Sphingosine Kinase-1 Enhances Resistance to Apoptosis Through Activation of PI3K/Akt/NF-Kb Pathway in Human Non–Small Cell Lung Cancer

Published OnlineFirst February 15, 2011; DOI: 10.1158/1078-0432.CCR-10-0720

Clinical Cancer Cancer Therapy: Preclinical Research

Sphingosine Kinase-1 Enhances Resistance to Apoptosis through Activation of PI3K/Akt/NF-kB Pathway in Human Non–Small Cell Lung Cancer

Libing Song1, Huaping Xiong2,4, Jun Li3,4, Wenting Liao1, Lan Wang2,4, Jueheng Wu2,4, and Mengfeng Li2,4

Abstract Purpose: The present study was to examine the effect of sphingosine kinase-1 (SPHK1) on chemotherapeutics-induced apoptosis in non–small cell lung cancer (NSCLC) cells, which is relatively insensitive to chemotherapy, and its clinical significance in NSCLC progression. Experimental Design: The correlation of SPHK1 expression and clinical features of NSCLC was analyzed in 218 paraffin-embedded archived NSCLC specimens by immunohistochemical analysis. The effect of SPHK1 on apoptosis induced by chemotherapeutics was examined both in vitro and in vivo, using Annexin V staining and TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) assays. Western blotting and luciferase analysis were performed to examine the impact of SPHK1 on the PI3K/Akt /NF-kB signaling. Results: The expression of SPHK1 was markedly increased in NSCLC and correlated with tumor progression and poor survival of patients with NSCLC. Upregulation of SPHK1 significantly inhibited doxorubicin- or docetaxel-induced apoptosis, associated with induction of antiapoptotic proteins Bcl-xl, c- IAP1, c-IAP2, and TRAF1. In contrast, silencing SPHK1 expression or inhibiting SPHK1 activity with specific inhibitor, SK1-I, significantly enhanced the sensitivity of NSCLC cells to apoptosis induced by chemotherapeutics both in vitro and in vivo. Moreover, we demonstrated that upregulation of SPHK1 activated the PI3K/Akt/NF-kB pathway, and that inhibition of the PI3K/Akt/NF-kB pathway abrogated the antiapoptotic effect of SPHK1 on NSCLC cells. Conclusions: Our results suggest that SPHK1 is a potential pharmacologic target for the treatment of NSCLC and inhibition of SPHK1 expression or its kinase activity might represent a novel strategy to sensitize NSCLC to chemotherapy. Clin Cancer Res; 17(7); 1–11. 2011 AACR.

Introduction cell lung carcinoma (SCLC) and non–small cell lung carcinoma (NSCLC), account for 90% of cases (2). Of the Lung cancer represents a leading cause of cancer-related NSCLC subtypes, the most common ones include squa- mortality and morbidity worldwide, with more than 1.23 mous cell carcinoma, large cell carcinoma, and adenocar- million new cases each year and the 5-year survival rate cinoma, with other subtypes occurring at relatively lower lower than 15% (1). Two major types of lung cancer, small frequencies (3). Currently available treatment strategies for NSCLC, including surgery, radiotherapy, chemotherapy, and photodynamic therapy, remain generally unsuccessful Authors' Affiliations: 1State Key Laboratory of Oncology in Southern (4). The 5-year survival rate for patients with stage IV China, Department of Experimental Research, Cancer Center; Depart- ments of 2Microbiology and 3Biochemistry, Zhongshan School of Medi- NSCLC is approximately 1%, with the median survival cine, Sun Yat-sen University; and 4Key Laboratory of Tropical Disease time of 7 months (5, 6). While SCLC usually reveals better Control (Sun Yat-sen University), Ministry of Education, Guangzhou, responsiveness to chemotherapy and radiation, NSCLC is Guangdong, PR China relatively insensitive to both therapeutic modalities (7). Note: Supplementary data for this article are available at Clinical Cancer Hence, identification of novel targets for more effective Research Online (http://clincancerres.aacrjournals.org/). anti-NSCLC strategies with minimal toxicity is urgent. L. Song, H. Xiong, and J. Li contributed equally to this work. Enzyme sphingosine kinase-1 (SPHK1) has been demon- Corresponding Author: Libing Song, State Key Laboratory of Oncology in strated to play a role in oncogenesis, and its biological Southern China and Departments of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou 510060, PR China. Phone: 86(20) function is associated with maintaining the balance 87343192; Fax: 86(20)87343171; E-mail: [email protected] or Meng- between prosurvival and apoptotic signaling (8). Accumu- feng Li, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou, Guangdong 510080, PR China; Phone: lated evidence has associated SPHK1 upregulation with 86(20)87332748; Fax: 86(20)87330209; E-mail: development of malignant phenotypes of various cancers. [email protected] such as proliferation, antiapoptosis, migration, angiogen- doi: 10.1158/1078-0432.CCR-10-0720 esis, and invasion (9–11). In 2000, Xia and colleagues first 2011 American Association for Cancer Research. reported the oncogenic potential of SPHK1, namely,

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Translational Relevance medium (Invitrogen) supplemented with 10% FBS (HyClone). Clinical data have demonstrated that non–small cell lung cancer (NSCLC) is relatively insensitive to che- Vectors and retroviral infection motherapy. Therefore, identification of minimally toxic An SPHK1 expression construct was generated by sub- targeting agents is urgent for the improvements of out- cloning PCR-amplified full-length human SPHK1 cDNA come of NSCLC. Herein, we report that upregulation of into the pMSCV plasmid. For depletion of SPHK1, 2 sphingosine kinase-1 (SPHK1) significantly inhibits human SPHK1-targeting siRNA sequences were cloned into doxorubicin- or docetaxel-induced apoptosis through pSuper-retro-puro to generate pSuper-retro-SPHK1-RNAi activation of PI3K/Akt/NF-kB pathway. In contrast, (s), respectively, and the sequences are RNAi#1: ablation of SPHK1 expression or inhibition of SPHK1 GGCTGAAATCTCCTTCACG; RNAi#2: GGGCAAGGCC- k kinase activity with specific inhibitor, SK1-I, signifi- TTGCAGCTC. pNF- B-luc and control plasmids (Clon- k cantly enhanced the sensitivity of NSCLC cells to apop- tech) were used to quantify NF- B activity. pBabe-Puro- k a k a tosis induced by chemotherapeutics both in vitro and in I B mut (plasmid 15291) expressing mutant I B was vivo. Taken together, our results suggested that SPHK1 from Addgene. Retroviral production and infection were targeting strategies might represent a new approach to performed as previously described (18). Stable cell lines sensitizing NSCLC to chemotherapy and improving the expressing SPHK1 or SPHK1 shRNAs were selected for m efficacy of anti-NSCLC treatment. 10 days with 0.5 g/mL puromycin.

Patients and tissue specimens Paraffin-embedded, archived NSCLC samples were obtained from 218 patients diagnosed with NSCLC overexpressing SPHK1 induced malignant transformation between January 2000 and October 2002 at the Department of NIH3T3 cells (12). The biological function of SPHK1 in of Pathology and Cancer Center at Sun Yat-sen University. protecting cancer cells from apoptosis induced by TNF-a, The histologic characterization and clinicopathologic sta- ionizing radiation, or anticancer drugs, due to increased ging of the samples were determined according to the WHO ceramide levels, has been documented (11, 13). Bonhoure criteria (3) and current International Union Against Cancer and colleagues demonstrated that ectopic overexpression TNM (tumor–node–metastasis) Classification (19). of SPHK1 conferred HL-60 leukemia cells resistance to Detailed clinical information of all patients is summarized doxorubicin- and etoposide-induced cell death (14). More- in Supplementary Table S1. Percentage tumor purity in over, SPHK1 has been shown to act as a sensor during sections adjacent to regions used for RNA extraction was imatinib-induced apoptosis of chronic myeloid leukemia estimated during routine histopathologic analysis. (CML) cells and protect LAMA84 cells from death by blocking the mitochondrial release of cytochrome c and Immunoblotting Smac/Diablo (15). The resistance to camptothecin or doc- Western blotting was performed according to standard etaxel in prostate cancer cells was also associated with methods as described previously (20), using anti-SPHK1 stimulation of SPHK1 activity (16). The above reports rabbit polyclonal antibody recognizing sphingosine kinase support a potential application of SPHK1 as a therapeutic 1 isoform 2 (Q9NYA1, MW 42.5 kDa; catalogue no. anticancer target. AP7237c; Abgent Inc.); anti-Akt, anti–p-Akt, anti-IKK, Our current study found that ablation of SPHK1 or anti–p-IKKa/b, anti-IkBa, anti-p-IkBa, anti–cleaved cas- inhibition of SPHK1 activity by specific inhibitor, SK1-I, pase-3, anti-PARP, anti–Bcl-xL, anti–c-IAP1, anti–c-IAP2, significantly sensitized NSCLC cells to apoptosis induced anti-TRAF1, anti–Bcl-2, anti-FLIP, and anti-Bim antibodies by chemotherapeutics both in vitro and in vivo. We also (Cell Signaling Technology); The membranes were stripped demonstrated that the antiapoptotic effect of SPHK1 on and reprobed with an anti–a-tubulin antibodies (Sigma) as NSCLC cells was associated with the activation of the NF- a loading control. kB and the PI3K/Akt pathways. Our findings suggest that inhibition of SPHK1 might represent a novel approach to Immunohistochemistry the treatment of NSCLC. Immunohistochemical (IHC) analysis was performed to study altered protein expression in 218 human NSCLC Materials and Methods tissues. The procedures were carried out similarly to previously described methods (21). The degree of immunos- Cell lines taining of formalin-fixed, paraffin-embedded sections was Primary normal lung epithelial cells (NLEC) were viewed and scored separately by 2 independent investiga- established according to a previous report (17) and cul- tors, who were blinded to the histopathologic features and tured in the keratinocyte serum-free medium (Invitrogen) patient data of the samples, and the scores were determined supplemented with epithelial growth factor and bovine by combining the proportion of positively stained tumor pituitary extract. Lung cancer cell lines, namely, A549, H- cells and the intensity of staining. Scores given by the 1299, PAa, 95D, and HLAMP, were maintained in DMEM 2 independent investigators were averaged for further

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comparative evaluation of the SPHK1 expression. The and 15. On day 17, animals were euthanized and tumors proportion of positively stained tumor cells was graded were excised, weighed, and subjected to pathologic exam- as follows: 0 (no positive tumor cells), 1 (<10% positive ination. tumor cells), 2 (10%–50% positive tumor cells), and 3 (>50% positive tumor cells). The cells at each intensity of Statistical analysis staining were recorded on a scale of 0 (no staining), 1 All statistical analyses were carried out using the SPSS (weak staining, light yellow), 2 (moderate staining, yellow- 13.0 statistical software package. Comparisons between ish brown), and 3 (strong staining, brown). The staining groups for statistical significance were performed with a index (SI) was calculated as follows: SI ¼ staining intensity 2-tailed paired Student’s t test. The c2 test was used to proportion of positively stained tumor cells. Using this analyze the relationship between SPHK1 expression and method of assessment, we evaluated the expression of clinicopathologic characteristics. Survival curves were SPHK1 in NSCLC by SI (scored as 0, 1, 2, 3, 4, 6, or 9). plotted using the Kaplan–Meier method and compared Cutoff values to define the high and low expression of by the log-rank test. Survival data were evaluated using SPHK1 were chosen on the basis of a measure of hetero- univariate and multivariate Cox regression analyses. P < geneity with the log-rank test statistics with respect to 0.05 was considered statistically significant in all cases. overall survival. An optimal cutoff value was identified. The SI score of 6 or above was used to define tumors with Results high expression and SI of 4 or less as tumors with low expression of SPHK1. SPHK1 expression is associated with the clinical features of NSCLC Luciferase reporter assay for NF-kB transcriptional Western blotting and real-time PCR analysis revealed that activity both protein and mRNA expressions of SPHK1 were mark- Cells were seeded in triplicates in 6-well plates (50,000 edly upregulated in multiple NSCLC cell lines, including cells/well) and allowed to settle for 12 hours. One hundred A549, 95D, PAa, H-1299, and HLAMP, in comparison with nanograms of pNF-kB-luciferase plasmid or control luci- those in 2 collections of NLEC (Fig. 1A and Supplementary ferase plasmid plus 10 ng pRL-TK Renilla plasmid (Pro- Fig. S1A). In 8 NSCLC tissue samples (T), SPHK1 expres- mega) was transfected into NSCLC cells, using the sion revealed more than 2-fold increases compared with Lipofectamine 2000 reagent (Invitrogen). Medium was that in the paired, normal, adjacent nontumorous tissues replaced after 6 hours and luciferase and Renilla signals (ANT), with each pair derived from a same patient, and were measured 48 hours after transfection using the Dual SPHK1 upregulation in these clinical NSCLC samples was Luciferase Reporter Assay kit (Promega) according to a further confirmed by IHC analysis (Fig. 1B and C and Sup- protocol provided by the manufacturer. plementary Fig. S1B and C). Taken together, our results indicated that SPHK1 was an enzyme upregulated in NSCLC. Xenografted tumor model and antitumor effect of Further evaluation of SPHK1 expression in paraffin- SK1-I in vivo embedded, archived clinical tumor tissue specimens Female BALB/c nude mice (4–5 weeks of age, 18–20 g) obtained from 218 cases of NSCLC, using IHC analysis were purchased from the Center of Experimental Animal of with an antibody against human SPHK1, showed that the Guangzhou University of Chinese Medicine and were level of SPHK1 protein significantly increased in all types of housed in barrier facilities on a 12-hour light/dark cycle. NSCLC (Fig. 2A) and strongly correlated with clinical All experimental procedures were approved by the Institu- staging (P ¼ 0.019), T classification (P ¼ 0.022), N classi- tional Animal Care and Use Committee of Sun Yat-sen fication (P ¼ 0.010), and M classification (P ¼ 0.027) of University. The BALB/c nude mice were randomly divided NSCLC patients (Supplementary Table S2). Log-rank and into 3 groups (n ¼ 5/group). One group of mice were Kaplan–Meier analysis tests demonstrated that overall sur- inoculated subcutaneously with A549/vector cells (1 106, vival time of patients with high expression of SPHK1 in suspended in 100 mL sterile PBS) per mouse in the right tumors was significantly shorter than that of the low oxter as control group. The other 2 groups were inoculated SPHK1 expression NSCLC group (P < 0.001; Fig. 2B). with A549/SPHK1 and A549/SPHK1 RNAi#1 cells (1 Furthermore, multivariate survival analysis indicated that 106, suspended in 100 mL sterile PBS), respectively. Tumor SPHK1 expression level was an independent prognostic volume was calculated using the equation (L W2)/2. In factor for the assessment of patient outcomes (Supplemen- the experiment testing the antitumor effect of SK1-I, the tary Tables S3 and S4). Moreover, the prognostic value of BALB/c nude mice (4–5 weeks of age, 18–20 g) were SPHK1 expression in different pathologic types of NSCLC implanted subcutaneously with A549 cells (2 106, sus- patients was examined. An adverse correlation between pended in 100 mL sterile PBS) to rapidly induce exponen- high SPHK1 expression in tumors and overall patient tially growing tumors. When tumors reached a volume of survival was clearly detected in all 3 NSCLC subtype 50 to 100 mm3, animals were randomly assigned to 3 groups, namely, squamous cell carcinoma (n ¼ 82; P < groups (n ¼ 5/group), followed by i.p. injection of 100 mL 0.001, log-rank; Fig. 2C, left), adneocarcinoma (n ¼ 91; P < vehicle (dimethyl sulfoxide, DMSO), SK1-I (50 mg/kg), or 0.01, log-rank; Fig. 2C, middle), and adenosquamous docetaxel (10 mg/kg), respectively, on days 1, 5, 9, 11, 13, carcinoma (n ¼ 42; P < 0.05, log-rank; Fig. 2C, right).

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Figure 1. Expression of SPHK1 is elevated in NSCLC. A, expression of SPHK1 protein in 2 NLEC collections and cultured NSCLC cell lines (A549, HLAMP, PAa, H-1299, and 95D), using a-tubulin as a loading control. B and C, Western blotting (B) and IHC (C) analysis of SPHK1 protein in primary NSCLC (T) and paired adjacent noncancerous lung tissues (ANT). Expression levels were normalized with a-tubulin.

Collectively, these data demonstrated that SPHK1 expres- treated with vehicle control (n ¼ 5, P < 0.01; P < 0.01). It is sion was linked to the clinical progression of NSCLC and noteworthy that no significant difference was detected in the might represent a valuable prognostic marker generally for body weights among nude mice in the 3 groups (data not NSCLC patients and specifically for patients with major shown). Collectively, our results suggest that SPHK1 may pathologic types of NSCLC. play an important role in the growth of NSCLC in vivo and that targeting SPHK1 through silencing its expression or SPHK1 plays an important antiapoptotic role in suppressing its enzyme activity may represent a potentially NSCLC in vivo effective strategy against NSCLC, either alone or in combi- The above finding that SPHK1 expression was signifi- nation with chemotherapy. cantly associated with the progression of NSCLC prompted Since clinical failure of chemotherapy is commonly and us to ask whether SPHK1 might play a role in NSCLC biologically attributable to the resistance of cancer cells to pathogenesis and therefore could be a novel therapeutic apoptosis, we further investigated whether SPHK1 target. To this end, a panel of A549 NSCLC cell lines were enhances the antiapoptotic activity of NSCLC cells in vivo. constructed to stably express either SPHK1 cDNA (A549/ As shown in Figure 3D, TUNEL (terminal deoxynucleotidyl SPHK1) or SPHK1 shRNAs (A549/SPHK1 shRNA; Fig. 3A) transferase–mediated dUTP nick end labeling) assay and inoculated in nude mice. After the tumor-bearing mice demonstrated that docetaxel induced far lower and higher were treated i.p. with docetaxel (10 mg/kg), a clinically degree of apoptosis in tumors derived from A549/SPHK1- commonly used chemotherapeutic agent against NSCLC, and A549/SPHK1 shRNA-NSCLC cells, respectively, than as shown in Figure 3B and Supplementary Figure S2A, the in the A549/vector control tumors. Also noteworthy is that volumes and weights of tumors formed by the A549/SPHK1 SK1-I treatment markedly elevated the number of apoptotic cells were significantly larger than those of tumors formed cells induced by docetaxel treatment. Taken together, our by vector control cells (n ¼ 5, P < 0.01; P < 0.01) whereas data suggested that SPHK1 may confer NSCLC cells resis- depletion of endogenous SPHK1 in A549 cells caused sig- tance to docetaxel-induced apoptosis and thus might repre- nificant inhibition of tumor growth in terms of both tumor sent a target for improving the efficacy of anti-NSCLC volume and weight (n ¼ 5, P < 0.01; P < 0.01; Fig. 3B and chemotherapy. Supplementary Fig. S2A). Moreover, we also examined the effect of a specific SPHK1 inhibitor SK1-I on the growth of Dysregulation of SPHK1 alters the sensitivity of xenografted NSCLC tumors. As shown in Figure 3C and NSCLC cells to apoptosis Supplementary Figure S2B, SK1-I treatment dramatically To further understand and characterize the antiapop- reduced the tumor size and weight as compared with those totic activity of SPHK1 in NSCLC cells, in vitro studies

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Figure 2. Overexpression of SPHK1 in archived NSCLC specimens. A, representative IHC images of SPHK1 expression in normal lung tissue and NSCLC specimens of different subtypes. B, survival curves of NSCLC patients with low versus high expression of SPHK1 (n ¼ 218; P < 0.001, log-rank test). C, statistical significance of the difference between curves for patients with high and low expression of SPHK1 within subgroups of squamous cell carcinoma (n ¼ 82; P < 0.001; left), adenocarcinoma (n ¼ 91; P < 0.01; middle), and adenosquamous carcinoma (n ¼ 42; P < 0.05; right).

were performed using NSCLC cell lines with SPHK1 Upregulation of SPHK1 activates NF-kB and PI3K/Akt overexpressed or silenced. As shown in Supplementary pathways Figure S3A, SPHK1-overexpressing 95D and A549 NSCLC To further investigate the molecular mechanism mediat- cells displayed significantly higher survival than the vec- ing the antiapoptotic effect of SPHK1, the levels of apop- tor control cells after treatment of various doses of dox- tosis relators Bcl-xl, c-IAP1, c-IAP2, FLIP, TRAF1, Bcl-2, and orubicin or docetaxel, whereas the number of dead cells Bim were examined. Western blotting and real time-PCR markedly increased when SPHK1 expression was silenced analysis revealed that at both mRNA and protein levels, by specific shRNA. Annexin V–binding and TUNEL assays antiapoptotic factors Bcl-xl, c-IAP1, c-IAP2, and TRAF1 showed that SPHK1 upregulation conferred NSCLC cells were significantly upregulated in the SPHK1-transduced resistance, and SPHK1 downregulation drastically A549 and 95D NSCLC cells and downregulated in enhanced their sensitivities, to chemotherapeutics SPHK1-knocked down cells, as compared with those in (Fig. 4A and B and Supplementary Fig. S3B and C). control cells, respectively (Fig. 5A and Supplementary Furthermore, the noted effect of SPHK1 on the apoptosis Fig. S4). As the above identified upregulated antiapoptotic was confirmed as decreased activating cleavages of PARP genes are known downstream targets of NF-kB, we further and caspase-3 induced by doxorubicin or docetaxel in tested whether the NF-kB activity was modulated SPHK1-overexpressing cells and a contrary effect in by SPHK1. NF-kB reporter assay showed that SPHK1 upre- SPHK1-knocked down cells (Fig. 4C and Supplementary gulation significantly increased, and in contrast, down- Fig. S3D). regulation of SPHK1 attenuated, NF-kB transcriptional

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Figure 3. The impact of SPHK1 expression on tumor growth in vivo. A, expression of SPHK1 was examined in indicated cells. a-Tubulin was used as a loading control. B, tumor volumes measured on the indicated days. Indicated cells (2 106) were injected s.c. in nude mice. When the mean tumor volume reached 50 mm3, mice were injected i.p. with docetaxel (10 mg/kg) every 2 days for 21 days. Data points are presented as the mean tumor volume SD. C, tumor volumes were measured on the indicated days. A549 vector cells (2 106) were injected in the right oxter of 15 nude mice. When the mean tumor volume reached 50 mm3, mice were randomized into 3 groups (n ¼ 5) and injected i.p. with vehicle (DMSO), docetaxel (10 mg/kg), or SK1-I (50 mg/kg) every 2 days for 17 days. Data points are presented as the mean tumor volume SD. D, representative immunofluorescent images (left) and quantification (right) of TUNEL-stained cells in indicated tumors. The numbers of TUNEL-positive cells were counted from 10 random fields and presented as percentages of total cell numbers. *, P < 0.05.

transactivating activity in A549 and 95D cells (Fig. 5B, left). NF-kB pathway was essential for the antiapoptotic function Consistently, the transactivation activity of NF-kB of SPHK1 in NSCLC cells (Fig. 5D). decreased in SK1-I–treated cells as compared with that in As the IKK/IkBa/NF-kB pathway could be activated by control cells (Fig. 5B, right). Western blotting analysis Akt (22), we then examined whether SPHK1 activated PI3K/ revealed that phosphorylation of IkBa and IKK signifi- Akt signaling. As shown in Figure 6A, the level of phos- cantly increased in SPHK1-overexpressing NSCLC cells phorylated Akt was indeed elevated in SPHK1-overpressing and reduced in SPHK1-knocked down cells (Fig. 5C, left) NSCLC cells in comparison with that in the control cells and in SK1-I–treated cells dose dependently (Fig. 5C, whereas SPHK1 downregulation or SPHK1 inhibition by right), indicating that the effect of SPHK1 on apoptosis SK1-I significantly decreased the phosphorylation level of resistance mainly due to the SPHK1 activity. Meanwhile, we Akt (Fig. 6A and B). Furthermore, Western blotting analysis found that overexpressing SPHK1 drastically increased and showed that the observed increases of phosphorylated IkBa knocked down SPHK1 reduced the cellular sphingosine-1- and IKK by overexpressing SPHK1 could be reversed by phosphate (S1P), which is produced by SPHK1, in both treatment of PI3K inhibitor LY294002 or a specific Akt A549 and 95D NSCLC cells (Supplementary Fig. S5). More- inhibitor (Akt inhibitor X), indicating that SPHK1- over, Annexin V–binding and TUNEL assays showed that mediated IKK/IkB/NF-kB activation might be through doxorubicin- or docetaxel-induced apoptosis of SPHK1- PI3K/Akt pathway (Fig. 6C). Moreover, SK1-I inhibition overexpressing cells dramatically increased when the activ- of SPHK1 significantly enhanced apoptosis of NSCLC cells ity of NF-kB was blocked by IkBa super-repressor (IkBa induced by doxorubicin or docetaxel. However, apoptosis mut), by treatment of NF-kB inhibitor (JSH-23), or by IKK only slightly increased when the NSCLC cells were treated inhibitor (Wedelolactone), indicating that the IKK/IkBa/ with SK1-I combined with PI3K inhibitor or Akt inhibitor,

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Figure 4. Antiapoptotic effect of SPHK1 in NSCLC in vitro. A, representative immunofluorescent images (left) and quantification (right) of TUNEL staining in vector-, SPHK1-, or SPHK1 shRNA-transduced A549 and 95D cells after docetaxel treatment (5 nmol/L) for 24 hours. Numbers are cells counted from 10 random fields. **, P < 0.01. B, immunofluorescent images (left) and quantification (right) of Annexin V/propidium iodide (PI) staining of vector-, SPHK1-, or SPHK1 shRNA-transduced A549 and 95D cells after docetaxel treatment (5 nmol/L) for 6 hours. Numbers are cells counted from 10 random fields. **, P < 0.01. C, Western blotting for proteolytic cleavage of procaspase-3 and PARP in vector-, SPHK1-, or SPHK1 shRNA-transduced A549 and 95D cells after docetaxel treatment (5 nmol/L) for 24 hours, using a-tubulin as a loading control.

suggesting that activation of PI3K/Akt was essential for the SPHK1 expression, and that SPHK1 upregulation sustains SPHK1-mediated protection against apoptosis (Fig. 6D). NSCLC cell survival and inhibits their sensitivity to apop- Taken together, our results indicated that the PI3K/Akt tosis inducers. We also demonstrated that inhibiting pathway may play a role in SPHK1-mediated activation SPHK1 activity with specific SPHK1 inhibitor or down- of NF-kB and prevention of apoptosis in NSCLC cells. regulating SPHK1 with RNAi might represent a novel strategy for the treatment of NSCLC. Discussion Increased resistance to apoptosis is a hallmark alteration in most types of cancers (23). Abrogation of proapoptotic The key findings made in our present study are that pathways has been demonstrated to be one of the progression of human NSCLC is related to an increase of events key to tumor development and progression, and

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Figure 5. SPHK1-mediated protection of apoptosis through activation of NF-kB and PI3K/Akt pathways. A, Western blotting of Bcl-xL, c-IAP1, c-IAP2, FLIP, TRAF1, Bcl-2, and Bim in indicated cells, using a-tubulin as a loading control. B, the effect of SPHK1 expression (left) and activity (right) on NF-kB activity in NSCLC cells, analyzed by luciferase reporter assay. *, P < 0.05. C, Western blotting for phosphorylated IKK (p-IKK), total IKK, phosphorylated IkBa (p-IkBa), and total IkBa in vector-, SPHK1-, or SPHK1 shRNA-transduced A549 and 95D cells (left) or in the SK1-I–treated A549/SPHK1 and 95D/SPHK1 cells (right), using a-tubulin as a loading control. The numbers under panel of the p-IkBa expression are quantification analyses of p-IkBa/IkBa ratio. D, quantification of TUNEL-positive (right) and Annexin Vþ/propidium iodide (PI; left) A549/SPHK1 and -95D/SPHK1 cells treated with doxorubicin or docetaxel combined with an IkB mutant (IkBmut), NF-kB inhibitor JSH-23 (30 mmol/L, NF-kB in), IKK inhibitor Wedelolactone (100 mmol/L, IKK in). *, P < 0.05. Error bars represent SD from 3 independent experiments.

impairments in apoptotic programming are tightly linked cancer type will bring new insights in developing more to the commonly seen failure of anticancer chemotherapy effective therapeutic strategies (24–26). Notably, in the and radiotherapy. Thus, clarification of the mechanisms current study, we found that SPHK1 plays an important modulating the apoptosis/survival process in a particular role in antiapoptosis of NSCLC that is relatively insensitive

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Figure 6. Upregulation of SPHK1 activates the PI3K/Akt pathway. A and B, Western blotting for phosphorylated Akt (p-Akt), total Akt, phosphorylated GSK-3b (p-GSK-3b), and total GSK-3b levels in vector-, SPHK1-, or SPHK1 shRNAi-transduced A549 and 95D cells (A) or in SK1-I treated A549/SPHK1 or 95D/SPHK1 cells (B), using a-tubulin as loading control. C, Western blotting for phosphorylated IKK (p-IKK), total IKK, phosphorylated IkBa (p-IkBa), and total IkBa in A549/SPHK1 and -95D/SPHK1 cells treated by vehicle (DMSO), PI3K inhibitor LY294002, or a specific Akt inhibitor (Akt inhibitor X), using a-tubulin as loading control. D, quantification of Annexin Vþ/propidium iodide (PI; right) or TUNEL-positive (left) A549/SPHK1 or 95D/SPHK1 cells treated with chemotherapeutics agent doxorubicin or docetaxel in combination with vehicle (DMSO), LY294002, Akt inhibitor X, Akt inhibitor X plus SK1-I, or LY294002 plus SK1-I. *, P < 0.05. Error bars, SD from 3 independent experiments.

to chemotherapy, both in vivo and in vitro. Ectopic expres- SK1-I (28). Prompted by the concern that NSCLC is gen- sion of SPHK1 in NSCLC cells dramatically enhances their erally insensitive to currently available chemotherapeutic resistance to apoptosis induced by doxorubicin or doce- agents, we asked whether SPHK1 plays a role in conferring taxel, 2 commonly used chemotherapeutics, whereas sup- NSCLC cells resistance to chemotherapeutics-induced pressing SPHK1 expression with shRNAs or inhibiting apoptosis during the treatment of NSCLC and thereby SPHK1 activity with a specific SPHK1 inhibitor, SK1-I, could be a target for the treatment of NSCLC. Strikingly, markedly abrogated the ability of NSCLC cells to resist inhibition of SPHK1 activity by SK1-I markedly sensitizes cytotoxic reagent-induced cell death, suggesting that NSCLC cells to the proapoptotic effect of chemotherapeu- SPHK1 activity contributes to sustaining the unwanted tics. Robustly, i.p. injection of SK1-I potently enhanced the survival of NSCLC cells under the treatment of chemother- tumor suppression effect of docetaxel, a clinically well- apeutics. established, proapoptotic chemotherapy drug against The balance between lipid mediators, such as S1P, breast, ovarian, and NSCLC (29, 30). Notably, the sphingosine, and ceramide, has been considered as a cel- unwanted toxicity of SK1-I appeared to be low as the body lular converter determining cell fate (27). Thus, key weight of treated mice was well kept and other signs of enzymes in this context, such as SPHK1 and SPHK2, which toxicity seemed to be absent in our study, despite that the regulate the S1P/ceramide conversion that contributes to exact toxicity profiles of SPHK1 targeting strategies are yet determining whether a cell proliferates or undergoes apop- to be pharmaceutically and clinically determined. tosis, could be potential targets for new anticancer drugs. It has been demonstrated that NF-kB plays important Recently, French and colleagues screened a library of syn- roles in the development of malignant phenotypes through thetic compounds using recombinant human SK1 as a bait multiple signaling pathways, and aberrant activation of NF- and identified a panel of inhibitors of SPHK1 including kB has been observed in variety of cancer types (31).

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A prominent mechanism linking NF-kB signaling to cancer types of NSCLC cells could also be modulated by upregu- progression is the abrogation of apoptosis (32). Numerous lated SPHK1. These issues are under further investigation in antiapoptotic proteins, such as Bcl-xL, c-IAPs, TRAFs, and c- the laboratory. Nevertheless, understanding the role of FLIP6, are transcriptionally regulated by the NF-kB (32, SPHK1 in NSCLC progression will not only advance our 33). Our current study found that several NF-kB regulated knowledge of the mechanisms underlying NSCLC survival, antiapoptotic proteins were upregulated in SPHK1-over- but also will help establish SPHK1 as a potential therapeu- expressing NSCLC cells and downregulated in SPHK1- tic target for the treatment of NSCLC. knocked down or -inhibited NSCLC cells, and that the transactivating activity of NF-kB could be stimulated by Disclosure of Potential Conflicts of Interest SPHK1 upregulation and suppressed by SPHK1 inhibition. On the other hand, although the IKK/IkB/NF-kB axis is No potential conflicts of interest were disclosed.. subjected to activation by a variety of distinct upstream signals, the data we obtained thus far suggest an involve- Grant Support ment of the PI3K/Akt pathway, the activation of which This study was supported by the Natural Science Foundation of China requires contribution of lipid mediators. Indeed, suppres- (nos. 81071647, 81071762, 81071780, 81030048, 30770836, 30771110, sion of PI3K/Akt signaling in NSCLC cells had little, if any, 30870963, 30872930, 30831160517); Program for New Century Excellent Talents in Universities (no. NCET-07-0877); the Science and Technology enhancing effect on SK1-I caused apoptosis, and such a lack Department of Guangdong Province, China (no. 8251008901000006, of additive effect between SPHK1 inhibition and PI3K 2008A030201006); Ministry of Education of China [nos. (2008)890 and inhibition suggests a seemingly linear relation of the two 200805580047]; and the Fundamental Research Funds for the Central Universities (no. 10ykzd03). The study was also supported by State Major signaling molecules along the action axis. Apparently, the Infectious Disease Research Program (China Central Government, molecular mechanism underlying SPHK1-mediated activa- 2009ZX10004-213, 2009ZX09103-041), Guangdong Recruitment Program tion of PI3K/Akt pathways, as well as its biological out- of Creative Research Groups and a key grant from the 985-III project. The costs of publication of this article were defrayed in part by the come, needs to be further delineated. In addition, several payment of page charges. This article must therefore be hereby marked other issues also remain to be addressed. For example, it advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate would be of great interest to know whether other pathways this fact. are also involved in mediating the antiapoptotic effect of Received March 23, 2010; revised September 24, 2010; accepted SPHK1 in NSCLC cells and what other malignant pheno- November 5, 2010; published OnlineFirst February 15, 2011.

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Sphingosine Kinase-1 Enhances Resistance to Apoptosis through Activation of PI3K/Akt/NF- κB Pathway in Human Non−Small Cell Lung Cancer

Libing Song, Huaping Xiong, Jun Li, et al.

Clin Cancer Res Published OnlineFirst February 15, 2011.

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