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. 2011 Sep 22;25(11):1858–1866. doi: 10.1210/me.2011-1048

Sphingosine Kinase 1 Is Overexpressed and Promotes Proliferation in Human Thyroid Cancer

Hongyu Guan 1, Liehua Liu 1, Junchao Cai 1, Juan Liu 1, Caisheng Ye 1, Mengfeng Li 1, Yanbing Li 1,✉
PMCID: PMC5417174  PMID: 21940753

Abstract

Sphingosine kinase 1 (SphK1), an oncogenic kinase, has been previously found to be elevated in various types of human cancer and play a role in tumor development and progression. Nevertheless, the biological and clinical significance of SphK1 in thyroid cancer is largely unknown. Here, we demonstrate that the expression of SphK1 is generally up-regulated in thyroid cancer and that its expression level is correlated with the degree of thyroid malignancy. Silencing SphK1 by specific RNA interference is able to suppress the proliferation of thyroid cancer cells, and SphK1 expression level is strongly associated with the expression of proliferation cell nuclear antigen in thyroid cancer tissues. Of particular note is that depletion of SphK1 results in dephosphorylation of protein kinase B and glycogen synthase kinase-3β and subsequent inactivation of β-catenin-T-cell factor/lymphoid enhancing factor transcriptional activity. Hence, taken together, our study has identified SphK1 as a proproliferative oncogenic kinase, an Akt/glycogen synthase kinase-3β/β-catenin activator, and probably a biomarker for thyroid cancer as well.

Sphingosine kinase 1 (SphK1), an evolutionarily conserved enzyme, has received increased attention during the past years because of its important role in various cellular processes, including cell proliferation, apoptosis suppression, inflammation, angiogenesis, and cell invasion (1, 2). Upon stimulation of a variety of agonists, such as TNF-α, SphK1 translocates from the cytosol to the plasma membrane and catalyzes the formation of sphingosine 1-phosphate (S1P) by phosphorylation of 1-OH of sphingosine (3, 4). S1P can be released from the cell to exert its biological effects through five G protein-coupled S1P receptors, namely S1P1–S1P5 (5). Of note, abrogation of S1P receptor signaling does not restrain the effect of SphK1 in some circumstances, suggesting that SphK1 can also act intracellularly (6, 7).

Accumulating evidence has suggested the important role of SphK1 in human tumorigenesis. NIH 3T3 fibroblast overexpressing SphK1 has been found to acquire transformed phenotypes and form tumors in mice, strongly indicating its oncogenic activity (8). It has been well documented that the expression of SphK1 is elevated in various types of human cancers, including glioma, head and neck squamous cell carcinoma, prostate cancer, salivary gland carcinoma, gastric cancer, and non-Hodgkin lymphomas (9–14). Intriguingly, the increased expression of SphK1 among some of these cancers has been associated with worse clinical outcome of patients (11–13). In vitro tumor growth was pronouncedly reduced when the cellular SphK1 activity was abolished via pharmacological and genetic inhibition (15, 16). Moreover, because SphK1 has been demonstrated as a sensor of chemotherapy in vivo, it is likely that chemotherapy combined with inhibition of SphK1 can synergistically act to induce tumor cell death (17). In thyroid cancer, a study by Bergelin et al. (18) suggests that SphK1 promotes the migration of the ML-1 thyroid follicular cancer cells in a protein kinase C- and ERK1/2-dependent manner. However, the expression level, biological functions, and potential roles of SphK1 remain largely unclear.

β-Catenin has been considered as a transcriptional regulator. Its nuclear accumulation leads to activation of target genes that are responsive to stimulation of T-cell factor/lymphoid enhancing factor (TCF/LEF) family (19, 20). Glycogen synthase kinase-3β (GSK-3β) negatively modulates the stabilization and nuclear translocation of β-catenin through Ser33/37/Thr41 phosphorylation and subsequent proteasome-mediated degradation (21). A key upstream regulator of GSK-3β is protein kinase B (Akt), a multifunction serine-threonine kinase, which phosphorylates Ser9 of GSK-3β and thereby induces its inactivation (22, 23). Given the regulatory role of SphK1 on Akt activation (15, 24), to begin to understand the role of SphK1 in the development and progression of thyroid cancer, in the current study, we examined the expression level of SphK1 and tested our hypothesis that Akt/GSK-3β/β-catenin axis may be involved in the proliferative function of SphK1 in thyroid cancer cells.

Results

SphK1 overexpression in thyroid cancer

To determine the clinical and functional relevance of SphK1 in thyroid cancer, we initially performed immunohistochemistry (IHC) assay to assess the expression of SphK1 in five pairs of thyroid cancer tissues (T) and their adjacent noncancerous thyroid tissues (N). As shown in Fig. 1A, the expression levels of SphK1 protein in cancer tissues were significantly higher than that in the adjacent N. Subsequently, we analyzed the expression of SphK1 protein in 10 cases of goiters, 16 cases of adenomas, and 42 cases of thyroid cancer using IHC. As shown in Fig. 1 and Table 1, the expression of SphK1 in thyroid cancer markedly increased as compared with that in either goiters or adenomas (P < 0.05 for both comparisons). Notably, most goiters (nine out of 10) and adenoma samples (14 out of 16) showed no detectable expression of SphK1, whereas SphK1 is highly expressed in 69% of thyroid cancers (29 out of 42). Interestingly, high SphK1 expression was observed in all ATC specimens tested (10 out of 10), as opposed to 59.1% (13 out of 22) or 60% (six out of 10) of papillary thyroid cancer or follicular thyroid cancer samples, respectively, exhibiting high-level expression of SphK1. Moreover, SphK1 expression was significantly correlated with tumor-node-metastasis staging (P = 0.009) and with T classification (P = 0.019) (Table 2). The expression level of SphK1 did not differ significantly by age, gender, or N classification (Table 2). These data suggest that SphK1 expression is associated with the grade of malignancy of thyroid cancer and that the observed up-regulation of SphK1 in thyroid cancer may play a biological role in the disease.

Fig. 1.

Fig. 1.

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The expression of SphK1 is elevated in thyroid cancer. A, IHC analysis of SphK1 in primary thyroid cancer (T) and paired noncancerous adjacent thyroid tissues (N). B, Representative images of IHC assays on SphK1 expression in thyroid tissue specimens from the studied cohort.

Table 1.

Expression of SphK1 in thyroid cancer

Tumor types Low SphK1 expression High SphK1 expression Total (%)
Goiter 9 1 1/10 (10)
Follicular adenoma 14 2 2/16 (12.5)
Papillary cancer 9 13 13/22 (59.1)
Follicular cancer 4 6 6/10 (60)
Anaplastic cancer 0 10 10/10 (100)
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Table 2.

Association between clinicopathologic parameters and level of SphK1 protein expression in thyroid cancer patients

Clinicopathologic variables Total SphK1 score P value
Age
    >45 yr (n = 24) 4.96 ± 3.75 0.463
    ≥45 yr (n = 18) 5.67 ± 3.25
Gender
    Male (n = 11) 4.00 ± 3.41 0.199
    Female (n = 31) 5.71 ± 3.50
TNM stage
    I and II (n = 26) 4.12 ± 3.10 0.009
    III and IV (n = 16) 7.13 ± 3.44
T classificarion
    T1 and T2 (n = 28) 4.32 ± 3.15 0.019
    T3 and T4 (n = 14) 7.14 ± 3.57
N classification
    N0 (n = 24) 5.13 ± 3.09 0.971
    N1 (n = 18) 5.44 ± 4.10
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Silencing of endogenous SphK1 suppresses the proliferation of thyroid cancer cells

To assess the role of SphK1 in thyroid cancer cell proliferation, we knocked down endogenous SphK1 by specific short hairpin RNA (shRNA) in WRO, FRO, and SW579 cells and examined its effect on cell growth. Depletion of SphK1 by shRNA was verified at both RNA and protein levels, and the results showed that neither of the two different shRNA used was able to significantly silence the expression of SphK1 (Fig. 2, A and B). Moreover, the enzymatic activities of SphK1 in SphK1-knocked down thyroid cancer cells were significantly decreased as compared with that of the vector control (Fig. 2C). 3-(4,5-Dimethylthiazol-2-yl) −2,5-diphenyltetrazolium bromide (MTT) and soft agar colony formation assays were performed to examine the effect of SphK1 on thyroid cancer cells proliferation. As shown in Fig. 2D, shRNA-transduced cells exhibited markedly decreased rate of proliferation in comparison with vector-transfected cells. Next, to further confirm the proproliferative role of SphK1 on thyroid cancer cells, we evaluated the effect of SphK1 knockdown on anchorage-independent growth, a characteristic of cellular transformation, of thyroid cancer cells. As shown in Fig. 2, E and F, indicated cells with SphK1 knocked down formed significantly fewer and smaller colonies than vector-control cells. These data suggest that SphK1 is involved in enhancing cell proliferation and might be associated with the transformed phenotype of thyroid cancer cells.

Fig. 2.

Fig. 2.

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Depletion of SphK1 abrogates the proliferation of thyroid cancer cells. Knockdown of SphK1 in WRO, FRO, and SW579 cell lines analyzed by real-time RT-PCR (A) and WB (B). For real-time RT-PCR assays, expression levels were normalized with GAPDH, and α-tubulin was used as a loading control for WB assays. C, SphK1 enzymatic activity in SphK1-knocked down thyroid cancer cells was significantly decreased. D, MTT assay was performed to evaluate the effect of SphK1 on the proliferation of indicated thyroid cancer cells at indicated time points. E, Representative images of anchorage-independent colonies formed by RNAi-transduced thyroid cancer cells or vector-control cells. F, Mean anchorage-independent colony numbers formed by indicated cells. For A, C, D, and F, results are expressed as mean ± sd (n = 3).

Proliferation cell nuclear antigen (PCNA) expression is correlated with SphK1 expression in thyroid primary cancer

To determine whether SphK1 regulates cell proliferation in clinical primary thyroid cancer specimens, the expression of PCNA, a marker of proliferating cells (25), was analyzed in the same T tested above. As shown in Fig. 3 and Table 3, in 42 cases of T samples, high-level coexpression of SphK1 and PCNA was exhibited in 24 cases (57.1%), and simultaneous low or lack of expression of both molecules was observed in 11 cases (26.2%). The Spearman correlation assay showed a significant statistical correlation between SphK1 and PCNA (r = 0.675, P < 0.001). These results strongly indicate that proliferation of thyroid cancer cells is associated with SphK1 expression.

Fig. 3.

Fig. 3.

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Correlation between SphK1 and PCNA expression in primary thyroid cancer. Images represent the correlation between SphK1 and PCNA expression in human thyroid cancer specimens.

Table 3.

Correlation between the expression of SphK1 and PCNA in thyroid cancer

Low PCNA expression High PCNA expression Total
Low SphK1 expression 11 2 13
High SphK1 expression 5 24 29
Total 16 26
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β-Catenin activity is inhibited in SphK1-knocked down thyroid cancer cells

Given the documented importance of β-catenin activation in tumor cell proliferation (26, 27), along with the observation that the β-catenin-TCF/LEF-induced transcription of cell cycle relevant genes (cyclin D1 and c-myc) was pronouncedly suppressed in SphK1- silenced cells (Fig. 4A), we next asked whether β-catenin signaling was regulated by SphK1 in thyroid cancer cells. As expected, the β-catenin-TCF/LEF transcriptional activity was inhibited markedly in response to depletion of SphK1 (Fig. 4B). Moreover, decreased nuclear translocation of β-catenin, a character of β-catenin inactivation, was observed in specific shRNA transduced thyroid cancer cells (Fig. 4C). Taken together, the proproliferative effect of SphK1 is likely mediated by β-catenin activation in thyroid cancer cells.

Fig. 4.

Fig. 4.

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Akt/GSK-3β/β-catenin signaling mediates the proproliferation role of SphK1 in thyroid cancer cells. A, Cellular mRNA levels of cyclin D1 and c-myc in indicated cells were analyzed by real-time RT-PCR using GAPDH as loading control. B, Indicated cells were transduced with the TOPflash or FOPflash firefly luciferase reporter plasmids, using cotransfected pRL-TK to normalize the luciferase values. Data presented as fold induction relative to vector-control cells. C, WB analysis of nuclear β-catenin protein in indicated cells. D, Enhanced phosphorylation of β-catenin at Ser33/37/Thr41 sites in SphK1-knocked down WRO, SW579, and FRO cells. E, Dephosphorylation of GSK-3β (Ser9) in response to silencing of SphK1. F, Effect of SphK1 depletion on phosphorylation of Akt at Thr308 and Ser473 was investigated by WB. For A and B, results derived from three independent experiments are expressed as mean ± sd, *, P < 0.05. For D–F, α-tubulin is presented as a loading control for WB assays.

SphK1 regulates β-catenin activation via Akt/GSK-3β signaling

We further investigated the underlying mechanism responsible for the β-catenin activation by SphK1. Phosphorylation of β-catenin by GSK-3β at Ser33/37/Thr41 residues is crucial for the degradation and inactivation of β-catenin (21). Indeed, we found that phosphorylation of β-catenin at Ser33/37/Thr41 sites is significantly enhanced in SphK1-down-regulated cells (Fig. 4D), suggesting that the effect of SphK1 on β-catenin is mediated by GSK-3β. Because GSK-3β activity is significantly inhibited through phosphorylation of Ser9 residue in the protein (22), we next investigated the phosphorylation of GSK-3β at Ser9. As shown in Fig. 4E, dephosphorylation of Ser9 of GSK-3β was observed in SphK1-silenced thyroid cancer cells. Thus, it is likely that SphK1 depletion leads to dephosphorylation and activation of GSK-3β, thereby facilitating phosphorylation of β-catenin. Several protein kinases, including Akt, p90RSK, p70S6K, protein kinase A, and protein kinase C, can phosphorylate Ser9 of GSK-3β in response to various stimuli (22, 23). Among these protein kinases, Akt, which acts as one of the downstream effectors of SphK1 (15, 24), is of specific interest. In line with the previous studies, depletion of SphK1 resulted in dephosphorylation of Akt in thyroid cancer cells (Fig. 4F). Together, these data demonstrated that silencing of SphK1 in thyroid cancer cell results in inactivation of Akt, activation of GSK-3β, and suppression of β-catenin dependent transcription.

Finally, to further confirm the regulatory role of SphK1 in Akt/GSK-3β/β-catenin signaling, we abrogated the enzymatic activity of SphK1 by using its specific inhibitor, 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SK-I) (28), and then examined the phosphorylation status of Akt, GSK-3β, and β-catenin. As expected, dephosphorylation of Akt and GSK-3β, and phosphorylation of β-catenin, were observed upon the treatment by SK-I (Fig. 5). Taken as a whole, Akt/GSK-3β/β-catenin signaling may be involved in mediating the proproliferative function of SphK1 in thyroid cancer cells.

Fig. 5.

Fig. 5.

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The effect of SphK1 inhibitor on Akt/GSK-3β/β-catenin pathway. The effect of SphK1 inhibitor on the Akt/GSK-3β/β-catenin signaling in WRO, FRO, SW579 thyroid cancer cells were assessed by WB.

Discussion

Our current study shows that SphK1 is overexpressed in thyroid cancer and associated with grade of malignancy. We also demonstrate that β-catenin signaling is involved in mediating the proproliferative function of SphK1 in human cancer cells.

As an oncogenic kinase, SphK1 has been found to be up-regulated in various human malignancies (9–14). Consistent with previous reports, our study showed that the expression of SphK1 is elevated in clinical specimens obtained from patients with thyroid cancer. In contrast to the low or undetectable levels in goiters and adenomas, SphK1 is markedly overexpressed in thyroid carcinomas. Of particular interest, the expression of SphK1 in well-differentiated, i.e. both follicular and papillary, thyroid cancers is significantly lower than that in poorly differentiated anaplastic cancer, indicating that its expression is correlated with malignancy grade. These results provide new insights in the molecular alterations that are associated with the development and progression of thyroid cancer.

Although a variety of stimuli, growth, and survival factors, such as TNF-α, platelet-derived growth factor, epidermal growth factor, TGF-β, vascular endothelial growth factor, and basic fibroblast growth factor in particular, can stimulate SphK1 activation (4), it remains unclear what mechanisms are underlying the overexpression of SphK1 in human cancers. Study by Paugh et al. (29) demonstrated that IL-1 could transcriptionally up-regulate SphK1 in glioblastoma cells. Also, in glioblastoma cells, hypoxic stress increases SphK1 mRNA and protein levels (30). To fully understand the pathogenesis of thyroid cancer, the mechanism that up-regulates SphK1 in thyroid cancer still needs to be further investigated.

Several lines of evidence have suggested proproliferative effect of SphK1 in human cancers. For instance, Dayon et al. (31) showed that SphK1 plays a central role in androgen-regulated prostate cancer cell growth. In addition, SK1-I, a potent SphK1 inhibitor, attenuated the growth of glioblastoma cells and lung cancer cells in vitro as well as in vivo (15, 32). Consistent with these studies, our results of MTT and soft agar assays demonstrate that depletion of SphK1 with specific shRNA markedly suppresses the growth of thyroid cancer cells, suggesting a possible requirement of SphK1 in uncotrolled thyroid cancer cell growth.

Notably, our findings showed that the activation of β-catenin signaling, a well-known proproliferative pathway (26, 27), is SphK1 dependent in the tested thyroid cancer models. Silencing of SphK1 in thyroid cancer cells drastically reduces both nuclear translocation of β-catenin and β-catenin-TCF/LEF transcriptional activity, thereby suppressing the transcription of cyclin D1 and c-myc. Akt can abrogate the activity of GSK-3β by phosphorylating its Ser9 residue (22, 23). GSK-3β inhibition leads to reduced levels of Ser33/37/Thr41 phospho-β-catenin, which in turn results in β-catenin stabilization (21). β-Catenin can then transport to nucleus, where it forms an active transcription factor complex with TCF/LEF and is capable of promoting target gene transcription (19, 20). These findings, together with published literature that has reported that SphK1 is correlated with activation of Akt (15, 24, 33), suggest that it is possible that the Akt/GSK-3β axis acts as the linkage between SphK1 and β-catenin signaling. Indeed, data presented in the current study suggest that SphK1 depletion inhibits the activity of Akt, which decreases the Ser9 phophorylation of GSK-3β and enhances its kinase activity, thus facilitating β-catenin phosphorylation and subsequent degradation and inactivation of β-catenin.

It is well accepted that aberrant activation of Akt plays a fundamental role in the development and progression of thyroid cancer (34, 35). Various mechanisms, including dysregulated activation of several upstream oncogenes and genetic alterations, have been proposed to explain the aberrant activation of Akt in thyroid cancer (34, 36). Here, we show that expression of oncogenic kinase SphK1 in thyroid cancer is involved in Akt activation. These results provide new insights into the complex regulatory mechanism for Akt activation in thyroid cancer. Several lines of studies have revealed that inhibition of Akt activity in thyroid cancer cells results in increased cell apoptosis and suppression of cell proliferation (37, 38). Intriguingly, small molecule SphK1 inhibitor could disrupt Akt signaling, suggesting a novel therapy strategy for thyroid cancer. So far, it is still not clear whether SphK1 activates Akt by direct or indirect interaction between two molecules. Delineating the mechanism by which SphK1 induces activation of Akt will improve our understanding of the oncogenic role of SphK1 in thyroid cancer.

In summary, we have found that SphK1 is overexpressed in thyroid cancer and promotes cell proliferation through Akt/GSK-3β/β-catenin signaling. With the hope that SphK1 may be a useful new target of the management of this cancer type, ongoing studies are investigating the mechanisms via which SphK1 is up-regulated and the mode of interaction between SphK1 and Akt.

Materials and Methods

Cell line and treatments

The WRO (follicular), FRO (anaplastic), and SW579 (poorly differentiated cancer with nuclear features of papillary cancer) thyroid cancer cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Sphingosine kinase inhibitor (SK-I; Calbiochem, La Jolla, CA), dissolved in dimethyl sulfoxide, was used to treat cells at indicated final concentrations and times (28).

Patients and tissue specimens

A total of 42 cases of paraffin-embedded thyroid cancer (22 cases of papillary thyroid cancer, 10 cases of follicular thyroid cancer, and 10 cases of anaplastic thyroid cancer), 10 cases of goiters, and 16 cases of adenoma samples, which had been clinically and histologically diagnosed at the First Affiliated Hospital of Sun Yat-sen University during year of 2005 to 2010, were obtained with previous patients' consents and approval from the Institutional Research Ethics Committee.

Immunohistochemistry

IHC analysis was performed to study altered protein expression in human T. The degree of immunostaining of SphK1 and PCNA proteins was examined and scored independently by two observers by combining both the proportion of positive staining tumor cells and the staining intensity as previously described (13). Scores representing the proportion of positively stained tumor cells was graded as: 0 (no positive tumor cells), 1 (<10%), 2 (10–50%), or 3 (>50%). The intensity of staining was determined as: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow brown), or 3 (strong staining, brown). The staining index was calculated as the product of staining intensity and percentage of positive tumor cells, resulting in scores of 0, 1, 2, 3, 4, 6, and 9. Cutoff values for SphK1 were chosen based on a measurement of heterogeneity using the log-rank test with respect to overall survival. We identified the optimal cutoff as: the staining index score of more than or equal to four was considered as high expression, and less than or equal to three as low expression.

Vectors and retroviral infection

For depletion of SphK1, two human siRNA sequences were cloned into pSuper-retro-puro to generate pSuper-retro-SphK1-RNAi#1 and pSuper-retro-SphK1-RNAi#2, respectively, and the sequences are RNAi#1, GGCTGAAATCTCCTTCACG and RNAi#2, GGGCAAGGCCTTGCAGCTC. Retroviral production and infection were performed as described previously (39, 40). The reporter plasmid containing wild (CCTTTGATC; TOPflash) or mutated (CCTTTGGCC; FOPflash) TCF/LEF binding site was purchased from Upstate Biotechnology (Lake Placid, NY).

Western blotting (WB)

WB was performed according to a standard method as described previously (13). The following primary antibodies were used: anti-SphK1 (Bethyl Laboratories, Montgomery, TX), antiphospho-Akt (Thr308), antiphospho-Akt (Ser473), antitotal-Akt, antiphopho-GSK-3β (Ser9), antitotal-GSK-3β, antiphospo-β-catenin (Ser33/37/Thr41), antitotal-β-catenin (Cell Signaling, Beverly, MA), and anti-α-tubulin (Sigma-Aldrich, St. Louis, MO). The nuclear extracts were prepared using the Nuclear Extraction kit (Active Motif, Tokyo, Japan) as the manufacturer instructed.

Sphingosine kinase activity assay

The activity of sphingosine kinase was quantified by using a commercial Sphingosine Kinase Activity Assay kit (Echelon Biosciences, Salt Lake City, UT) as the manufacturer instructed.

RNA extraction and real-time RT-PCR

RNA extraction, RT, and real-time PCR were performed as described previously (41). The primers selected are as the following: SphK1 forward, 5′-GTATGAATGCCCCTACTTGG-3′ and reverse, 5′-AACACACCTTTCCCATCCT-3′; cyclin D1 forward, 5′-AACTACCTGGACCGCTTCCT-3′ and reverse, 5′-CCACTTGAGCTTGTTCACCA-3′; c-myc forward, 5′-TTCGGGTAGTGGAAAACCAG-3′ and reverse, 5′-CAGCAGCTCGAATTTCTTCC-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-GACTCATGACCACAGTCCATGC-3′ and reverse, 5′-AGAGGCAGGGATGATGTTCTG-3′.

Cell growth assay

In vitro cell growth was measured using the MTT method; 5 × 103 cells were plated per well in 96-well plates and cultured overnight. Cell proliferation was evaluated with MTT assay every 24 h over a period of 6 d. Each assay was repeated three independent times in triplicates.

Soft agar colony formation assay

Two milliliters of 0.66% agar medium was added to each well of six-well plates to form bottom agar. Fifty thousand cells were mixed with 2 ml of 0.33% agar medium and then were layered onto the bottom agar and incubated at 37 C in 5% CO2, and 0.5 ml of culture medium was added every week to keep the soft agar from drying and to supply nutrition. After 3 wk of culture (2 wk for FRO cells), we calculated the numbers of colonies with a Zeiss microscope (Carl Zeiss, Jena, Germany).

Reporter assay

For the reporter assay, cells were seeded in triplicates in 24-well plates and allowed to settle for 24 h. Eight hundred nanograms of TOPflash plasmid or FOPflash plasmid plus 10 ng of pRL-TK renilla plasmid were transfected into the cells using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Forty-eight hours after transfection, Dual-Luciferase reporter assays were performed according to the manufacturer's instructions (Promega, Madison, WI). Results are presented as means ± sd of three independent experiments performed in triplicates.

Statistical analysis

All statistical analyses were carried out using the SPSS 11.0 statistical software package. Bivariate correlation between SphK1 and PCNA expression was calculated as Spearmans rank correlation coefficients. Association of SphK1 protein expression with clinicopathological parameters was assayed using Mann-Whitney U test. All values represent at least three independent experiments and are expressed as the means ± sd. The differences between two experimental conditions were compared on a one-to-one basis using the Student's t tests. P < 0.05 was considered statistically significant.

Acknowledgments

This work was supported by The Natural Science Foundation of China Grants no. 81070659 and no. 81001190; the Guangdong Recruitment Program of Creative Research Groups; the National Science and Technique Major Project 201005022-2; the Key Science and Technique Research Project of Guangdong Province 2010B030600003; Research Fund for the Doctoral Program of Higher Education of China no. 2009171110054; the Natural Science Foundation of Guangdong Province of China Grant no. 12510089010000300; the Science and Technique Research Project of Guangzhou Municipality, Guangdong Province, China no. 2010J-E521; and the Foundation for the Author of Excellent Doctoral Dissertation of Guangdong Province, China no. 80000-3226201.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
Akt
Protein kinase B
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
GSK-3β
glycogen synthase kinase-3β
IHC
immunohistochemistry
MTT
3-(4,5-dimethylthiazol-2-yl) −2,5-diphenyltetrazolium bromide
N
noncancerous thyroid tissue
PCNA
proliferation cell nuclear antigen
RNAi
RNA interference
shRNA
short hairpin RNA
SK-I
2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole
S1P
sphingosine 1-phosphate
SphK1
sphingosine kinase 1
T
thyroid cancer tissue
TCF/LEF
T-cell factor/lymphoid enhancing factor
WB
Western blotting.

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