Sphingosine kinases (SK) regulate the balance between proapoptotic ceramides and mitogenic sphingosine-1-phosphate (S1P); however, the functions of the two isoenzymes (SK1 and SK2) in tumor cells are not well defined. Therefore, RNA interference was used to assess the individual roles of SK1 and SK2 in tumor cell sphingolipid metabolism, proliferation, and migration/invasion. Treatment of A498, Caki-1, or MDA-MB-231 cells with siRNAs specific for SK1 or SK2 effectively suppressed the expression of the target mRNA and protein. Ablation of SK1 did not affect mRNA or protein levels of SK2 and reduced intracellular levels of S1P while elevating ceramide levels. In contrast, ablation of SK2 elevated mRNA, protein, and activity levels of SK1 and increased cellular S1P levels. Interestingly, cell proliferation and migration/invasion were suppressed more by SK2-selective ablation than by SK1-selective ablation, showing that the increased S1P does not rescue these phenotypes. Similarly, exogenous S1P did not rescue the cells from the antiproliferative or antimigratory effects of the siRNAs. Consistent with these results, differential effects of SK1- and SK2-selective siRNAs on signaling proteins, including p53, p21, ERK1, ERK2, FAK, and VCAM1, indicate that SK1 and SK2 have only partially overlapping functions in tumor cells. Overall, these data indicate that loss of SK2 has stronger anticancer effects than does suppression of SK1. Consequently, selective inhibitors of SK2 may provide optimal targeting of this pathway in cancer chemotherapy. Mol Cancer Res; 9(11); 1509–19. ©2011 AACR.
A large body of data show that sphingolipids, in particular ceramides, sphingosine, and sphingosine-1-phosphate (S1P), act as signaling molecules to regulate biological processes including apoptosis, proliferation, and stress responses (1–3). In contrast to proapoptotic ceramides and sphingosine, S1P induces cell proliferation and migration/invasion and promotes survival (4), acting through cell surface S1P receptors (5) or intracellularly as a second messenger (6). Therefore, the enzymes that interconvert sphingolipids have promise as new targets for cancer therapy.
As the key enzymes that catalyze the phosphorylation of sphingosine to S1P, sphingosine kinases (SK) are the checkpoint for S1P production (7). Two isoenzymes, SK1 and SK2, have been identified in human cells (8, 9). Although they share homology and catalyze the production of S1P, it is unclear whether their cellular functions are redundant in tumor cells. SK1 is mainly localized in the cytosol and migrates to the plasma membrane upon activation (10), which is a critical step for its oncogenic effects (11). SK1 is frequently overexpressed in tumor tissues compared with normal tissues (12), and overexpression in NIH3T3 cells promotes growth (4). Conversely, downregulation of SK1 in colon adenocarcinoma cells decreases the expression of COX-2 which is a pathogenic factor in colon carcinogenesis (13). Reports indicate that SK1 regulates motility, growth, and chemoresistance of MCF-7 cells (14) and progression through the cell cycle (15). Furthermore, depletion of SK1 in HEK 293 cells abrogates epidermal growth factor–induced migration (16). Serum S1P levels in SK1−/− mice are decreased by 50% and tissue S1P levels are normal, suggesting that SK1 deficiency can be at least partially compensated for by SK2 (17).
The roles of SK2 in cancer cells are not fully understood. Overexpression of SK2 was suggested to promote apoptosis (18); however, this likely reflects increased expression of its BH3 domain because a kinase-inactive mutant is similarly proapoptotic (19). Interestingly, apoptosis results in S1P production by SK2 which promotes macrophage survival allowing clearance of dead cells (20). This is associated with caspase-1–mediated cleavage of SK2 resulting in its secretion and generation of extracellular S1P (21). In tumor cells, downregulation of SK2 inhibits the proliferation of glioblastoma cells (22) and eliminates epidermal growth factor–induced migration of MDA-MB-453 cells (16). Also, the growth of SK2-deficient MCF-7 tumor xenografts is significantly delayed in mice (23). A nuclear localization sequence in SK2 likely accounts for its presence in the nucleus, cytosol, and endoplasmic reticulum under different circumstances (19). These different subcellular localizations may allow unique functions of SK1 and SK2 (24, 25). In SK2−/− mice, plasma S1P levels are reduced by only 25%, indicating a greater contribution from SK1 (26).
Small-molecule sphingosine kinase inhibitors have been described (12, 27), and a SK2-selective inhibitor, ABC294640, has been shown to have antitumor activity in mouse tumor models (28–30). Because sphingosine kinases are being increasingly considered as targets for new anticancer drugs, it is critical that the roles of SK1 and SK2 in cancer cells are defined. Therefore, we have used isoenzyme-selective siRNAs to assess the roles of SK1 and SK2 in sphingolipid metabolism, proliferation, and migration/invasion in human tumor cells.
Materials and Methods
Cell lines and reagents
A498 kidney adenocarcinoma, Caki-1 kidney clear cell carcinoma, and MDA-MB-231 breast adenocarcinoma cells were from the American Type Culture Collection and cultured in Minimum Essential Media, McCoy's 5A, and Dulbecco's Modified Eagle's Media, respectively, supplemented with 10% FBS and 50 μg/mL gentamicin in a 37°C, humidified, 5% CO2, 95% air environment. Silencer® validated siRNAs were purchased from Ambion Inc. as follows: SK1 (siSK1, #1181 targeting exon 3 of NM_021972), SK2 (siSK2, #1587 targeting exon 7 of NM_020126), ERK1 (siERK1, #142304 targeting exon 7 of NM_002746), and nontargeted siRNA (negative control, siNC). Additional siRNAs were purchased from Santa Cruz Biotechnology, Inc., and consisted of pools of 3 siRNAs targeting different coding regions of SK1 (sc-44114) or SK2 (sc-39225). All media, supplements, and primers were from Invitrogen. S1P and ω-(7-nitro-2-1,3-benzoxadiazol-4-yl)(2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol (NBD-sphingosine) were purchased from Avanti Polar Lipids, Inc. All other chemicals were from Sigma-Aldrich. Polyclonal chicken antibodies were raised against a unique SK1 peptide sequence (CVEPPPSWKPQQMPPPEE) and a unique SK2 sequence (EAEEQQDQRPDQELT), respectively, by Aves Lab Inc.
Briefly, 18 μL of Lipofectamine 2000 (Invitrogen) was mixed with 1000 μL Opti-MEM–reduced serum media and incubated for 15 minutes at room temperature. siRNA was added to a final concentration of 50 nmol/L in 6 mL of medium, incubated for 20 minutes, and then added to 5 mL of antibiotic-free growth media in 10-cm dishes along with 6 × 105 cells.
Total RNA was extracted and purified using RNeasy columns (Qiagen), and 1 μg was used to synthesize cDNA (total volume = 20 μL) with the SuperScript III Kit (Invitrogen) following manufacture's protocol. Human 18S rRNA and glyceraldehyde–3–phosphate dehydrogenase (GAPDH) were used as endogenous control transcripts for normalization of the target transcripts. Aliquots (2 μL) of the reverse transcription reaction were used in quantitative PCR (qPCR; 20 μL) in the presence of the following target-specific primers for amplification:
SK1: 5′-TGAGCAGGTCACCAATGAAG-3′; 5′-TGTGCAGAGACAGCAGGTTC-3′
SK2: 5′-GGAGGAAGCTGTGAAGATGC-3′; 5′-GCAACAGTGAGCAGTTGAGC-3′
ERK1: 5′-CAACATGAAGGCCCGAAACTACC-3′; 5′-TAACATCCGGTCCAGCAGGTCAAG-3′
ERK2: 5-′TACACCAACCTCTCGTACATCG-3′; 5′-CATGTCTGAAGCGCAGTAAGATT-3′
18S: 5′-TTGGAGGGCAAGTCTGGTG-3′; 5′-CCGCTCCCAAGATCCAACTA-3′
GAPDH: 5′-TGCACCACCAACTGCTTAGC-3′; 5′-GGCATGGACTGTGGTCATGAG-3′
SYBR Green Supermix and a MyiQ Real-Time PCR system (Bio-Rad) were used for qPCR with cycling parameters of 95°C for 5 minutes; 45 cycles of 95°C for 45 seconds, and 60°C for 1 minute. A dissociation profile was generated for each run to verify specificity of the amplification. The average cycle threshold (Ct) value for each group was determined and normalized by the endogenous control Ct value. The relative percentage expression was calculated using the following equation: % relative expression = 2(−ΔCt) × 100, where Δ Ct represents the difference in normalized Ct for the knockdown groups versus control (siNC).
At room temperature, cells grown on glass coverslips were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and then incubated sequentially with the following: 10% goat serum for 1 hour, chicken SK1 or SK2 antibodies in 1% goat serum for 1 hour, and fluorophore-conjugated chicken secondary antibody (Invitrogen) for 1 hour. Coverslips were mounted with a 4′,6-diamidino-2-phenylindole–containing reagent and examined with a Nikon Eclipse E800 microscope and NIS-Elements software.
SK1 activity assay
Cellular SK1 activity was measured using a high-performance liquid chromatography (HPLC)-based assay described previously (28). Briefly, cell lysates were incubated with NBD-sphingosine in SK1-selective assay buffer, and the product (NBD-S1P) was resolved by reversed phase HPLC with fluorescence detection. The area under the curve for the NBD-S1P peak was compared with quantified as a measure of SK1 activity.
Protein isolation and Western blot analyses
Cells were washed 3 times with ice-cold PBS, harvested, and centrifuged at 1,000 × g for 5 minutes. Cell pellets were lysed with 100 μL of lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 1% NP-40, pH = 7.4, and protease and phosphatase inhibitors) by vortexing. Supernatants were prepared by centrifugation at 5,000 × g for 30 minutes at 4°C, and protein concentrations were determined using the BCA Kit (Pierce). Equal amount of protein (30 μg per lane) was fractionated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Immunoblotting was carried out with the following primary antibodies (Cell Signaling except noted): anti-AKT, anti-pAKT, anti-ERK1/2, anti-pERK1/2, anti-p21, anti-p53, anti-FAK, anti-pFAK (Y397), anti–vascular cell adhesion molecule 1 (VCAM1; Santa Cruz Biotechnology), anti-β-actin; and HRP-conjugated secondary antibodies. The immunocomplexes were visualized by the enhanced chemiluminescence method. Protein expression was quantified by densitometric scanning of the BIOMAX XAR films after normalization to β-actin using the NIH ImageJ Program.
Cell proliferation assays
Cells were transfected with siRNAs in 96-well plates (5,000 cell per well), and then at varying times, the cells were washed with PBS, fixed with 10% trichloroacetic acid, washed with PBS, stained with sulforhodamine-B, and destained with 1% acetic acid. The cell-bound dye was then dissolved in 10 mmol/L Tris, and the absorbance at 560 nm was measured using a Spectramax M5 spectrophotometer (Molecular Devices). In certain experiments, 1 μmol/L (final) S1P was added to the culture medium 48 hours after transfection.
Cells were transfected as described above and then harvested by trypsinization, washed, and fixed with 75% ethanol at 4°C. The fixed cell were washed with PBS, resuspended, and stained with DNase-free RNase A (0.4 mg/mL) and propidium iodide (0.05 mg/mL) in PBS. Cell-cycle analyses were conducted using a FACStarplus flow cytometer (BD Biosciences).
Sphingolipid mass measurements
At 72-hour posttransfection, cells were harvested and washed 3 times with PBS. The cell pellets were subjected to sphingolipid profiling by HPLC-MS (mass spectrometry) by the Lipidomics Core facility at MUSC as described elsewhere (31).
Migration and invasion assays
Cells were harvested by trypsinization 48 hours after siRNA transfection, washed with PBS, and suspended in serum-free media. For migration assays, 50,000 cells were placed in a Transwell insert (24-well format, 8-μm pore; BD Biosciences). For invasion assays, matrigel-precoated inserts were used. In both cases, the inserts were placed into the 24-well plates with serum-containing growth medium and incubated under normal culture conditions for 4 hours (migration) or 24 hours (invasion). To assess the ability of exogenous S1P to rescue cell migration, 1 μmol/L S1P was added to the culture medium of the siRNA-treated cells for the final 24 hours before harvest. The assays were then conducted as described above, except that 1 μmol/L S1P and 0.1% fatty acid–free bovine serum albumin were added to both the top and bottom compartments of the Transwells. The nonmigrating cells were removed using cotton swabs, and cells that had migrated to the underside of the inserts were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells were counted in 5 random squares for each well using a light microscope, and results are presented as the average number of migrated/invaded cells per field.
SK1 and SK2 are selectively depleted by isoenzyme-specific siRNAs
To compare the functions of the endogenous SK1 and SK2, we used siRNAs to knockdown the expression of SK1 and/or SK2 in 3 human cancer cell lines, A498, Caki-1, and MDA-MB-231. The relative expression levels of SK1 and SK2 measured by qPCR are expressed relative to SK1 in A498 cells (Fig. 1A). The data indicate that A498 cells have the highest levels of mRNA encoding SK1 and SK2 and that transcript levels of SK1 are considerably higher than those for SK2 in all 3 cell lines. For all of the following knockdown experiments, cells transfected with siNC are defined as the control (i.e., 100% expression). As shown in Fig. 1B, mRNA levels of the targeted sphingosine kinase isoenzyme in A498 cells were substantially decreased by 48 hours after transfection and remained suppressed for at least 72 hours. For A498 cells, at 72 hours posttransfection, SK1 mRNA levels in cells treated with either siSK1 or siSK1 + siSK2 were reduced approximately 70%, whereas siSK1 treatment did not affect levels of SK2 mRNA. Conversely, mRNA for SK2 was reduced approximately 85% in cells treated with either siSK2 or siSK1 + siSK2. Surprisingly, cells treated with siSK2 contained dramatically elevated levels of mRNA for SK1. Expression analyses in Caki-1 and MDA-MB-231 cells provided similar results in that the targeted sphingosine kinase isoenzymes were effectively depleted. Importantly, knockdown of SK2 resulted in elevated expression of SK1 mRNA in Caki-1 and MDA-MB-231 cells, although the magnitudes of the responses were less than that observed with the A498 cells. In all of the transfection experiments, the cells remained attached to the plates for at least 72 hours.
Chicken antibodies against peptides specific for human SK1 and SK2 were made to analyze their protein levels. This was necessary because several commercially available sphingosine kinases antibodies failed to provide us with interpretable data by immunoblotting or immunocytochemical analyses. The newly developed antibodies were poor for immunoblotting but provided excellent selectivity and sensitivity in immunofluorescence staining experiments (Supplementary Fig. S1), suggesting a need for epitope structure for immunorecognition. Using these antibodies, we quantified and confirmed the 70% to 85% decreases in the protein levels of the targeted sphingosine kinase isoenzymes in siRNA-transfected A498 cells (Fig. 2A and B). Importantly, the expression of SK1 protein was elevated approximately 40% in cells treated with siSK2. To further confirm the effects of the siRNA, SK1 enzymatic activity was measured in lysates from siRNA-transfected A498 cells using a fluorescence-based HPLC assay that we have previously described (28). As indicated in Fig. 2C, siSK1 strongly reduced SK1 activity, whereas siSK2 treatment increased SK1 activity by approximately 70%. The activity of SK2 was below the detection limit for the assay for all samples. Overall, the data confirm that the siRNAs are effective for selective depletion of the targeted sphingosine kinases isoenzymes and that SK1 expression appears to be sensitive to the expression level of SK2.
Effects of sphingosine kinase knockdown on sphingolipids
Sphingosine kinases convert sphingosine to S1P; however, it is well established that the metabolism of sphingolipids is a dynamic process (32), so that an altered flux through sphingosine kinase may modulate several sphingolipids. As shown in Fig. 3, SK1-selective knockdown resulted in elevated levels of total ceramides (total Cer; approximately 70% increase from control) and all of the individual ceramide species measured, that is, dihydro-C16-ceramide (DHC16-Cer), C24-ceramide (C24-Cer), C20:1-ceramide (C20:1-Cer), C20-ceramide (C20-Cer), C18-ceramide (C18-Cer), and C14-ceramide (C14-Cer). Also, the level of S1P was decreased as expected, but there was a small decrease in sphingosine, indicating that the sphingosine was likely being converted to ceramides by ceramide synthase. In marked contrast, the selective knockdown of SK2 did not substantially affect the levels of most ceramide species, causing only a slight increase in total ceramide levels. Interestingly, selective knockdown of SK2 resulted in significantly elevated S1P levels, and this was largely abrogated when SK1 was simultaneously suppressed in the double knockdown cells. The medium from cells grown in serum-free medium was analyzed by HPLC-MS; however, the levels of extracellular S1P were below the detection limit of the assay (data not shown). Combined with the qPCR results described above, it seems likely that the elevated S1P level arises from the increased expression of SK1 in the SK2 selectively depleted cells. These data indicate that SK1 is the dominant isoenzyme in A498 cells for the synthesis of S1P (on a total S1P mass basis).
Knockdown of sphingosine kinase suppresses cell proliferation
Because sphingosine kinases catalyze the production of mitogenic S1P, we evaluated the effects of their knockdown on cell-cycle progression and proliferation. As shown in Fig. 4A, SK1 and/or SK2 knockdown resulted in an increase in the percentage of A498 cells in the G1 phase of the cell cycle, with corresponding decreases in the S and G2–M phases. Responses to siSK2 were somewhat greater than responses to siSK1, and combined addition of both siRNA did not further arrest the cells beyond what occurred with siSK2 alone. Moreover, the addition of 1 μmol/L S1P to the siRNA-transfected cells did not overcome the cell-cycle arrest (Fig. 4B). These results suggest that both SK1 and SK2 promote cell-cycle progression, although removal of SK2 appears to be more suppressive. Interestingly, knockdown of SK1 and/or SK2 did not promote a significant level of apoptosis in these A498 cells (<1% of cells in all cases). Therefore, the accumulation of ceramides, particularly in the SK1 selectively depleted cells, is not sufficient to drive these cells into apoptosis. Similar responses were seen in Caki-1 and MDA-MB-231 cells (Fig. 4B and C). It is notable that responses of MDA-MB-231 cells, which have mutated p53, indicate that cell-cycle arrest induced by sphingosine kinase knockdown is not dependent on p53.
Consistent with the cell-cycle data, knockdown of either SK1 or SK2 reduced the proliferation of A498 cells for at least 96 hours after transfection (Fig. 4D), with inhibition being much stronger as a result of SK2-selective ablation. Similar results were obtained upon siRNA transfection of Caki-1 and MDA-MB-231 cells (Fig. 4E and F). It is noteworthy that selective depletion of SK2 completely blocked the proliferation of the cells despite the elevated expression of SK1 and the concomitant elevation of S1P levels in these cells. As with the cell-cycle studies, addition of exogenous S1P did not rescue A498 cells from inhibition of proliferation caused by SK1 and/or SK2 depletion, indicating that intracellular S1P is involved in these processes. In contrast with extracellular S1P, intracellular S1P functions as a secondary messenger independent of S1P G-protein–coupled receptors (33). While the mechanisms have not been fully characterized, potential targets for intracellular S1P have been discussed (34, 35). Because of the physical properties of S1P, extracellular S1P penetrates into cells only very poorly (36), which prevents it from contributing to the intracellular S1P pool. Therefore, it is not surprising that exogenous S1P did not rescue sphingosine kinase–ablated cells, and this is consistent with other recent reports (37, 38). This is a critical point in terms of drug development in that it predicts that pharmacologic inhibition of S1P production in tumor cells will block proliferation even in the presence of plasma S1P. The combined results indicate that SK2 is necessary for optimal proliferation of tumor cells whereas SK1 plays a lesser role.
Signaling effects of sphingosine kinase knockdown
The roles of SK1 and SK2 in signaling pathways that regulate cell-cycle progression were assessed in a series of immunoblots using samples prepared from siRNA-treated A498 cells. As shown in Fig. 5A, at 72 hours posttransfection, SK1 selectively ablated cells had elevated p21 levels, which slows progression through the cell cycle at the G1 to S-phase (39), whereas the SK2-selective and dual knockdown cells showed slight decreases in p21 levels. p53 protein levels revealed expression patterns identical to those for p21. These results suggest that cell-cycle arrest by selective depletion of SK2 is p53 independent, which is consistent with cell-cycle arrest in cell lines with either wild-type or mutated p53.
As markers of cell survival and proliferation, AKT and extracellular signal-regulated kinase (ERK) were also analyzed. Levels of pAKT were decreased dramatically by depletion of either SK1 or SK2 (Fig. 5B), whereas no further decrease was observed in the double knockdown group. None of the siRNA treatments affected the AKT protein levels. Thus, SK1 and SK2 appear to redundantly regulate signaling through AKT. The effects of sphingosine kinase depletion on ERK were complex. Surprisingly, compared with controls, all 3 knockdown groups had greatly elevated pERK1 and decreased pERK2 levels, with the response to siSK2 being quantitatively larger than the response to siSK1. Interestingly, treatment with either siSK1 or siSK2 increased levels of ERK1 protein while decreasing levels of ERK2 protein. qPCR analyses confirmed that SK2-selective knockdown increased ERK1 mRNA to a larger degree than did selective knockdown of SK1 (Fig. 5B). Similarly, the reductions in ERK2 protein levels were accompanied by decreases in expression of ERK2 at the message level. To determine the requirement for ERK1 for cell-cycle arrest in the sphingosine kinase knockdown cells, siRNA targeting ERK1 was cotransfected with siSK1 and/or siSK2 into A498 cells. Data shown in Fig. 5C indicate that ablation of ERK1 did not alter responses to either sphingosine kinase siRNA. Taken together, the data suggest that SK2 has a greater role than SK1 in regulating the expression and phosphorylation of ERK1 and promoting the expression and phosphorylation of ERK2.
Knockdown of sphingosine kinase suppresses cell migration and invasion
Because S1P can also induce cell migration (40), we investigated the effects of sphingosine kinase knockdown on FBS-induced migration and invasion (through Matrigel). As shown in Fig. 6A, both migration and invasion were more strongly attenuated by SK2-selective and double knockdown than by SK1-selective knockdown in A498 cells. Similar results were obtained in the migration and invasion assays using MDA-MB-231 cells (Fig. 6B); however, Caki-1 cells were poorly migratory, so the effects of the sphingosine kinase siRNAs could not be assessed in these cells. Furthermore, exogenous S1P did not rescue the SK1 and/or SK2 knockdown cells from inhibition of migration/invasion (Fig. 6C). To examine possible mechanisms for these effects, we assessed the levels of pFAK (Y397), FAK, and VCAM1 by immunoblotting. As shown in Fig. 6D, all 3 knockdown groups had decreased levels of pFAK (Y397), which plays a key role in promoting cell migration (41). However, the expression of total focal adhesion kinase (FAK) protein was markedly increased in the SK2-selective and double knockdown cells. These 2 groups of cells also showed increased electrophoretic mobility of VCAM1, which may reflect inhibition of its glycosylation—an important step for promigration function of VCAM1 (42, 43). In contrast, SK1-selective knockdown did not alter VCAM1 electrophoretic mobility, although its expression level was slightly decreased. These results suggest that SK2 may regulate cell migration by altering VCAM1 glycosylation. Overall, the data suggest that SK2 plays a more important role in the regulation of cancer cell migration and invasion than does SK1.
It is well known that S1P production by sphingosine kinases can promote cell proliferation and inhibit apoptosis, making sphingosine kinases potential cancer therapeutic targets. Most of the emphasis has been focused on SK1, particularly since the demonstration that overexpression can transform cells (44). Although SK2 has the same ability as SK1 to produce S1P, different kinetic properties and subcellular localizations of these isoenzymes may provide them with distinct functions in cell signaling. Although SK2 has been drawing more attention recently, it is still not clear whether SK1 and SK2 have redundant, overlapping, or opposing functions in human cancer cells. Resolution of this ambiguity is essential for the design of sphingosine kinase–targeted drugs, that is, the preference for isoenzyme-selective or dual sphingosine kinase inhibitors must be defined. To this end, we have used RNA interference to assess the activities of SK1 and SK2 in tumor cells. In the present studies, A498 kidney cancer cells were chosen as the primary model because they have relatively high levels of SK1 mRNA (45) and normal kidney tissue has high expression of SK2 mRNA (9). Another kidney cell line (Caki-1) was used to confirm phenotypic effects of SK1 and SK2 ablation in this tissue type. We included the breast cancer cell line MDA-MB-231 to broaden the relevance of the studies and to compare signaling patterns in a cell line with mutated p53 (A498 and Caki-1 cells both contain wild-type p53). Furthermore, MDA-MB-231 cells are reported to be highly metastatic (46).
Transient knockdown of expression of target mRNAs provides an advantage over the use of stably overexpressing cells and cells from transgenic knockout mice in that compensatory changes in the expression of the alternate isoenzyme can be examined. This is particularly important in the study of sphingosine kinases because SK1 expression appears to be regulated by SK2; however, the actions of elevated SK1 do not compensate phenotypically for the loss of SK2. Attempts to make stable knockdowns of SK1 and SK2 using short hairpin RNAs were unsuccessful in that no clones expressed SK2 mRNA levels lower than approximately 30% of control (data not shown). This implies that survival of the tumor cells requires at least some expression of SK2. Another issue in studies of sphingosine kinases is that commercially available antibodies for SK1 and particularly for SK2 performed poorly in immunoblotting and immunohistochemical experiments. Therefore, we generated sphingosine kinase isoenzyme–specific chicken antibodies that have excellent specificity and selectivity in immunocytochemical experiments (Supplementary Fig. S1); however, these antibodies also are inadequate for immunoblotting studies.
Our studies suggest a one-way regulation of the expression of SK1 and SK2. Specifically, knockdown of SK1 did not influence levels of SK2 at either the mRNA or the protein level in A498 cells. Conversely, knockdown of SK2 significantly induced the expression of SK1 mRNA, protein, and enzymatic activity, and this is associated with substantial increases in the cellular levels of S1P. However, it is critical to note that this response did not rescue the cells from inhibition of proliferation, migration, or invasion because of ablation of SK2. Furthermore, addition of S1P to the culture medium did not overcome the antiproliferative or antimigratory effects of SK2 depletion. This indicates that neither SK1 nor extracellular S1P can functionally compensate for loss of SK2 expression and/or activity. The lack of redundancy was also shown by the observations that depletion of SK2 always produced a greater suppression of tumor cell activities (proliferation, migration/invasion). Therefore, it seems clear that SK2 plays unique roles that cannot be replaced by SK1, and this is consistent with recent observations by others (22, 23, 47). The mechanism for induction of SK1 expression in response to siSK2 is under further study and appears to be time sensitive and quantitatively associated with the level of SK2. For example, SK2-directed siRNAs from Santa Cruz Biotechnology, which target different sequences than the Ambion siRNA, were less efficient in the selective depletion of SK2 and cross-reacted with SK1 to produce a small decrease in its mRNA levels (Supplementary Fig. S2A). Although knockdown of SK1 and SK2 were not as complete with the Santa Cruz siRNA, they did confirm that proliferation was much more strongly suppressed by SK2 knockdown than by SK1 knockdown (Supplementary Fig. S2B).
Lipidomic analyses also indicated differences between SK1 and SK2 knockdown cells. Not only was there reduced S1P following SK1 depletion, but also the levels of ceramides were increased in these cells. Conversely, the selective ablation of SK2 left most of the ceramide species unchanged. These divergent effects of SK1 and SK2 knockdown on sphingolipid profiles and cell responses may reflect their different subcellular localizations and consequently different subcellular pools of S1P. Unlike SK1 which is predominantly cytosolic, SK2 is localized in nucleus with a small portion in the cytosol or membranes depending on the cell type as well as endoplasmic reticulum (19, 48, 49). It is possible that when the cytosolic SK1 is ablated, the cytosolic pool of SK2 compensates its loss. However, if SK2 is ablated, SK1 cannot compensate to provide a local organelle (e.g., nuclear) pool of S1P because of lack of targeting. Similarly, exogenous S1P likely does not rescue sphingosine kinase–ablated cells because it does not localize correctly in the cells. Thus, the subcellular distribution of S1P, and perhaps ceramides, may provide a mechanism for the lack of redundancy between SK1 and SK2. The data further indicate that the local pool of S1P produced by SK2 is more important for promoting cell proliferation and migration than that by cytosolic S1P produced from SK1.
Current models suggest that S1P activates certain signal pathways, including PI3K/AKT, by binding to the cell surface receptors, (50). In addition, sphingosine kinases have been shown to activate the RAS/ERK pathway by a G-protein–coupled receptor–independent pathway (51). The current data indicate that both SK1 and SK2 are necessary for optimal AKT phosphorylation. In addition, our results show an interesting divergence in the regulation of ERK1 and ERK2 by sphingosine kinases in that depletion of either sphingosine kinase isoenzyme markedly reduced ERK2 expression and activation but increased ERK1 expression. To investigate the impact of the increased ERK1 on the cell cycle, siERK1 was cotransfected with siSK1 and siSK2. The data indicate that ablation of ERK1 does not affect cell-cycle arrest induced by ablation of SK1 or SK2. The observation that ablation of SK2 results in stronger downregulation of ERK2 and proliferation than does ablation of SK1 agrees with the previous studies showing that suppression of ERK1 alone is not sufficient to block cell replication (52, 53).
The combined data indicate that sphingosine kinase isoenzyme–selective ablation differentially affects sphingolipid profiles, signaling, proliferation, migration, and invasion. For SK1-selective ablation, reduction of S1P and elevation of ceramides may remove the growth-stimulatory signaling through S1P receptors, as the cytosolic pool of S1P is more likely to be secreted. In contrast, selective depletion of SK2 may remove the nuclear pool of S1P, which is quantitatively smaller than the SK1-derived pool and this downregulates several key signaling proteins, including ERK2, pAKT, p53, and p21. On the basis of our results and those of others (18, 19), SK2 has 2 opposing aspects that affect cell proliferation. Specifically, the enzymatic formation of S1P is proproliferation, whereas the BH3 domain is proapoptotic. When expressed at a normal level, SK2 makes localized S1P (most likely in the nucleus) which plays an important role in regulating cancer cell proliferation and migration/invasion. Conversely, when SK2 is overexpressed by genetic or pharmacologic manipulation, which may happen via a feedback mechanism after cells are treated with a SK2-selective inhibitor (Supplementary Fig. S3), the BH3 domain of SK2 provides a magnified proapoptotic stimulus that overshadows its proliferative activity. Therefore, blocking the enzymatic activity of SK2 while maintaining its expression, thereby allowing its BH3 domain to promote cell death, may provide the maximum anticancer activity.
Several sphingosine kinase inhibitors have been described in recent years, including ABC294640, which is an SK2-selective inhibitor that competes with sphingosine (28). ABC294640 has antiproliferative and antitumor activity (28–30), as well as anti-inflammatory activity, in a variety of cellular and animal models, supporting the hypothesis that SK2 is the preferred target over SK1 for new anticancer agents. Because circulating S1P is a major regulator of vascular and immune systems (54), selective inhibition of SK2 should be less toxic in vivo than inhibition of SK1 or dual inhibition of both isoenzymes. Clinical trials of ABC294640 are in progress to test this hypothesis.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This work was supported by NIH 1R01CA122226.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The authors thank Dr. Yuri Peterson who helped to design the epitopes of antibodies.
Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).
- Received July 12, 2011.
- Revision received August 15, 2011.
- Accepted August 29, 2011.
- ©2011 American Association for Cancer Research.