Oncogene (2014) 33, 5559–5568 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE Enhanced expression of receptor 1 contributes to oncogenic signalling by sphingosine kinase 1

DH Pham1,2, JA Powell1, BL Gliddon1, PAB Moretti1, A Tsykin1,2, M Van der Hoek3, R Kenyon3, GJ Goodall1,2,4 and SM Pitson1,2,4

Sphingosine kinase 1 (SK1) is a lipid kinase that catalyses the formation of sphingosine-1-phosphate (S1P). Considerable evidence has implicated elevated cellular SK1 in tumour development, progression and disease severity. In particular, SK1 has been shown to enhance cell survival and proliferation and induce neoplastic transformation. Although S1P has been found to have both cell- surface G--coupled receptors and intracellular targets, the specific downstream pathways mediating oncogenic signalling by SK1 remain poorly defined. Here, using a expression array approach, we have demonstrated a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through enhancing expression of 1 (TFR1). We showed that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression, resulting in increased transferrin uptake into cells. Notably, we also found that SK1 activation and localization to the plasma membrane, which are critical for its oncogenic effects, are necessary for regulation of TFR1 expression specifically through engagement of the S1P G-protein coupled receptor, S1P2. Furthermore, we showed that blocking TFR1 function with a neutralizing inhibits SK1-induced cell proliferation, survival and neoplastic transformation of NIH3T3 fibroblasts. Similar effects were observed following antagonism of S1P2. Together these findings suggest that TFR1 has an important role in SK1-mediated oncogenesis.

Oncogene (2014) 33, 5559–5568; doi:10.1038/onc.2013.502; published online 25 November 2013 Keywords: expression profiling; DNA microarray; neoplastic transformation; sphingosine kinase; transferrin receptor

INTRODUCTION In this study, we describe a novel mechanism whereby SK1 Sphingosine kinase 1 (SK1) catalyses the formation of sphingosine- regulates cell survival and proliferation through control of 1-phosphate (S1P), a bioactive phospholipid that has important (TFR1) expression. We show that elevated roles in a wide variety of cellular processes, including calcium levels of SK1 enhanced both total and functional TFR1 expression mobilization, proliferation, apoptosis, angiogenesis, inflammatory resulting in higher rates of transferrin (Tf) uptake into cells. We responses and cytoskeletal rearrangement.1 Elevated levels of also demonstrate that it is specifically the phosphorylated, plasma SK1/S1P have been shown to enhance cell survival and membrane-localized, oncogenic form of SK1 that mediates the proliferation,2,3 and there is now substantial evidence impli- effects on TFR1 expression via engagement of the S1P receptor, cating an important role of SK1 in the development, progression S1P2. Furthermore, we show that blocking TFR1 function with and severity of various cancers.4 This includes findings that SK1 a neutralizing antibody inhibited SK1-induced cell proliferation expression levels are elevated in a variety of human solid tumours and survival, suggesting that TFR1 has an important role in and correlates with poor patient survival,5,6 that deregulation of SK1-induced cell proliferation, survival and transformation. SK1 has an important role in both acute and chronic myeloid leukaemia4 and that overexpression of SK1 in NIH3T3 fibroblasts induces full neoplastic cell transformation.3 Furthermore, RESULTS inhibition of SK1 by genetic or pharmacological approaches has Expression profiling of gene regulation by SK1 been shown to significantly reduce tumour growth in mice7–10 To identify regulated in response to elevated SK1 and also sensitize tumour cells to other chemotherapeutics.11–13 expression, we used a cell line with doxycycline-inducible SK1 We have previously shown that the oncogenic signalling by SK1 expression17 to conduct analysis by DNA micro- is dependent on both its activation and translocation from the array. Using this inducible system, we were able to examine cytosol to the plasma membrane.14,15 These data suggest immediate early genes regulated in response to a subtle increase that translocation of SK1 to the plasma membrane places the in cellular SK1 overexpression, which is more likely to resemble enzyme in close proximity with its substrate and thereby enables the in vivo level of SK1 previously detected in human cancers.18 localized production of S1P either to be released to act on We reasoned that this approach would enhance the likelihood of the S1P cell-surface receptors15 or to regulate intracellular identifying direct and physiologically relevant target genes of SK1 signalling targets,16 which subsequently mediate the oncogenic and reduce the possibility of nonspecific secondary effects due to effects of this enzyme. However, the molecular mechanism by high and long-term overexpression. which SK1 exerts its oncogenic effects has yet to be clearly SK1 overexpression was induced with doxycycline in HEK293 elucidated. cells to levels up to 10-fold higher than endogenous (Figure 1a);

1Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, SA, Australia; 2School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia; 3Adelaide Microarray Facility, SA Pathology, Frome Road, Adelaide, SA, Australia and 4School of Pharmacy and Medical Science, University of South Australia, Adelaide, SA, Australia. Correspondence: Professor SM Pitson, Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, 5000 SA, Australia. E-mail: [email protected] Received 28 May 2013; revised 10 October 2013; accepted 19 October 2013; published online 25 November 2013 TFR1 contributes to oncogenic signalling by SK1 DH Pham et al 5560

Figure 1. Gene regulation in response to elevated SK1 expression. (a) SK1 activity in cells following induction of SK1 overexpression by 0.5 ng/ml doxycycline. (b) Heat plot of significantly regulated genes at 6 and 12 h following induction of SK1 overexpression (replicate analyses shown), listed in the order of average Log2 fold change at 12 h. The 30 genes with the greatest increase or decrease in fold changes are shown. See Supplementary Table S1 for the full list of 191 genes with significant regulation, as well as for further details. (c) Expression of FLAG-tagged wild-type SK1 and inactive SK1G82D after 6 h of induction with 0.5 ng/ml doxycycline was assessed by immunoblot of cell lysates with anti-FLAG and SK1 activity assays. (d) Validation by qPCR of differential regulation of TFRC, FUS and SFPQ at 6 and 12 h following induction of wild-type SK1 (K) and SK1G82D (J) expression. Data for TRFC represent the mean±s.e.m. from three independent experiments, each analysed in triplicate. Data for FUS and SFPQ are mean±range from two independent experiments, each analysed in triplicate. *Po0.05 compared with SK1G82D.

RNA was isolated at 6 and 12 h after induction, and then DNA additional microarray analyses using HEK293 cells induced to microarray analyses were performed comparing the gene expres- overexpress the catalytically inactive SK1G82D variant19 to a similar sion pattern with that of uninduced cells. Even though SK1 low level as that used for wild-type SK1 (Figure 1c). Low overexpression was only moderately higher than endogenous overexpression of SK1G82D, however, did not result in significant levels, this was sufficient to result in statistically significant regulation of any gene. regulation of 191 genes at 6 or 12 h (Figure 1b and We next sought to validate the microarray studies. As some of Supplementary Table S1). Of these, 107 genes were upregulated the main biological effects of elevated cellular SK1 are enhanced and 84 were downregulated. To eliminate SK1 protein effects that cell survival, proliferation and induction of neoplastic transforma- were not dependent on its catalytic activity, we performed tion, genes known to be involved in these processes that were

Oncogene (2014) 5559 – 5568 & 2014 Macmillan Publishers Limited TFR1 contributes to oncogenic signalling by SK1 DH Pham et al 5561 among those regulated in the microarray studies were selected for further examination of their gene regulation by qPCR. From this validation we identified three genes, TFRC, FUS and SFPQ, that were differentially regulated by SK1, and not SK1G82D (Figure 1d and Supplementary Table S1), suggesting that they are regulated by elevated cellular SK1 activity. TFRC encodes for TFR1, a membrane-associated glycoprotein that mediates iron uptake by binding to iron-loaded Tf.20 Notably, elevated TFR1 has been widely reported in a variety of human tumours, often correlating with tumour grade and patient outcome.21–26 Furthermore, high TFR1 expression enhances cell growth and tumour formation in mice,27,28 and inhibition of TFR1 by either genetic, pharmacological or antibody-neutralization approaches has been shown to reduce cancer cell proliferation, tumour growth and metastases in mice.29–32 Together, these findings raised the possibility that TFR1 may have a role in SK1-mediated oncogenesis.

SK1-mediated changes in TFR1 mRNA and protein depend on SK1 phosphorylation and plasma membrane localization To further confirm that SK1 regulates TFR1 expression, we induced cells to overexpress moderate levels of SK1, and then assessed total cellular TFR1 protein levels by western blotting, along with TFR1 mRNA by qPCR (Figure 2, left panels). The results showed that enhanced SK1 resulted in a twofold increase in both TFR1 protein and mRNA compared with that in uninduced control cells. Notably, TFRC expression was upregulated by wild-type SK1 and moderately downregulated by SK1G82D in both microarray and qPCR analyses (Figure 1). As SK1G82D is known to act in a dominant- negative manner and block endogenous SK1 activation,19 these data suggested that TFRC may be regulated in response to SK1 activation. SK1 activation by phosphorylation at Ser225 and its subsequent translocation to the plasma membrane are crucial for oncogenic signalling by this enzyme.15 We have previously shown that following high level overexpression, SK1 becomes phosphorylated, irrespective of the presence of cell agonists,14 Figure 2. SK1 mediates increased TFR1 mRNA and protein expres- and thus can drive constitutive oncogenic signalling. Here, we sion and is dependent on SK1 phosphorylation and localization to examined whether SK1 was also constitutively phosphorylated the plasma membrane. Cells were cultured for 16 h either in the following moderate overexpression, and found that indeed it was absence or in the presence of doxycycline to induce expression of wild-type SK1, non-phosphorylatable SK1S225A, or, constitutively, (Figure 2d). Thus, we further examined whether TFR1 expression pm-S225A was dependent on this type of SK1 regulation. To do this we first plasma membrane localized, non-phosphorylatable SK1 . Cells were harvested and lysates were prepared for TFR1 protein generated HEK293 cell lines with inducible expression of SK1S225A pm-S225A analysis by immunoblotting with anti-TFR1 antibodies, which were (the non-phosphorylatable human SK1) and SK1 (the quantified relative to tubulin expression (a), TFR1 mRNA analysis by constitutively plasma membrane-localized, non-phosphorylatable qPCR (b), SK1 activity assays (c) and immunoblot analysis of SK1).15 We then induced expression of SK1S225A and SK1pm-S225A to phospho-SK1 and SK1 overexpression by FLAG detection (d). Data the same level as used previously for wild-type SK1, as determined represent the mean±s.e.m. of six experiments. *Po0.001 and by cellular SK1 activity (Figure 2c, centre and right panels, **Po0.0001, compared with control (Ctl) cells. Dotted lines indicate respectively), and performed TFR1 qPCR and western blot analysis where lanes from the same immunoblots have been spliced to assess the effect on TFR1 expression (Figure 2). In contrast to the together to aid interpretation. effects of wild-type SK1, TFR1 mRNA and protein expression levels were unaltered following induction of SK1S225A expression. total TFR1 in cells, it was unclear whether this resulted in a However, expression of the plasma membrane-localized version subsequent increase in TFR1 function via enhanced presentation of this SK1 variant, SK1pm-S225A, resulted in a twofold increase in of this receptor at the cell surface and concomitant enhanced Tf TFR1 mRNA as well as in protein expression to a similar extent as uptake. Therefore, we examined the functional effects of increased that seen in wild-type SK1. These data indicate that the SK1 on cell surface presentation of TFR1 and Tf uptake via phosphorylation and subsequent plasma membrane localization fluorescence microscopy. Control (uninduced) cells showed very of SK1 necessary for oncogenic signalling by this enzyme are low cell surface presentation of endogenous TFR1 (Figure 3a). critical for SK1-induced regulation of TFR1 expression. Following the induction of SK1 expression, however, a clear increase in the levels of TFR1 at the cell surface was observed SK1 induces TFR1 cell-surface expression and enhances transferrin (Figure 3a). This effect was further confirmed by examining the uptake level of Tf uptake in these cells as a direct measure of functional TFR1 is a that, when presented on the TFR1. Consistent with an increase in total and cell surface TFR1 extracellular surface of the plasma membrane, can bind to iron- expression, there was also an approximately twofold increase in Tf loaded Tf and mediate iron uptake into cells via .20 uptake into the cells expressing SK1 compared with control cells Return of TFR1 to the cell surface occurs via the recycling (Figure 3b). Together, these results indicate that SK1 over- endosomal compartment, where considerable TFR1 can reside.33 expression enhances not only total TFR1 expression but also Thus, although we demonstrated that elevated SK1 enhanced subsequent functional TFR1 presentation on the cell surface.

& 2014 Macmillan Publishers Limited Oncogene (2014) 5559 – 5568 TFR1 contributes to oncogenic signalling by SK1 DH Pham et al 5562

Figure 4. Exogenous S1P increases TFR1 protein expression in a dose-dependent manner. HEK293 cells were cultured in the presence of increasing concentrations of S1P for 16 h. TFR1 protein expression was then examined in cell lysates by immunoblot analysis with anti-TFR1 antibodies and quantified relative to tubulin expression. Data represent the mean±s.e.m. of three independent experiments. *Po0.05 and **Po0.01, compared with cells not treated with S1P.

inhibitors to these various S1P receptors, and the effects on TFR1 expression were examined. The results showed that VPC- 23019, an inhibitor of S1P1 and S1P3, had no effect on SK1- induced TFR1 protein expression (Figure 5a). In contrast, however, treatment of cells with JTE-013, an inhibitor of S1P2, effectively blocked TFR1 expression induced by SK1 in HEK293 cells (Figure 5a). As expected, JTE-013 had no effect on SK1 activity Figure 3. SK1 enhances cell-surface TFR1 expression and increases (Figure 5a). Similar results were also observed in NIH3T3 cells, Tf uptake into cells. Cells were either treated or not treated with where JTE-013 effectively blocked induction of TFR1 expression by 4 ng/ml doxycycline for 16 h to induce SK1 to approximately 20-fold exogenous S1P (Figure 5b). over endogenous levels. (a) Cell-surface TFR1 expression was As JTE-013 has been suggested to also be an antagonist of 36 37 detected in unpermeabilized cells using anti-TFR1 antibodies S1P4, and may have other off-target effects, we further (green); cells were then subsequently permeabilized and increased examined the involvement of S1P2 by utilizing siRNA knock- expression of total SK1 in cells was confirmed using anti-SK1 down of S1P in cells expressing SK1 and then examined its effect antibodies (red). Images are representative of more than 300 cells 2 examined. (b) To examine Tf uptake, cells were incubated with Alexa on TFR1 mRNA and protein expression. The results again show 568-labelled Tf for 30 min at 4 1C. Unbound Tf was then removed that TFR1 expression induced by SK1 was blocked in cells and cells were incubated at 37 1C for 10 min and the internalization following knockdown of S1P2 (Figure 5c). Taken together, these of Tf (red) was observed and quantified. Cell nuclei were visualized results indicate that SK1 regulates TFR1 expression via the S1P2 by staining with DAPI (blue). Data represent the mean±s.e.m. of receptor. four independent experiments. *Po0.005 compared with control cells. Inhibition of TFR1 ablates SK1-induced cell proliferation, survival and neoplastic transformation Exogenous S1P regulates TFR1 expression Overexpression of SK1 has been shown to enhance cell 3,15 We previously showed that activation and subsequent transloca- proliferation and survival and induce neoplastic transformation. tion of human SK1 to the plasma membrane results in both As described above, TFR1 has also been associated with cancer. increases in intracellular S1P and enhanced release of S1P into the Therefore, we investigated whether TFR1 has a role in oncogenic extracellular environment.14,15 Therefore, to assess the roles of signalling by SK1 through the use of a TFR1 neutralizing antibody 38,39 intra- and extracellular S1P we next examined the effect on TFR1 previously shown to block the biological functions of TFR1. 3,15,40 expression following the addition of exogenous S1P to cells. The Consistent with previous studies, SK1 overexpression results (Figure 4) show a dose-dependent increase in TFR1 protein enhanced cell proliferation (Figure 6a) and protected cells from expression in cells treated with S1P. Notably, this increase in TFR1 serum-deprivation-induced apoptosis (Figure 6b). Strikingly, how- protein expression was detectable at very low concentrations of ever, this SK1-induced cell proliferation and survival was blocked S1P; as low as 10 nM (Figure 4), suggesting the involvement of S1P in the presence of the TFR1 neutralizing antibody. Notably, the cell-surface receptor(s) in this process. TFR1 antibody showed little effect on cell proliferation and survival in the absence of SK1 overexpression, suggesting that its effects were specific for SK1-mediated signalling. Comparable SK1 regulates TFR1 expression via S1P2 findings were also observed following siRNA-mediated knock- To directly examine whether SK1 regulates TFR1 by acting through down of TFR1 (Figure 6c), further supporting the role of TFR1 in the S1P cell-surface receptor(s), we used isoform-selective S1P SK1-mediated mitogenic signalling. receptor inhibitors. The HEK293 cells used in this study express We next examined the role of TFR1 in SK1-induced neoplastic 34,35 three of the S1P receptor subtypes: S1P1, S1P2 and S1P3. Thus, transformation by performing focus formation assays using cells were induced to overexpress SK1 in the presence of NIH3T3 cells ectopically expressing SK1 in the presence of a

Oncogene (2014) 5559 – 5568 & 2014 Macmillan Publishers Limited TFR1 contributes to oncogenic signalling by SK1 DH Pham et al 5563

Figure 5. SK1 mediates increased TFR1 expression via S1P2.(a) SK1-inducible cells were treated with 10 mM VPC-23019 (antagonist of S1P1/ S1P3) or with 1 mM JTE-013 (antagonist of S1P2), and then cultured for 16 h either in the absence or in the presence of doxycycline to induce SK1 expression. TFR1 expression was then analysed in cell lysates by immunoblotting with anti-TFR1 antibodies and quantified relative to tubulin expression. As expected, neither VPC-23019 nor JTE-013 had any effect on SK1 activity. Data represent the mean±s.e.m. of three independent experiments. (b) NIH3T3 cells were treated with 1 mM S1P for 16 h in the presence of 1 mM JTE-013 or vehicle control (Ctl) and TFR1 expression was analysed in cell lysates by immunoblotting with anti-TFR1 antibodies. Tubulin expression was determined as a loading control. Data are representative of three independent experiments. (c) SK1-inducible cells were transfected with either control siRNA or S1P2 siRNA for 48 h and then subsequently induced or not induced with doxycycline for 16 h. TFR1 expression was then analysed in cell lysates by immunoblotting with anti-TFR1 antibodies and quantified relative to tubulin expression. SK1 activity was also assessed in cell lysates, whereas S1P2 expression was determined by immunoblotting with anti-S1P2 antibodies. Data represent the mean±s.e.m. of six independent experiments. *Po0.01 and **Po0.001. Dotted lines indicate where lanes from the same immunoblot have been spliced together to aid interpretation.

TFR1 neutralizing antibody. Consistent with previous studies, our between the expression of SK1 and that of TFR1 (Figure 8). results showed that SK1 overexpression in NIH3T3 cells induced Remarkably, this correlation was evident in studies encompassing the formation of numerous foci. The number of SK1-induced foci, a broad range of solid tumours (Figure 8a), as well as in large however, was significantly reduced in the presence of the TFR1 studies examining specific solid cancers (Figures 8b and c), and neutralizing antibody compared with isotype control antibody also in acute myeloid leukaemia (Figure 8c). Together, these (Figure 7b), suggesting that TFR1 has an essential role in SK1- findings support a pathophysiological role for SK1 in the induced neoplastic transformation. regulation of TFR1 expression in human cancer. As our findings indicate a role of S1P2 in SK1-induced TFR1 expression, we next examined the effect of antagonism of this S1P receptor on SK1-induced neoplastic transformation. Blockade of DISCUSSION S1P2 with JTE-013 resulted in a significant reduction in the number Significant steps towards understanding the molecular mechan- of SK1-induced foci (Figure 7c). Together, these findings suggest isms of cellular regulation by SK1 have been made in the past few that the S1P2/TFR1 pathway is essential in SK1-induced neoplastic years through the identification that SK1 activation and transloca- transformation. tion from the cytosol to the plasma membrane is crucial for oncogenesis mediated by this enzyme.14,15,41 Indeed, localization of SK1 to the plasma membrane, where its substrate, sphingosine, Elevated SK1 expression correlates with elevated TFR1 expression is enriched, appears critical for agonist-induced S1P generation in diverse human cancers and for the pro-proliferative, pro-survival and oncogenic effects of Our in vitro findings prompted us to examine the significance of SK1.15 The downstream mechanisms whereby SK1 leads to TFR1 regulation by SK1 in human cancer. Analysis of gene tumourigenesis, however, are still being elucidated. In attempts expression data from a diverse range of human cancer tissues to understand these downstream targets of SK1, we have revealed a small, but highly significant positive correlation examined genes that are differentially regulated by SK1 using a

& 2014 Macmillan Publishers Limited Oncogene (2014) 5559 – 5568 TFR1 contributes to oncogenic signalling by SK1 DH Pham et al 5564

Figure 6. Targeting TFR1 blocks SK1-induced cell proliferation and survival. (a) Cell proliferation and (b) apoptosis were assessed in SK1- inducible cells cultured in serum-free medium for 24 h either with SK1 induction by doxycycline, or not, and in the presence of either anti-TFR1 neutralizing or isotype control antibodies. Data represent the mean±s.e.m. of four independent experiments. (c)siRNA knockdown of TFR1 was performed for 48 h prior to induction of SK1 expression, or not, and cell proliferation was assessed in serum-free medium 24 h later. Data represent the mean±s.d. of three independent experiments. *Po0.05 and **Po0.005.

genome-wide DNA microarray approach. Rather than use a constitutive overexpression system, which has the potential to lead to non-physiologic effects, we chose a more subtle and controlled system to examine the immediate effects of moderate (close to physiological) inducible expression of SK1 on gene regulation. One of the genes identified in this study that was upregulated by SK1 expression was TFRC, the gene that encodes TFR1, which has been widely identified as an attractive anti-cancer tar- 20,31,32 get. We demonstrated that SK1 expression, via S1P2, upregulated TFR1 mRNA and protein levels, which manifested in enhanced TFR1 cell surface levels and Tf uptake. Although these Figure 7. TFR1 neutralizing antibodies or S1P2 antagonism attenu- findings were supported by positive correlations in the expression ates SK1-induced neoplastic transformation. NIH3T3 cells stably levels of SK1 and TFR1 in a broad range of human cancers expressing SK1 or vector control cells (a) were assessed for (Figure 8), the mechanism(s) mediating this upregulation of TFR1 neoplastic transformation by focus formation in the presence of by SK1 and S1P2 awaits further examination. In most tissues, TFR1 either anti-TFR1 neutralizing or control antibodies (b), or the S1P2 expression is also controlled by iron availability with fewer antagonist JTE-013 (c). Cells were cultured for 3 weeks with medium receptors expressed when iron is abundant and more when iron containing either 10 mg anti-TFR1 neutralizing control antibodies (b), is scarce.20 This regulation of TFR1 expression is post- 0.25 mM JTE-013 or DMSO vehicle control (c) replaced every 3–4 days. transcriptionally controlled by means of the well-characterized Foci were scored after fixing with methanol and staining with methyl violet. Data represent the mean±s.e.m. of three indepen- interaction between iron-regulatory and the iron- dent experiments. *Po0.05 and **Po0.001. responsive elements in the 3’ untranslated regions of iron- related TFR1 mRNAs.42 Interestingly, TFR1 expression is known to be also regulated by AP-1, a transcription factor known to be 43 48 activated by S1P, raising the possibility that SK1 could enhance activation of the S1P2 receptor, suggesting that SK1/S1P may TFR1 transcription via this mechanism. c-Myc has also been increase TFR1 transcription through the accumulation of hypoxia- reported to activate TFR1 transcription during tumourigenesis,27,44 inducible factor-1a. Clearly, further studies are required to and notably c-Myc has been shown to be downregulated at both investigate these possibilities. the mRNA and protein levels in mice that lack SK1.7 Interestingly, We have previously shown that activation of SK1, through however, MYC was among the 84 genes downregulated in phosphorylation and subsequent translocation from the cyto- response to SK1 overexpression in our studies (Figure 1b and plasm to the plasma membrane, is required for oncogenic Supplementary Table S1), suggesting that c-Myc is unlikely to have signalling of this enzyme.14,15 Notably, here we showed that the a role in TFR1 expression induced by SK1. TFR1 expression is also non-phosphorylatable SK1 (SK1S225A), which does not induce regulated by other mechanisms, including the hypoxia-inducible neoplastic cell transformation,15 does not upregulate TFR1. In factor-1a, which is typically activated under hypoxic conditions.45 contrast, the oncogenic plasma membrane-localized non- Interestingly, SK1 was recently shown to enhance hypoxia- phosphorylatable SK1 (SK1pm-S225A) did enhance TFR1 inducible factor-1a stability and function.46,47 Notably, this is expression. This is a clear demonstration that plasma membrane consistent with another study that demonstrated that S1P localization of SK1, which normally occurs after phosphorylation- increases hypoxia-inducible factor-1a protein stability through mediated activation, is necessary to regulate TFR1 expression.

Oncogene (2014) 5559 – 5568 & 2014 Macmillan Publishers Limited TFR1 contributes to oncogenic signalling by SK1 DH Pham et al 5565

Figure 8. Human cancer tissue expression analysis identifies a positive correlation between SK1 and TFR1 expression in diverse human cancers. Statistical analysis was conducted on large public microarray data sets based on samples derived from cancer patients. Linear models were fitted to log2 intensities corresponding to SPHK1 (SK1) and TFRC (TFR1) genes in each data set using lm function in R. Examples of this analysis from two of the larger data sets, Bittner GSE2109 (a) and Hatzis GSE25066 (b), are shown, whereas the results of statistical analysis of these, and two other large data sets, Wouters GSE14468 and Lee GSE13507, are provided in (c).

Our findings that SK1-induced TFR1 expression is mediated via SK1G82D suggests that this was not simply a result of the presence the cell-surface receptor S1P2 were initially somewhat surprising of unfolded SK1 protein. Interestingly, studies have indicated that as, in contrast to the positive effects of S1P1 and S1P3 receptors on various HSPs are overexpressed in a wide variety of human 55–57 cell survival and proliferation, S1P2 has been generally implicated tumours and can promote cancer cell proliferation and in decreased cell proliferation and survival in a number of cell survival.58,59 Furthermore, inhibition of HSPs by either genetic or 49 À / À types. Consistent with this, S1P2 mice are prone to develop chemotherapeutic approaches has been shown to induce B-cell lymphomas at elevated age (18–24 months),50 which apoptosis in various cancer cells and reduce tumour growth in suggests a tumour suppressor role for this receptor. In contrast, various xenograft mouse models.60–63 Thus, it is possible that however, but consistent with our findings, other recent evidence enhanced HSP expression may be a physiological response to supports a role for S1P2 in contributing to carcinogenesis. For elevated SK1 expression. example, S1P2 mRNA was found to be elevated in Wilms tumours In summary, we have identified TFR1 as a novel downstream and appears to have an oncogenic role in this neoplasm via target of SK1, which appears to be important in oncogenic induction of cyclooxygenase-2 expression and subsequent signalling by this enzyme. The observation that blocking TFR1 51 pro-proliferative, pro-survival effects. S1P2 has also been function prevented SK1-mediated cellular proliferation, survival shown to enhance Bcr-Abl1 protein phosphorylation and and neoplastic transformation suggests that TFR1 may represent a stability, which contributes to oncogenic signalling and imatinib downstream target for SK1-mediated tumourigenesis. Our results resistance in chronic myeloid leukaemia.52 Furthermore, recent provide new insight into the mechanisms of regulation by which studies have demonstrated that S1P2 can have a role in enhancing SK1/S1P exerts its oncogenic effects by enhancing TFR1 expres- tumour metastasis via suppression of BRMS1 (breast carcinoma sion via signalling through the S1P2 receptor. metastasis suppressor 1) expression.10 It should be noted, however, that, in contrast to this previous work, BRMS1 was one of the 104 genes upregulated by SK1 expression in our study MATERIALS AND METHODS (Supplementary Table S1). Generation of expression constructs and cell lines In addition to the identification of TFR1 as a downstream Constructs for tight doxycycline-inducible expression of wild-type human effector of SK1, our microarray studies also suggest that a SK1,54 non-phosphorylatable SK1 (SK1S225A) and non-phosphorylatable SK1 pm-S225A considerable number of genes were regulated by moderate mutant that constitutively localizes to plasma membrane (SK1 ), all containing C-terminal FLAG-epitope tags and AU-rich destabilizing overexpression of active SK1. In particular, a number of genes 17,64 encoding for heat shock proteins (HSPs) were elevated by elements, were described previously. The inducible construct for expression of the catalytically inactive human SK1 (SK1G82D)19 with a overexpression of wild-type SK1, but not the inactive SK1G82D. C-terminal FLAG-epitope tag was generated by subcloning into pcDNA5/ HSPs are known to function as chaperones to assist in protein 17 53 FRT/TO-SK1-AU following digestion with BamHI and NotI. folding, assembly, degradation and translocation. Although SK1 Flp-In T-Rex HEK293 cells (Invitrogen, Carlsbad, CA, USA) were cultured, 54 has been shown to be inherently unstable, our findings that transfected, selected, induced with doxycycline and harvested as these proteins were not upregulated by expression of the inactive described previously.17 S1P, VPC-23019 and JTE-013 were purchased

& 2014 Macmillan Publishers Limited Oncogene (2014) 5559 – 5568 TFR1 contributes to oncogenic signalling by SK1 DH Pham et al 5566 from Cayman Chemical (Ann Arbor, MI, USA). Cell proliferation was Quantitative RT-PCR (qPCR) determined by BrdU incorporation, and apoptosis was measured via DAPI QPCR analyses for TFR1 were performed with RNA preparations from the 15 staining as described previously. Anti-human TFR1 neutralizing antibody samples used in the microarray experiments or with RNA samples [RVS10] and isotype control antibodies (Abcam, Cambridge, UK) were used prepared from subsequent independent experiments. Briefly, cDNA was to examine the role of TFR1 on cell proliferation and apoptosis of these synthesized from 2 mg of total RNA by using an Omniscript RT (Qiagen) human cells. and oligo(dT). TFR1 qPCR analyses were performed with 2 Â Quantitect For generation of NIH3T3 cells expressing SK1, we constructed plasmids SYBR Green PCR Master mix reagent (Qiagen) with primers 50-ACCCATTCGT expressing C-terminally FLAG-tagged SK1 and also enhanced green GGTGATCAAT-30 (forward) and 50-CGTTTCCAACTGCCCTATGA-30 (reverse) fluorescent protein (eGFP) via an internal ribosome entry site (IRES). To (Geneworks, Adelaide, SA, Australia). Data were acquired using Rotor-Gene do this, the pcDNA3-IRES-eGFP vector was generated by subcloning the 6000 software 1.7 (Qiagen) and expression of mRNA was normalized 65 IRES-eGFP cassette from pMSCV-IRES-eGFP into pcDNA3 following against glyceraldehyde 3-phosphate dehydrogenase. digestion with EcoR1 and NotI. The cDNA encoding FLAG-tagged SK154 was then subcloned into this vector following digestion with EcoR1. NIH3T3 cells were cultured, transfected and pooled stable lines selected as siRNA knockdown described previously,66 except that following a week of selection cells were Cells were transfected with either siRNA duplex oligonucleotides targeting sorted for GFP expression using fluorescence-activated cell sorting to S1P (siGENOME SMARTpool siRNA M-003952-03; Dharmacon, Lafayette, increase the number of cells expressing SK1. Focus formation assays were 2 CO, USA), TFR1 (ON-TARGETplus SMARTpool siRNA L-003941-00; Dharma- performed as previously described.3 Purified rat anti-mouse TFR1 con) or respective non-targeting negative control pools using Lipofecta- neutralizing antibody and purified rat IgG isotype control antibody mine RNAiMAX reagent (Invitrogen) according to the manufacturer’s (Biolegend, San Diego, CA, USA) were used to examine the role of TFR1 protocol. Expression of S1P and TFR1 was analysed by immunoblot with on neoplastic growth of these murine cells. 2 anti-S1P2 (Cayman) and anti-TFR1 (Zymed) antibodies, respectively.

Immunoblot analysis Immunofluorescence Equal amounts of protein were separated by SDS–PAGE and transferred To examine TFR1 cell surface expression, cells were plated into poly-L-lysine- onto nitrocellulose membranes, which were then blocked overnight with coated coverslips and incubated for 24 h, and SK1 expression was induced phosphate-buffered saline (PBS) containing 5% (w/v) skimmed milk with doxycycline for a further 16 h. The cells were then fixed for 15 min with powder (SMP) and 0.1% (w/v) Triton X-100 at 4 1C. SK1, SK1G82D, SK1S225A 4% paraformaldehyde, blocked with 2% bovine serum albumin in PBS for and SK1pm-S225A were detected via their FLAG-epitope tags with the 10 min and then incubated with 2 ng/ml anti-TFR1 antibody [RVS10] monoclonal M2 anti-FLAG antibody (Sigma, St Louis, MO, USA). To detect (Abcam) in PBS containing 2% bovine serum albumin for 1 h at room TFR1, blots were blocked with PBS containing 5% skimmed milk powder temperature. To also examine total SK1 expression, the cells were and 0.2% Tween-20 and then detected using monoclonal anti-TFR1 permeabilized with 0.1% Triton X-100 in PBS for 15 min and then incubated antibody (Zymed Laboratories, South San Francisco, CA, USA) and HRP- with anti-SK1 antibodies.14 The immunocomplexes were detected with conjugated anti-mouse IgG (Thermo Scientific, Waltham, MA, USA) using Alexa-488-conjugated anti-mouse (for anti-TFR1) or Alexa-594-conjugated an enhanced chemiluminescence kit (ECL, GE Healthcare, Little Chalfont, anti-rabbit IgG (for anti-SK1) (Thermo Scientific). Coverslips were mounted UK). Blots were washed and then re-probed with monoclonal anti-a- with Dako fluorescent mounting medium. Membranes TFR1 and SK1 were tubulin antibody (Abcam) for loading controls. visualized via fluorescence microscopy performed using an Olympus BX-51 microscope. All images were acquired at room temperature at  60 magnification using analySIS Five Life Science Research software (Olympus, RNA preparation, microarray and data analysis Tokyo, Japan). Microarray studies were performed on cells that were either uninduced or induced to express SK1 proteins with 0.5 ng/ml doxycycline. RNA was Transferrin uptake assay extracted at 6 and 12 h after induction of SK1 expression by TRIzol extraction (Invitrogen) followed by RNAeasy column prep (Qiagen, Hilden, To examine Tf uptake, cells were plated onto poly-L-lysine-coated coverslips Germany) according to the manufacturer’s specifications. RNA samples and incubated for 24 h, and then SK1 expression was induced with were run on Bioanalyser (Agilent, Santa Clara, CA, USA) before committing doxycycline for 16 h. After induction, cells were serum starved for 1 h and to arrays to ensure high-quality RNA integrity. incubated at 4 1C for 5 min to block nonspecific internalization of Tf, and Compugen human 19 000-oligonucleotide library microarrays were then incubated with 50 ng/ml Alexa 568-conjugated Tf (Molecular Probes, performed at the Adelaide Microarray Facility. Briefly, cDNA probes were Eugene, OR, USA) for 30 min at 4 1C. Unbound Tf was then removed with prepared by incubating RNA with anchored polyT(V)N. Samples were mixed cold serum-free medium and then cells were either immediately fixed for with Superscript II and aminoallyl dNTP mix in Superscript II buffer 15 min with 4% paraformaldehyde or incubated in serum-free medium at containing dithiothreitol, incubated at 42 1C and then hydrolysed. The 37 1C for 10 min, washed with cold serum-free medium and then fixed in cDNAs were purified using QIAquick PCR purification kit (Qiagen) according paraformaldehyde. Coverslips were mounted with Dako fluorescent to the manufacturer’s protocol, dried, dissolved and incubated with Cy3 or mounting medium. Tf uptake was visualized via fluorescence microscopy Cy5 dyes (GE Healthcare). The labelled cDNAs were again purified using a as described above. QIAquick PCR purification kit, mixed with hybridization buffer, dried, dissolved in saline-sodium citrate containing formamide, denatured and then hybridized to arrays overnight. The arrays were washed, dried and SK1 activity scanned with a Genepix 4000B Scanner (Axon Instruments, Sunnyvale, CA, SK1 activity was measured as described previously,72 using conditions USA). Gene expression patterns in these cells were examined at various time selective for SK1. points following induction of SK1 or variants, and compared with zero time point just before induction. All arrays were carried out in dual-colour mode using two biological replicates for each time point, with dye-swap replicates also used for each comparison. CONFLICT OF INTEREST Image analysis, spot identification and intensity extraction were The authors declare no conflict of interest. performed using the Spot software package.67 Morphological opening background correction was applied to mean spot signal intensities.67 Analysis was performed in R with LIMMA package of BioConductor.68 Intensity-dependent global lowes normalization was performed and data in ACKNOWLEDGEMENTS individual arrays were scaled to each other.69 The linear models allowed This work was supported by the Fay Fuller Foundation, a University of Adelaide analysis of the data set as a whole, resulting in improved efficiency. Postgraduate Scholarship (to DHP), Senior Research Fellowships (508098 and Differentially expressed mRNAs were identified using a moderated t statistic 1042589) and Project Grant (626936) from the National Health and Medical Research approach and P-values were calculated from these t statistics.70 The P-values Council of Australia (to SMP). We thank Julia Zebol for technical assistance and were adjusted for multiple testing by controlling the false-discovery rate.71 Andrew Bert for assistance with Figure preparation.

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