability and translation (Bomsztyk et al., 2004; Lee et al., 2007). The expression of hnRNP K is aberrantly increased in numerous cancers, including nasopharyngeal carcinoma (NPC; Pino et al., 2003; Carpenter et al., 2006; Hatakeyama et al., 2006; Roychoudhury and Chaudhuri, 2007; Chen et al., 2008). The tumorigenic activity of hnRNP K is conferred through its ability to increase proliferation (Lynch et al., 2005), clonogenic potential (Notari et al., 2006) and metastasis (Inoue et al., 2007). These effects may be due, at least in part, to the ability of hnRNP K to upregulate c-myc expression through the c-myc internal ribosome entry segment (IRES; Evans et al., 2003; Notari et al., 2006). hnRNP K is a nucleocytoplasmic shuttling protein and primarily located in the nucleus (Michael et al., 1997); however, cytoplasmic accumulation of hnRNP K via ERKmediated phosphorylation of hnRNP K serine-284 and -353 has been reported in cervical carcinoma HeLa cells (Habelhah et al., 2001) and chronic myelogenous leukemia cells (Notari et al., 2006). Cytoplasmic translocation of hnRNP K is critical to its ability to regulate translation (Notari et al., 2006) and promote migration of fibrosarcoma cells (Inoue et al., 2007). Importantly, cytoplasmic hnRNP K, but not nuclear hnRNP K, has been significantly correlated with poor prognosis in NPC patients (Chen et al., 2008). However, the regulation and function of cytoplasmic hnRNP K in NPC have not yet been fully elucidated. Thymidine phosphorylase (TP; also termed plateletderived endothelial cell growth factor (PD-ECGF) or gliostatin) plays an important role in nucleoside metabolism. Additionally, both angiogenic and chemotactic properties have been attributed to TP, which has been shown to inhibit tumor cell apoptosis (reviewed in Liekens et al., 2007). TP is overexpressed in various cancers, including NPC, and its expression levels have been shown to have prognostic value (Liekens et al., 2007; Chen et al., 2008). The increased enzymatic activity of TP in cancer cells is utilized to activate capecitabine for treatment of metastatic colorectal and breast carcinomas (Liekens et al., 2007), and to increase the sensitivity of NPC cells to the capecitabine intermediate prodrug, 50 -DFUR (Chen et al., 2008).

Regulation of TP by ERK-regulated hnRNP K in NPC L-C Chen et al

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TP expression is transcriptionally controlled by the transcription factors, Sp1 and STAT (Goto et al., 2001; Zhu et al., 2002; Yao et al., 2005), and posttranscriptional regulation of TP mRNA stability has been proposed in response to interferons (Schwartz et al., 1998; Yao et al., 2005). However the molecular mechanisms underlying the posttranscriptional regulation have not yet been established. Although relatively rare in Caucasians, NPC is relatively common in the southeastern region of China. NPC is sensitive to radiation therapy, and more advanced disease is treated using a combination of radiotherapy and chemotherapy. The survival rate of NPC is B92% at 1 year and B50% at 5 years, with 20–25% of patients displaying distant metastases after treatment with the current regimes (Lee et al., 1992; Tse et al., 2007). Thus, it is essential that we investigate more sensitive and comprehensive approaches for developing new or supplementary therapeutic methods against NPC. Elucidation of the mechanisms underlying TP activation in NPC may offer a rational strategy for the design of more effective therapies against this disease. In this study, we show that serum deprivation of NPC cells leads to upregulation of TP expression through the stabilization of its mRNA. Mechanistically, we show that ERK-dependent phosphorylation of hnRNP K triggers its accumulation in the cytoplasm, where it binds to the mRNA of TP, increasing its half-life and thereby increasing the protein levels of TP. Finally, we show that hnRNP K-mediated upregulation of TP reduces apoptosis of NPC cells under hypoxic conditions. Collectively, these results strongly suggest that ERK-mediated cytoplasmic accumulation of hnRNP K stabilizes the mRNA of TP, thereby increasing the resistance of NPC cells to hypoxia-induced apoptosis.

Results TP is strongly induced in serum-deprived NPC cells We recently showed that serum deprivation of the NPCTW02 cell line increases TP protein levels, thereby sensitizing the cells to drug-induced apoptosis (Chen et al., 2008). To elucidate the mechanisms responsible for the elevated TP expression levels observed in NPC, we herein first confirmed that the induction of TP protein by serum deprivation was consistently observed in three NPC cell lines, NPC-TW01, -TW02 and -TW04 cells (Supplementary Figure S1a–c), and cells subjected to serum deprivation did not undergo apoptosis, as shown by analysis of the sub-G1 population (Supplementary Figure S1d). As acidosis was previously shown to induce TP expression in the absence of serum (Griffiths et al., 1997), we additionally examined the pH values of media from cell cultures grown with and without serum. The pH values of conditioned serum-free media of NPC cells sampled at 24, 48 and 72 h were 7.6, 7.6 and 7.7, respectively, whereas those of serum-grown cultures were 7.5, 7.2 and 6.7, respectively (data not shown), indicating acidosis has no apparent effect on

serum deprivation-induced TP expression. Accordingly, we propose that serum deprivation, which may mimic growth factor deficiency, is sufficient to induce TP expression in NPC cells. TP mRNA stability is increased under conditions of serum deprivation We next examined the mechanisms that could be involved in the above-described serum deprivationinduced TP activation. We first used the protein synthesis inhibitor, cycloheximide, to examine the halflife of TP proteins, and found no obvious difference in NPC-TW02 cells cultured with or without serum (Supplementary Figure S2). In contrast, quantitative RT–PCR revealed that the levels of TP mRNA were slightly elevated at 8 h after serum deprivation, 7.7-fold higher than control levels at 24 h, and 4.5-fold higher than control levels at 48 h (Figure 1a). The higher levels of TP mRNA under serum deprivation conditions may result from either transcriptional activation or posttranscriptional regulation. To distinguish between these two levels of regulation, we transfected a TP gene promoterreporter construct into NPC cells, cultured the cells with or without serum and examined reporter activity at 4, 8, 16 and 24 h. TP promoter activity was not significantly induced under serum deprivation conditions, suggesting that the increased expression of TP in serum-starved cells did not occur at the transcriptional level (Figure 1b). Consistent with this hypothesis, nuclear run-on assays revealed that the levels of newly synthesized TP mRNA were similar in cells cultured with and without serum (Figure 1c). Accordingly, we next examined the possibility of posttranscriptional regulation of TP levels. To examine the half-life of TP mRNA in cells cultured with and without serum, we treated NPC cells with actinomycin D to block de novo RNA synthesis, and then measured the persistence of the existing TP mRNA by quantitative RT–PCR at 0.5, 1, 2 and 4 h after treatment. As shown in Figure 1d, our results revealed that serum deprivation led to substantial stabilization of the TP mRNA. Specifically, the halflives of TP mRNA were 8.2 h and 3.1 h in serum-starved and control cells, respectively. These results suggest that the serum deprivation-triggered upregulation of TP is due to mRNA stabilization, not protein stabilization or transcriptional activation. The CURE sequence of TP and hnRNP K are crucial for serum-deprivation-induced stabilization of the TP mRNA The TP mRNA contains a CU-rich element (CURE) sequence (CCCCCCUCGCCC) in its 30 translated region (Figure 2a); this sequence exhibits high homology to other CURE-like sequences (Schwartz et al., 1998). Several studies have shown that CURE sequences function posttranscriptionally to regulate the mRNA stability of the CURE-containing mRNA (Levy et al., 1996; Czyzyk-Krzeska and Bendixen, 1999; Waggoner and Liebhaber, 2003; Wang et al., 2006; Emerald et al., 2007). To confirm that the CURE sequence in TP functions in its posttranscriptional regulation, we Oncogene


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Figure 1 Thymidine phosphorylase (TP) mRNA stability is enhanced by serum deprivation. (a) Kinetics of TP mRNA in cells grown under serum deprivation. Nasopharyngeal carcinoma (NPC)-TW02 cells were cultured with or without serum, and RNA was collected at 1, 2, 4, 8, 24 and 48 h. The levels of TP mRNA were determined by quantitative RT–PCR. Results are presented as the mean±s.d. from three experiments. (b) TP promoter activity examined under serum deprivation. The TP promoter construct, pGL3-TP (encompassing nucleotides 1327 to þ 71) was transfected into NPC-TW02 cells, which were then cultured with or without serum. Cells were collected at the indicated time points, and TP promoter activity was determined from luciferase activity (normalized versus that of b-galactosidase). No significant difference in TP promoter activity was evident in cells cultured with and without serum. (c) Nuclear run-on assay. Nuclei prepared from cells cultured with or without serum were incubated with [a-32P]UTP. Run-on transcripts were hybridized to 5 mg of TP cDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA or control vector. (d) Increase of TP mRNA half-life by serum deprivation. The levels of TP mRNA in cells cultured with or without serum for 48 h were measured following treatment with actinomycin D for 0.5, 1, 2 and 4 h.

inserted three copies of wild-type and mutated TP CURE into the 30 -UTR of a firefly luciferase reporter gene, generating pMIR-CURE (CURE) and pMIRCUREmut (CUREmut), respectively (Figure 2b; also see Supplementary materials and methods). We then examined luciferase activity in NPC-TW02 cells that were transfected with pMIR-CURE or pMIR-CUREmut, and then cultured with or without serum. As shown in Figure 2c, luciferase activities were 2-fold and 1.5-fold higher in pMIR-CURE-transfected cells versus pMIR-CUREmut-transfected cells under serum deprivation and normal serum conditions, respectively. These findings indicate that the TP CURE sequence is involved in the expressional activation of TP, and that serum deprivation enhances this activation (P ¼ 0.01). TP expression in the cancer cells of NPC patients has been significantly correlated with cytoplasmic accumulation of hnRNP K (Chen et al., 2008), which has been associated with the posttranscriptional control of CURE-containing mRNA (Bomsztyk et al., 2004; Ostareck-Lederer and Ostareck, 2004; Wang et al., 2006). To assess the possible role of hnRNP K in regulating TP mRNA stability under serum deprivation conditions, we examined whether hnRNP K knockdown affected luciferase activity from a CURE-containing reporter construct. As shown in Figure 2d, luciferase Oncogene

activity was reduced by B50% in cells transfected with the hnRNP K siRNA compared to control siRNA, suggesting that hnRNP K increased the expression of a reporter gene carrying TP CURE in vitro. Next, we investigated whether TP expression is physiologically regulated by hnRNP K. TP protein and mRNA levels were examined in serum-deprived NPC cells transfected with hnRNP K siRNA. As shown in Figure 2e, very little TP protein was detected in cells treated with hnRNP K siRNA in the presence and absence of serum. Similarly, TP mRNA levels were dramatically reduced (by 88%) in cells treated with hnRNP K siRNA under serum deprivation conditions, compared to cells transfected with control siRNA (Figure 2e). To confirm that hnRNP K physically binds to the TP mRNA during serum deprivation, we conducted an immunoprecipitation RT–PCR assay using an anti-hnRNP K antibody. As shown in Figure 2f, a higher level of TP mRNA was detected in the anti-hnRNP K-immunoprecipitated complexes from serum-deprived cultures compared to serum-grown cells, whereas no TP mRNA was detected using a negative control immunoglobulin G (IgG) antibody in either condition. These in vitro and in vivo analyses strongly suggest that the binding of hnRNP K to the CURE sequence of TP is involved in stabilizing the TP mRNA under conditions of serum deprivation.


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Figure 2 Interaction between thymidine phosphorylase (TP) CU-rich element (CURE) and heterogeneous nuclear ribonucleoprotein K (hnRNP K) is critical for TP mRNA stability. (a) Comparison of CU-rich mRNA sequences. (b) Schematic representation of TP CURE (pMIR-CURE) and its mutant (pMIR-CUREmut). The predicted CURE sequence is underlined and the mutated sequence is noted at the bottom. (c) Effects of TP CURE on luciferase activity were assessed in cells that were transfected with pMIR-CURE (CURE) or pMIR-CUREmut (CUREmut) and thereafter cultured with or without serum for 48 h; pSV2-gal was used as the transfection control. Effects of CURE on luciferase activity were determined as the activity ratio of CURE/CUREmut. (d) Effects o f hnRNP K knockdown on the TP CURE reporter construct were assessed in cells transfected with control siRNA (C) or hnRNP K siRNA (K). Forty-eight hours after siRNA transfection, cells were transfected with pMIR-CURE and then cultured without serum for 48 h. Relative luciferase activity was determined as fold decrease compared with control siRNA-transfected cells, and was normalized versus b-galactosidase activity. The levels of hnRNP K were determined by western blotting; actin was measured as a loading control. (e) Induction of TP protein and mRNA was assessed in cells treated with control siRNA (C) or hnRNP K siRNA (K). Forty-eight hours after siRNA transfection, cells were cultured with or without serum. The induction of TP protein was determined by western blotting 48 h later. Tubulin was measured as a loading control. Relative amounts of TP mRNA were measured by quantitative RT–PCR and normalized with regard to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. (f) TP mRNA associated with endogenous hnRNP K was detected in cells cultured with or without serum for 48 h. TP mRNA was determined by immunoprecipitation RT–PCR using anti-hnRNP K (K) or immunoglobulin G (IgG) negative control (C) antibody. The hnRNP K levels of the immunoprecipitates and 5% input are shown. The actin level of the 5% input was used as a loading control.

Cytoplasmic accumulation of hnRNP K is induced by serum deprivation The expression levels of hnRNP K were similar in NPCTW02 cells cultured with and without serum (Figure 2f), indicating that the changes in TP mRNA stability were not due to induction of hnRNP K expression in serumstarved cells. However, the cytoplasmic levels of hnRNP K were previously found to be significantly elevated in cancer cells of NPC patients, and these levels were found to correlate with TP expression (Chen et al., 2008), suggesting that translocation of hnRNP K may be involved in the induction of TP. To assess the effect of

serum deprivation on hnRNP K localization, we cultured NPC-TW01, -TW02 and -TW04 cells with or without serum for 48 h and used immunofluorescence microscopy to examine the cellular localization of hnRNP K. As shown in Figure 3a, hnRNP K was highly localized to the nucleus when all three NPC cell lines were cultured with serum. In contrast, cytoplasmic localization of hnRNP K was clearly observed in cells grown under conditions of serum deprivation. Quantitative analysis showed that the integrated fluorescence intensity of cytoplasmic hnRNP K but not the nuclear hnRNP K was significantly increased under serum Oncogene


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deprivation conditions (Po0.001). We further confirmed the cytoplasmic accumulation of hnRNP K by using nuclear/cytoplasmic fractionation approach. Consistently, the levels of cytoplasmic hnRNP K in

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NPC-TW01, -TW02 and -TW04 cells cultured without serum were increased by approximately 1.4-, 1.5- and 1.4-fold, respectively, compared to the serum-grown cultures (Figure 3b). These observations indicate that


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cytoplasmic translocation of hnRNP K is induced by serum deprivation in NPC cells. ERK signaling drives cytoplasmic localization of hnRNP K Cytoplasmic accumulation of hnRNP K has been shown to be controlled by ERK kinase-dependent phosphorylation of hnRNP K and inhibited by MEK inhibitor, PD98059 in cervical carcinoma HeLa cells (Habelhah et al., 2001) and chronic myelogenous leukemia cells (Notari et al., 2006). To determine the role of the ERK signaling pathway in inducing the cytoplasmic translocation of hnRNP K and the subsequent induction of TP expression, we examined the activation status of ERK kinases, ERK1/2 and ERK5, in cells cultured with or without serum, using western blot analysis. As shown in Figure 4a, ERK1/2 kinase was activated at 48 h in serum-deprived NPC cell lines, and this effect was significantly correlated to TP induction. However, the expression of ERK5 was hardly detectable under both conditions, implying that ERK5 seems unlikely to participate in hnRNP K translocation in all the three NPC cell lines examined. Moreover, the cytoplasmic translocation of hnRNP K was blocked in cells treated with PD98059, a pharmacological inhibitor of MEK (Figure 4b). The localization of hnRNP K in serumgrown cells was highly restricted to the nucleus, with 89.9% of the hnRNP K proteins being detected in the nucleus compared to only 10.1% in the cytoplasm. Significantly more cytoplasmic hnRNP K (27.2%; Po0.001) was observed after 48 h of serum deprivation, and this effect was selectively reduced by PD98059 treatment (from 27.2 to 12.6%; Po0.01). In contrast, as shown in Figure 4c, serum-grown cells failed to show a significant reduction in cytoplasmic hnRNP K following PD98059 treatment (10.1–7.6%; P ¼ 0.24). ERK signaling-mediated hnRNP K translocation was further confirmed through the use of two mutants: an hnRNP K phosphomimetic S284/353D mutant in which the ERK phosphoacceptor sites (serine 284 and 353) were mutated to aspartic acids (Habelhah et al., 2001), and the nonphosphatable serine to alanine (S284/353A) mutant (Habelhah et al., 2001). The localization of hnRNP K proteins was examined in serum-deprived NPC cells, transiently expressing recombinant enhanced green fluorescent protein (EGFP)-tagged wild-type or dominant-negative S284/353A mutant hnRNP K

proteins, and the cytoplasmic proportions of the various hnRNP K proteins were determined by GFP positivity. As shown in Figure 5a, although the majority of the expressed hnRNP K proteins were localized within the nucleus, the cytoplasmic proportion was significantly larger for recombinant wild-type hnRNP K (14.7%, Po0.001) compared to the dominant-negative S284/ 353A mutant hnRNP K (6.3%). To additionally demonstrate that ERK-mediated phosphorylation is able to drive hnRNP K translocation from the nucleus to the cytoplasm, NPC cells cultured with serum were transfected with constructs expressing recombinant EGFP-tagged wild-type, dominant-negative S284/353A or dominant-active S284/353D hnRNP K. As shown in Figure 5b, the cytoplasmic proportion of dominantactive S284/353D mutant hnRNP K (11.6%) was significantly larger than that of wild-type (4.5%) or dominant-negative S284/353A mutant (4.5%) hnRNP K proteins (Po0.001 in both cases). Taken together, our results suggest that activation of ERK signaling is critical for the cytoplasmic localization of hnRNP K proteins in NPC cells. Induction of TP expression by cytoplasmic localization of hnRNP K Given our results indicating that ERK-mediated cytoplasmic localization of hnRNP K is correlated with hnRNP K-mediated TP induction, we next tested whether ERK-dependent phosphorylation of hnRNP K is essential for serum deprivation-induced upregulation of TP. Serum-grown or -starved NPC cells were treated with various concentrations (2, 10 and 50 mM) of PD98059 for 48 h. As shown in Figure 6a, we observed dose-dependent inhibition of TP proteins and mRNA in PD98059-treated cells cultured under serum deprivation conditions, as shown by both western blotting and quantitative RT–PCR analyses. Notably, PD98059 did not affect the basal level of TP expression in serumgrown cells (Figure 6a). Accordingly, phospho-ERK1/2 was activated under serum deprivation, which was inhibited by PD98059 treatment in a dose-dependent manner. In addition, TP protein and mRNA levels were induced 1.7- and 1.6-fold in serum-grown cells transfected with the dominant-active S284/353D mutant hnRNP K, but not with the wild-type or dominantnegative S284/353A mutant proteins (Figure 6b). Consistent with the requirement of ERK signaling activation

Figure 3 Serum deprivation conditions trigger cytoplasmic accumulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in nasopharyngeal carcinoma (NPC) cells. (a) Three NPC cell lines, NPC-TW01, -TW02 and -TW04, were cultured on glass coverslips with and without serum for 48 h. The localization of hnRNP K was visualized after cell fixation followed by immunostaining with an anti-hnRNP K antibody. Nuclei were visualized by 40 -6-diamidino-2-phenylindole (DAPI) staining. Scale bar ¼ 20 mm. The inset shows a higher magnificent image (scale bar ¼ 5 mm). The right panel shows the quantitative results of subcellular fluorescence intensity of hnRNP K. The integrated intensity in the nucleus and in the cytoplasm of individual cells was determined using the average of 300–500 cells. *Po0.001. (b) Cytoplasmic accumulation of hnRNP K by western blotting. Three NPC cell lines, NPC-TW01, -TW02 and -TW04, were cultured with and without serum for 48 h. Cytoplasmic and nuclear compartments were isolated and the presence of hnRNP K, Sp1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by western blotting. Sp1 and GAPDH were acted as loading controls as well as nuclear and cytoplasmic markers, respectively. The amount of nuclear and cytoplasmic hnRNP K proteins was normalized with the amount of Sp1 and GAPDH, respectively, and the resulting values from the serum-grown cultures were set as 1.0. Oncogene


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for TP induction under serum deprivation, our results collectively indicate that activation of ERK signaling is critical for the cytoplasmic accumulation of hnRNP K, which subsequently regulates TP expression. hnRNP K and TP prevent hypoxia-induced apoptosis in NPC cells Previous studies have suggested that the catalytic product of TP, 2-deoxy-D-ribose, prevents hypoxiainduced apoptosis (Kitazono et al., 1998; Ikeda et al., 2006). To determine whether overexpression of TP is beneficial for the survival of cancer cells undergoing hypoxia, we incubated NPC-TW02 cells stably expressing high levels of recombinant TP in 2% O2 for 72 h, thereby mimicking hypoxic conditions. Apoptosis was measured based on phosphatidylserine externalization, using Annexin V staining. As shown in Figure 7a, fewer apoptotic cells (a 60% reduction) were observed in NPC-TW02 cells stably expressing high levels of recombinant TP, relative to parental cells. To confirm that upregulation of endogenous TP protein also increases resistance to hypoxia, we next cultured NPCTW02 cells under serum deprivation and exposed them to hypoxia. Consistent with our hypothesis, apoptosis was reduced 4.8-fold in serum-free cultures compared to serum-grown cultures (Figure 7b). We then used TP knockdown cells to examine the effects of TP on apoptosis in serum-deprived cells. Significantly more apoptotic cells were detected in NPC-TW02 cells transfected with TP siRNA compared with control siRNA-transfected cells (a 1.8-fold increase; Po0.05; Figure 7b). However, no significant change of hypoxiainduced apoptosis was detected in serum-grown NPCTW02 cells subjected to TP knockdown (Supplementary Figure S3), indicating that basal-level TP expression in serum-grown NPC cells was not sufficient to protect the cells against hypoxia. Next, we studied the involvement of the TP regulator, hnRNP K, in protection against hypoxia-induced apoptosis. As shown in Figure 7c, apoptotic cells were increased 2.2-fold after knockdown of hnRNP K

Figure 4 ERK signaling drives cytoplasmic accumulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K). (a) Analysis of the ERK signaling pathway. Three nasopharyngeal carcinoma (NPC) cell lines, NPC-TW01, -TW02 and -TW04, were cultured with or without 10% serum and harvested at 48 h. Phosphorylated ERK1/2, total ERK1/2 and ERK5 were analysed by western blotting using anti-phospho ERK1/2, anti-ERK1/2 and anti-ERK5 antibodies. NPC-TW02 cells transiently expressing myc-tagged ERK5 were acted as an ERK5 positive control. (b) The effect of ERK signaling inhibition on the localization of hnRNP K. NPC-TW02 cells were cultured with and without serum and/or the MEK inhibitor, PD98059, for 48 h. The localization of hnRNP K was visualized by immunostaining with an anti-hnRNP K antibody, and nuclei were visualized by 40 ,6-diamidino-2-phenylindole (DAPI) staining. The inset shows an enlarged image of a selected cell. Scale bar ¼ 47.62 mm. (c) The summary of the cytoplasmic proportions of hnRNP K proteins in cells cultured under the indicated conditions. *Po0.001 and **Po0.01. The inhibitory effect of PD98059 on phosphorylation of ERK1/2 was determined by western blotting. Oncogene


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in serum-starved NPC cells. However, knockdown of hnRNP K in serum-grown cells did not affect hypoxia-induced apoptosis. Our data collectively suggest that hnRNP K is activated by serum deprivation-mediated ERK signaling, which protects NPC cells against hypoxia-induced apoptosis through TP induction.

Discussion Previous studies have shown that hnRNP K directly interacts with nucleic acids and regulates gene expression at multiple levels (Bomsztyk et al., 2004; Lee et al., 2007). TP acts as an angiogenic factor and mediates inhibition of tumor cell apoptosis, thereby contributing

Figure 5 Phosphorylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is critical for cytoplasmic accumulation of hnRNP K. (a) Confocal microscopy of nasopharyngeal carcinoma (NPC)-TW02 cells expressing recombinant enhanced green fluorescent protein (EGFP)-tagged wild-type (wt) and dominant-negative S284/353A mutant (A) hnRNP K proteins, following culture for 48 h without serum. The right panel shows the cytoplasmic proportions of EGFP-tagged hnRNP K proteins observed in the various cells. (b) Confocal microscopy of NPC-TW02 cells expressing recombinant EGFP-tagged wild-type (wt), dominant-negative S284/ 353A (A), and dominant-active S284/353D (D) mutant hnRNP K proteins, following 48 h of culture with 10% serum. The right panel shows the cytoplasmic proportions of EGFP-tagged hnRNP K proteins in the various cells. Scale bars are as indicated. *Po0.001.

Figure 6 ERK-mediated cytoplasmic heterogeneous nuclear ribonucleoprotein K (hnRNP K) is critical for thymidine phosphorylase (TP) induction. (a) Effect of the MEK inhibitor, PD98059, on TP induction. The indicated doses of PD98059 were added to nasopharyngeal carcinoma (NPC) cells cultured with or without serum for 48 h. The levels of TP were determined by quantitative RT–PCR (upper panel) and western blotting (lower panel). The inhibition of ERK1/2 phosphorylation by PD98059 was determined by western blotting. (b) Effects of wild-type, S284/353A mutant, and S284/353D mutant hnRNP K proteins on TP induction. NPC cells transiently transfected with constructs encoding myc-tagged wild-type (wt), dominant-negative S284/353A mutant, and dominantactive S284/353D mutant hnRNP K, or empty vector (pcDNA3.1), were cultured with 10% serum for 48 h. The relative levels of TP were determined by quantitative RT–PCR (upper panel) and western blotting (lower panel). Following normalization with regard to tubulin levels, fold induction of TP proteins was calculated based on the intensity of TP proteins in cells transfected with various recombinant hnRNP K versus that in cells transfected with empty vector. Recombinant myc-tagged and total hnRNP K levels were determined by western blotting using anti-c-myc and anti-hnRNP K antibodies, respectively. *Po0.05; ** and *** indicate endogenous and recombinant myc-tagged hnRNP K proteins, respectively. Oncogene


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Figure 7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K) confers resistance to hypoxia-induced apoptosis via thymidine phosphorylase (TP) induction. (a) Clones stably expressing high levels of TP are resistant to hypoxia-induced apoptosis. Parental nasopharyngeal carcinoma (NPC)-TW02 cells and clones stably overexpressing TP (TW02/TP) were cultured under normoxia or hypoxia (2% O2) for 72 h. The percentage of apoptotic cells was measured by Annexin V staining. Overexpression of TP prevents cell death (by B60%). All data are presented as the mean±s.d. from at least three experiments. *Po0.001. Levels of recombinant myctagged TP proteins were examined by western blotting, with actin as the loading control. (b) NPC-TW02 cells cultured with or without serum, or additionally transfected with TP siRNA (TP) or control siRNA (C), were incubated in 2% O2 for 72 h. Apoptosis was measured by Annexin V staining. All data are presented as the mean±s.d. from at least three experiments. *Po0.05. (c) Expression of hnRNP K protects serum-starved cells against hypoxia-induced apoptosis. NPC-TW02 cells cultured with or without serum, or additionally transfected with hnRNP K siRNA (K) or control siRNA (C), were incubated in 2% O2 for 72 h. Apoptosis was measured by Annexin V staining. All data are presented as the mean±s.d. from at least three experiments. *Po0.05. The levels of hnRNP K and TP were determined by western blotting using anti-hnRNP K and anti-TP antibodies, respectively. The actin level in each cell lysate was used as the internal control. (d) Model for hnRNP K-mediated TP regulation, showing that hnRNP K is a key molecule in the control of TP expression under stress conditions. On exposure of cells to a stress such as growth factor deficiency, hnRNP K is phosphorylated by activated ERK and is differentially accumulated in the cytoplasm. Cytoplasmic hnRNP K binds to the CU-rich element (CURE) sequence of the TP mRNA; this association contributes to prolonging the half-life of the TP mRNA, thereby increasing TP protein levels. This hnRNP K-mediated elevation of TP expression allows tumor cells to survive better under adverse conditions, such as hypoxia.

to tumor progression (reviewed in Liekens et al., 2007). Cytoplasmic accumulation of hnRNP K is significantly associated with high TP expression in NPC, and high levels of both proteins are correlated with shorter survival and distant metastasis in NPC patients (Chen et al., 2008). In this study, we evaluated the role of hnRNP K in TP regulation and anti-apoptosis in NPC cells. We showed for the first time that TP expression is upregulated through mRNA stabilization, which occurs via binding of hnRNP K to the CURE sequence of TP. Moreover, cytoplasmic localization of hnRNP K is controlled by ERK-dependent phosphorylation, which is required for TP induction. Importantly, hnRNP Kmediated elevation of TP expression increases the resistance of NPC cells to apoptosis induced by hypoxia. Our findings collectively show that ERK-mediated cytoplasmic hnRNP K accumulation regulates TP mRNA stability, providing a mechanistic explanation for previous studies correlating cytoplasmic hnRNP K levels with TP overexpression in NPC patients. Oncogene

Poorer survival has been associated with both increased cytoplasmic hnRNP K in patients with NPC (Chen et al., 2008) and decreased nuclear hnRNP K in Dukes C colorectal cancer (Carpenter et al., 2006). Cytoplasmic localization of hnRNP K is required for BCR/ABL oncogenic activity in chronic myelogenous leukemia (Notari et al., 2006), and has been shown to promote cellular migration in fibrosarcoma cells (Inoue et al., 2007). Cytoplasmic hnRNP K also regulates posttranscriptional activity; it has been shown to directly stimulate IRES-dependent c-myc translation (Evans et al., 2003; Notari et al., 2006) and stabilize the gastrin mRNA (Lee et al., 2007). Accordingly, cytoplasmic hnRNP K may elevate tumorigenic potential by altering the expression of its target genes at the posttranscriptional level. In this study, we identified the presence of a CURE sequence in the TP mRNA. Several other CURE-containing genes, such as vascular endothelial growth factor (Levy et al., 1996), MAP3K7IP2 (Wang et al., 2006) and a-tropomyosin (Lin et al., 2007), have been shown to be upregulated


1913

under adverse conditions (hypoxia, and the presence of nitric oxide and arsenite, respectively) via changes in mRNA stability and/or translational regulation. Consistent with these results, we herein show that hnRNP K induces TP expression under serum deprivation conditions through its interaction with the CURE sequence of the TP mRNA (Figure 2). Binding of the TP CURE sequence to hnRNP K was also observed with RNA electrophoretic mobility shift assay (REMSA) experiments (Supplementary Figure S4). Specifically, a TP CURE RNA-protein complex formed under serum deprivation conditions was competed off by unlabeled CURE, and supershifted by anti-hnRNP K antibodies. Thus, our results strongly support a novel function of cytoplasmic hnRNP K in the stabilization of TP mRNA through the binding of hnRNP K to the CURE element at the 30 end of the TP mRNA. Although TP gene expression has been shown to be regulated by the Sp1 and STAT transcription factors (Zhu et al., 2002; Yao et al., 2005), the mechanisms of TP mRNA stabilization induced by interferon-a, -b and -g have yet not been fully elucidated (Schwartz et al., 1998; Yao et al., 2005). We herein show that TP is a new target for cytoplasmic hnRNP K, providing insight into the mystery of TP mRNA stabilization and perhaps explaining the regulation of TP mRNA stability in response to interferon-a, -b and -g. Cytoplasmic accumulation of hnRNP K is responsive to intracellular and extracellular stimuli and is regulated by ERK-mediated phosphorylation of hnRNP K (Habelhah et al., 2001; Notari et al., 2006). This study validates the importance of ERK-mediated phosphorylation of hnRNP K in NPC cells. Under serum deprivation conditions, ERK signaling was activated in NPC cells (Figure 4a). Although ERK signaling was inactive in cells cultured with serum, the phosphomimetic mutation of ERK phosphoacceptor sites increased the cytoplasmic proportion of hnRNP K in these cells (Figure 5b). Moreover, TP was induced by ERK-dependent cytoplasmic hnRNP K accumulation and forced expression of phosphomimetic S284/353D mutant hnRNP K, but not by forced expression of dominant-negative S284/353A mutant hnRNP K. Using EGFP-tagged hnRNP K and its mutant proteins to determine the cellular location, a smaller proportion of cytoplasmic hnRNP K (Figure 5) as compared to the endogenous protein (Figure 4c) was observed under our experimental conditions. This might be due to the nature of EGFP, which is prone to localize to the nucleus resulting in the overall reduction of cytoplasmic proportion of EGFP-tagged hnRNP K (Seibel et al., 2007). Taken together, our findings indicate that cytoplasmic accumulation of hnRNP K is controlled by ERK signaling in NPC cells, which is critical for the induction of TP expression. Given that ERK signaling is one of the stress-related pathways, and its hyperactivation is a hallmark of cancer (Katz et al., 2007), it would be interesting to explore other CURE-containing mRNAs that are regulated by ERK signaling-mediated translocation of cytoplasmic hnRNP K, as they could also be involved in cancer.

It is intriguing to elucidate the mechanisms involved in the serum deprivation-activated ERK signaling in NPC cells. ERK signaling is also known to be triggered by growth factors such as EGF. EGF stimulation can induce cytoplasmic translocation of a ribonucleoprotein major vault protein (Kim et al., 2006), and regulates hnRNP K to stabilize gastrin mRNA (Lee et al., 2007). We have performed experiments to evaluate if EGF also induces ERK-mediated hnRNP K translocation in NPC cells. Our results indicated that secreted EGF was undetectable in culture media collected from serumdeprived NPC cells (Supplementary Figure S5a). In addition, neither cytoplasmic translocation of hnRNP K nor sequential TP expression could be induced by addition of recombinant EGF (Supplementary Figure S5b). Moreover, EGF receptor kinase inhibitor, AG1478, had minimal effect on serum deprivationinduced hnRNP K translocation (Supplementary Figure S5b). Therefore, EGF signaling was unlikely to play a role in hnRNP K translocation under our experimental conditions. Hypoxia is a general stress in various solid tumors and limits tumor growth (Dewhirst et al., 2008). Increased tumor cell resistance to hypoxia may therefore contribute to tumor progression. TP has been shown to be a survival factor capable of protecting cells from hypoxia-induced apoptosis (Kitazono et al., 1998; Ikeda et al., 2006), and it has been significantly associated with cytoplasmic accumulation of hnRNP K and poorer survival in patients with NPC (Chen et al., 2008). Accordingly, we herein propose that hnRNP K participates in tumorigenesis through TP induction, further contributing to tumor cell survival under stress conditions (hypoxia). This hypothesis is strongly supported by our observation of increased hypoxia-induced apoptosis after knockdown of hnRNP K and TP in NPC cells cultured without serum. Our results collectively suggest that ERK-dependent cytoplasmic hnRNP K induces TP expression through mRNA stabilization, possibly by helping tumor cells survive in a microenvironment where growth factors and oxygen are often limited (Figure 7d). This hypothesis supplies a reasonable explanation for the shorter survival seen in NPC patients with higher levels of cytoplasmic hnRNP K and TP. However, future studies will be required to confirm the clinical significance of this proposed ERK-dependent translocation mechanism. Given that overexpression of TP in NPC is associated with poor prognosis (Chen et al., 2008), TP may be seen as a potential drug target for treatment of NPC, as previously shown for metastatic colorectal and breast carcinoma (Liekens et al., 2007). Accordingly, clarification of the exact mechanism of TP expression may improve patient stratification and facilitate the development of novel therapeutic strategies. In addition, as hnRNP K is an upstream regulator of TP, it may also be a potential drug target. In sum, our studies highlight a novel role for ERKmediated cytoplasmic accumulation of hnRNP K, in that it induces TP protein expression through mRNA stabilization. Oncogene


1914 Materials and methods Cell culture, plasmid construction, transfection, luciferase reporter assay, cell death analysis, cycloheximide chase, western blotting, nuclear run-on assay, REMSA, compartmentalization and ELISA Please refer to Supplementary data for details. Quantitative and immunoprecipitation RT–PCR RNA samples from whole cells and immunoprecipitates were isolated using TRIzol (Invitrogen, Carlsbad, CA, USA). Immunoprecipitates were precipitated from 1 mg of whole cell lysates in NP40 lysis buffer using an hnRNP K antibody. Mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a negative control. Reverse transcription of RNA (1 mg) was performed using ImProm-II (Promega, Madison, WI, USA) and Oligo(dT)15 primers (Promega). The primers used to amplify the TP cDNA were 50 GCAAGGTGCCAATGAT and 50 -CTCCACGAGTTTCTT ACTG, and those used to amplify the internal control cDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were as previously described (Tsai et al., 2006). Quantitative RT– PCR was performed on a Light-Cycler (Roche Diagnostics), according to the manufacturer’s instructions, using the FastStart DNA Master SYBR Green I reagent (Roche Diagnostics, Mannheim, Germany). The results for TP were normalized to those of GADPH. For mRNA half-life assessment, actinomycin D (5 mg/ml) was added after 48 h, cells were cultured with or without serum, and RNA was prepared at the indicated times. RNA interference NPC-TW02 cells were transfected with 50 nmol/l dsRNA duplexes and 50 mg dsRNA transfection reagents (TransITTKO; Mirus Bio Corporation, Madison, WI, USA) according to the manufacturer’s protocol. SMARTpool reagents, including four 21-bp RNA duplexes targeting TP and hnRNP K, were purchased from Dharmacon (Lafayette, CO, USA), and negative control siRNA was synthesized by Research Biolabs (Singapore, Singapore). The oligonucleotide sequences are presented in Supplementary Table S1. At 48 h after transfection, the medium containing the siRNA complexes was replaced with fresh serum-containing or serum-free medium. After a further 48 h of culture, cells were harvested and cell extracts were prepared and subjected to western blotting to confirm that the transfected RNA duplexes had the ability to knock down target gene expression.

Immunofluorescence microscopy All NPC cells were grown on glass coverslips for 24 h, followed by incubation with either serum-containing or serum-free medium for 48 h. Endogenous hnRNP K was detected with a specific antibody, and sequentially incubated with a fluorescein isothiocyanate-conjugated secondary antibody (BD Transduction Laboratories, Lexington, KY, USA). For analysis of recombinant hnRNP K, NPC-TW02 cells were transiently transfected with various EGFP-tagged hnRNP K constructs, and then incubated in medium with or without serum for 48 h. Nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Poole, UK) during the secondary antibody incubation. The localization of hnRNP K was observed by confocal fluorescence microscopy with a Leica TCS SP2 confocal laser scanning microscope (Leica Microsystems Inc.). For each assay, images for 300–500 cells were acquired using the same settings. Based on the images, the integrated fluorescence intensities (mean fluorescence intensities area) of hnRNP K proteins in the nuclei and the entire cell bodies of individual cells were measured using the Leica Qwin software (Leica Microsystems Inc.). The cytoplasmic proportion (%) of hnRNP K was calculated by subtracting the hnRNP K intensity in the nucleus from that in the entire cell body. For analysis of ectopic hnRNP K, NPCTW02 cells transiently transfected with various EGFP-tagged hnRNP K constructs were incubated in the medium with or without serum for 48 h. Images for 50–100 GFP-expressing cells in each experiment were acquired and the cytoplasmic proportion of EGFP-hnRNP K was analysed as described above. Statistical analysis All statistical analyses were performed using the SPSS 13.0 statistical software package. The data were analysed with the Student’s t-test. Differences were considered significant at a level of Po0.05. Conflict of interest The authors declare no conflict of interest. Acknowledgements Grant support was provided by the Ministry of Education, Taiwan (to Chang Gung University), the National Science Council, Taiwan (grants NSC 94-2314-B-182A-188, 94-3112B-182-005 and 95-2320-B-182-001 to Y-S Chang) and Chang Gung Memorial Hospital, Taiwan (grants CMRPD150961 and CMRPG360221 to Y-S Chang). Special thanks to Chung Shan Medical University for supporting CL Liang to attend the 12th International EBV Symposium.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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