Induction of serine hydroxymethyltransferase 2 promotes tumorigenesis and metastasis in neuroblastoma

High-risk neuroblastoma (NB) remains an extremely difficult subgroup to cure and is associated with MYCN amplification. Serine hydroxymethyltransferase 2 (SHMT2) regulates serine metabolism in a myc-dependent manner; it is upregulated in several cancers and is associated with tumor aggressiveness. Akt-2, an important regulator of MYCN via the PI3K/Akt pathway, induces metastatic potential in NB. The association between SHMT2 and PI3K/Akt in hepatocyte regeneration has been well established but its mechanistic interaction in cancer has yet to be clearly elucidated. Herein, we evaluated the exact role of SHMT2 on the PI3K/Akt pathway, in addition to NB tumorigenesis and metastatic potential in vitro. SHMT2 gene expression and overall survival (OS) were assessed. Two human NB cell lines were examined. SHMT2 silencing and overexpression were performed. The downstream effects were analyzed with immunoblotting, RT-qPCR and functional assays were performed. We found SHMT2 gene expression is associated with decreased OS and MYCN amplification. SHMT2 protein and mRNA expression are increased in MYCN-amplified cells. SHMT2 expression has a direct interaction with Akt-2 and MYCN. Induction of SHMT2 increased cellular proliferation, colony formation and cellular migration and SHMT2 expression was increased in metastatic NB cells. We conclude that SHMT2 regulates N-Myc via phosphorylation of Akt-2 and plays an important role in NB tumorigenesis by contributing to cell growth, migration, colony formation and metastasis in vitro.


INTRODUCTION
Neuroblastoma (NB) is a pediatric tumor derived from neural crest cells. It is the most common pediatric, extracranial solid tumor. The high-risk group of NB remains one of the most difficult subgroups of NB to treat with resistance to therapeutic regimens and high disease relapse. Children with high-risk NB tumors have a long-term survival rate of less than 50%. Highrisk disease is classified based on imaging stage, patient age, histology, presence of diploidy, 11q aberrations or MYCN amplification [1]. Surgical resection is a viable treatment option for patients with low-risk NB. However, high-risk NBs require multi-modality treatment including chemotherapy, radiation, stem-cell transplant and recently, immunotherapy. Despite these treatment advances, highrisk NB has a low disease-free survival and increased rate of relapse or remission with many tumors developing chemotherapy and radiation resistance [2].
Altered metabolism is a key feature of cancer cells and allows cells to survive in stressful conditions. Glucose and glutamine metabolism are commonly reprogrammed pathways with mutations involving the MYC family. MYCN encodes for the oncoprotein N-Myc. MYCN amplification and overexpression in NB is associated with increased proliferation and enhanced malignant potential, while knockdown of MYCN has been shown to result in tumor growth arrest and apoptosis in NB [3]. Increased MYC gene expression affects several aspects of metabolism such as glycolysis, mitochondrial function and serine metabolism [4].
Oncotarget 33 www.oncotarget.com Serine plays an important role in one-carbon metabolism as a one-carbon donor to the folate cycle [5]. Altered serine metabolism has been linked to several cancer types and has been shown to be affected in a myc-dependent manner by mitochondrial serine hydroxymethyltransferase 2 (SHMT2) [4]. SHMT2 converts serine to glycine in the mitochondria and is induced by HIF1α and MYC in hypoxia to promote cell survival. Upregulation of SHMT2 is found in many types of cancers, including lymphoma, glioma, cholangiocarcinoma and breast cancer [6,7]. In both glioma and breast cancer, SHMT2 is associated with tumor aggressiveness and is an independent predictor of prognosis [8,9].
Although little is known about the exact role of SHMT2 in NB, a recent report found an association between SHMT2, N-Myc expression and high-risk NB [10]. A commonly deregulated pathway involving N-Myc in high-risk NB is the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which regulates angiogenesis by stabilizing N-myc [11]. In addition, Akt-2 is an important regulator of NB metastatic potential. While the exact molecular interaction between SHMT2 and PI3K/Akt in malignancy has not yet been elucidated, another recent study demonstrated that increased expression of SHMT2 in hepatocytes led to physiologic Akt activation via PI3K [12]. Given the association between SHMT2 and PI3K/ Akt in hepatocytes, and the importance of the PI3K/Akt pathway in NB angiogenesis and metastasis, SHMT2 may play a critical role in NB tumorigenesis and metastasis via the PI3K/Akt pathway. In the present study, we sought to determine the exact role of SHMT2 in the PI3K/Akt pathway and the effect of SHMT2 on NB tumorigenesis and metastasis in vitro.

SHMT2 expression is associated with MYCN amplification and decreased overall survival
Given the role of SHMT2 as an independent predictor of prognosis in breast cancer and glioma, we sought to determine the potential relationship between SHMT2 gene expression and overall survival in NB patients [8,9]. Using the R2 genomics analysis application to evaluate the Kocak database, consisting of 649 NB patients, the relationship between SHMT2 expression and overall survival (OS) in NB was examined. The optimal cut-off value for high versus low expression is calculated in the R2 database using a KaplanScan, a logrank test that identifies the most significant expression value for survival analysis. The optimal gene expression cut-off value for high versus low SHMT2 expression in the Kocak database was 19713.8 (Supplementary Figure 1). As shown in Figure 1A, increased SHMT2 expression was associated with a significant decrease in OS in NB patients, with an OS of 39% in patients with high SHMT2 expression compared to an OS of 80% in patients with low SHMT2 expression. In addition, the relationship between MYCN amplification and SHMT2 expression in NB patients was also analyzed. We found an increased SHMT2 expression in patients with MYCN amplification compared to those without MYCN amplification ( Figure 1B). SHMT2 mRNA and protein expression were evaluated in four NB cell lines: two MYCN-amplified cell lines, BE(2)-C and SK-N-DZ, and two non-MYCN-amplified cell lines, SK-N-AS and SK-N-SH. SHMT2 mRNA expression was assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Both MYCN-amplified cell lines, BE(2)-C and SK-N-DZ, demonstrated a 2.4 to 23.4-fold increase in SHMT2 mRNA expression compared to the non-MYCN-amplified cell lines, SK-N-AS and SK-N-SH ( Figure 1C). SHMT2 protein expression was evaluated using immunoblotting and quantified with densitometry analysis. SHMT2 protein expression was increased in both of the MYCN-amplified cell lines, BE(2)-C and SK-N-DZ, and decreased in the non-MYCN-amplified cell lines, SK-N-AS and SK-N-SH ( Figure 1D). Densitometry analysis demonstrated a 1.8 to 4.5-fold increase in SHMT2 protein expression in MYCNamplified cells compared to the non-MYCN-amplified cells ( Figure 1E).

SHMT2 regulates N-Myc via decreased activation of Akt-2
In order to assess the potential role of SHMT2 in NB in vitro, stable cell lines with SHMT2 silencing and SHMT2 overexpression were created. The MYCNamplified NB cell line, BE(2)-C, and the non-MYCNamplified cell line, SK-N-AS, were used. Both cell lines were transfected with shRNA targeting SHMT2 (shSHMT2) and non-target control shRNA (shCTL), as well as a SHMT2 overexpression plasmid, pCo-SHMT2, to create stably transfected cell lines. Antibiotic selection was performed for two weeks after transfection and successful transfection was confirmed using RT-qPCR (Figure 2A, 2B) and immunoblotting ( Figure 2C, 2D).
The effects of SHMT2 silencing and overexpression on AKT-2 and MYCN mRNA expression were evaluated using RT-qPCR. In the non-MYCN-amplified cell line, SK-N-AS, SHMT2 silencing decreased AKT-2 mRNA expression by 1.2-fold, while SHMT2 overexpression increased AKT-2 mRNA expression by 1.6-fold ( Figure  2A). In the MYCN-amplified cell line BE(2)-C, SHMT2 silencing decreased AKT-2 mRNA expression by 2.6fold, while SHMT2 overexpression increased AKT-2 mRNA expression by 1.3-fold ( Figure 2B). SHMT2 silencing increased MYCN mRNA expression by 1. Immunoblotting was performed to evaluate the effects of SHMT2 silencing and overexpression on protein expression in both BE(2)-C and SK-N-AS cells. SHMT2 silencing decreased phosphorylated Akt-2 (pAkt-2) expression and SHMT2 overexpression increased pAkt-2 expression in both cell lines ( Figure 2C and 2D; top panel). Densitometry analysis showed a 1.3-fold decrease in SK-N-AS pAkt-2 protein expression and 1.6-fold decrease in BE(2)-C pAkt-2 protein expression with SHMT2 silencing ( Figure  2D, bottom panel). While SHMT2 silencing decreased N-Myc protein expression, the opposite effect was seen on MYCN mRNA expression in the BE(2)-C cell line.
Given the incongruent findings between the effects of SHMT2 silencing and overexpression on mRNA and protein expression in the MYCN-amplified cell line, BE(2)-C, further analysis was performed. As described in the subsequent findings, SHMT2 overexpression was noted to increase cellular proliferation and SHMT2 silencing decreased cellular proliferation, suggesting shCTL, shSHMT2 and pCo-SHMT2 cells may have been at different cell-cycle phases at 48 hours when mRNA and protein samples were initially collected. In order to account for potential variations in cell cycle, BE(2)-C shCTL, shSHMT2 and pCo-SHMT2 cells were plated on a 6-well plate at 0.25 × 10 6 cells/well. Protein and mRNA were collected at exactly 24 hours after plating and RT-qPCR and immunoblotting were performed. Findings are summarized in Figure 3. At 24 hours, SHMT2 silencing decreased AKT-2 mRNA expression by 2.0-fold and . SHMT2 silencing did not impact Akt-2 protein expression. SHMT2 overexpression increased pAkt-2 expression, but had no effect on Akt-2 expression. Densitometry analysis, reported as a ratio of each protein band density relative to the density of each ß-actin control band (protein density: ß-actin density), confirmed immunoblotting findings in BE(2)-C cells (bottom panel). SHMT2 silencing resulted in pAkt-2 protein expression 1.6 times lower than control. SHMT2 overexpression increased pAkt-2 protein expression by 1.3-fold. SHMT2 silencing and overexpression had no effect on Akt-2 protein expression. SHMT2 silencing decreased N-Myc protein expression by 1.3-fold and SHMT2 overexpression decreased N-Myc protein expression by 1.8-fold.
Oncotarget 36 www.oncotarget.com nearly eliminated MYCN mRNA expression, decreasing it by 500-fold ( Figure 3A). SHMT2 overexpression increased AKT-2 and MYCN mRNA expression by 1.5 and 1.2-fold, respectively. Similar findings were seen with immunoblotting. As seen in Figure 3B, SHMT2 silencing completely inhibited N-Myc protein expression at 24-hours. SHMT2 overexpression increased N-Myc protein expression by 2.3-fold at 24 hours. Taken together, these findings suggest SHMT2 silencing inhibits N-Myc expression via decreased activation of Akt-2.
To further elucidate the mechanistic relationship between SHMT2 and MYCN-amplification, studies were performed on two additional NB cell lines, SK-N-DZ (MYCN-amplified) and SK-N-SH (non-MYCN amplified). SHMT2 silencing and overexpression were performed in the same manner as previously described. RT-qPCR and immunoblotting were performed to assess the effects of SHMT2 silencing and overexpression on AKT-2 and MYCN mRNA expression as well as pAkt-2, N-Myc and c-Myc protein expression. SHMT2 silencing resulted in a 1.2-fold decrease in AKT-2 and 1.1-fold decrease in MYCN mRNA expression and SHMT2 overexpression resulted in a 1.2-fold increase in AKT-2 and a 1.3-fold increase in MYCN mRNA expression in the SK-N-DZ cell line (Supplementary Figure 2A). Whereas SHMT2 silencing did not affect AKT-2 and MYCN mRNA expression and SHMT2 overexpression decreased both AKT-2 and MYCN mRNA expression by 1.4-fold and 1.2-fold, respectively, in the SK-N-SH cell line (Supplementary Figure 2D).
Next, the effects of SHMT2 silencing and overexpression on protein expression were examined. SHMT2 silencing decreased N-Myc protein expression by 1.4-fold and pAkt-2 protein expression by 1.1-fold, whereas SHMT2 overexpression increased N-Myc protein The protein expression of pGSK-3α/β, a downstream target of Akt-2, was evaluated to confirm successful inhibition of Akt-2. As shown in Supplementary Figure 3C, the non-MYCN amplified cell line, SK-N-AS, was more susceptible to Akt-2 inhibition, with a 1.4 and 2.9-fold decrease in pGSK-3α/β expression in both SHMT2 silencing and overexpression cells, respectively. Interestingly, treatment with CCT129830 in the BE(2)-C shSHMT2 cells resulted in a 12.5-fold decrease in pGSK-3α/β expression compared to a 2.9fold decrease with SHMT2 silencing alone, suggesting SHMT2 silencing increased the effectiveness of Akt-2 inhibition (Supplementary Figure 3F). In addition, SHMT2 overexpression resulted in a 1.6-fold increase in pGSK-3α/β expression in untreated cells and a 1.9fold increase in pGSK-3α/β expression in cells treated with CCT129830, suggesting SHMT2 overexpression cells were able to compensate for Akt-2 inhibition (Supplementary Figure 3F). Taken together, the effects of Akt-2 inhibition on both MYCN-amplified and non-MYCN amplified NB cells further supports the finding that SHMT2 affects both c-Myc and N-Myc protein expression via Akt-2.

SHMT2 promotes cellular proliferation, colony formation and migration in vitro
The impact of SHMT2 silencing and overexpression on cellular proliferation was assessed using CCK-8 assays. Cellular proliferation of shCTL, shSHMT2 and pCo-SHMT2 cells in both the SK-N-AS and BE(2)-C cell lines was assessed at 24, 48 and 72 hours. As seen in Figure 4A and 4B, SHMT2 silencing significantly impaired cellular proliferation by 1-fold in both cell lines at 72 hours (p < 0.05), while SHMT2 overexpression enhanced cellular proliferation by 1-fold in both cell lines at 72 hours (p < 0.05).
To evaluate the potential role of c-Myc expression on NB tumor function, the effects of SHMT2 silencing and overexpression on colony formation in the non-MYCN amplified cell line, SK-N-SH, were examined. Cells were plated at 1000 cells/well on a 6-well plate in triplicate and colony formation was assessed at 7 days. There were fewer colonies seen with SHMT2 silencing and larger colonies seen with SHMT2 overexpression. However, there was no significant difference in colony formation between shCTL and shSHMT2 cells, but there was a significant decrease in colony formation in pCo-SHMT2 cells compared to shCTL (shCTL 280.7 ± 36 colonies vs 231.8 ± 10 colonies, p = 0.001) (Supplementary Figure 4A). However, this is likely due to pCo-SHMT2 cells overgrowing the plate and forming larger colonies compared to shCTL and shSHMT2 cells, as seen in the representative images. (Supplementary Figure 4B). These findings suggest SHMT2 silencing and overexpression affected colony formation via N-Myc or c-Myc and that there was no functional effect on the non MYCN-amplified cell line, SK-N-SH, which lacks c-Myc.

Metastatic NB cells demonstrate increased SHMT2 mRNA and protein expression
To further evaluate the role of SHMT2 on NB metastatic potential, the LM2 cell line was evaluated. The LM2 cell line, which was established in our laboratory previously [13], is a highly aggressive NB cell line with propensity to metastasize that was obtained from BE(2)-C cell liver metastases after splenic injection for two cycles. In order to assess for SHMT2 mRNA and protein expression, RT-qPCR and immunoblotting were performed. Figure 6A demonstrates a 2.3-fold increase in SHMT2 mRNA expression in the metastatic cell line, LM2, compared to parental BE(2)-C cells. Immunoblotting also demonstrated a 2.7-fold increase in SHMT2 protein expression in the LM2 cells compared to parental BE(2)-C cells ( Figure 6B and 6C). This result indicated that SHMT2 may affect the tumor cell spread in vitro. Therefore, the R2 genomics analysis platform was used to analyze SHMT2 expression in NB patients based on stage. Figure  6D demonstrates increasing SHMT2 expression with increasing stage (excluding 4S), with the highest SHMT2 expression seen in patients with stage 4, metastatic disease. Together these results indicate SHMT2 plays an important role in NB metastasis in vitro and that SHMT2 expression is increased in patients with metastatic NB.
In summary, SHMT2 plays an important role in NB tumorigenesis. Based on the findings above, SHMT2 increases N-Myc mRNA and protein expression via phosphorylation/activation of Akt-2, leading to increased cellular proliferation, colony formation, migration and metastasis (Figure 7).

DISCUSSION
NB is the most common pediatric, extracranial, solid tumor, accounting for approximately 15% of pediatric cancer deaths [1]. Poor prognostic factors in children with NB include: age greater than 18 months at the time of diagnosis, unfavorable histology, increased vascularization and MYCN gene amplification. A common deregulated metabolism pathway is the MYC gain-offunction pathway. Increased MYC gene expression affects several aspects of metabolism, including: glycolysis, mitochondrial function and serine metabolism. Serine metabolism is affected in a MYC-dependent manner by the mitochondrial enzyme SHMT2 [4]. Upregulation of SHMT2 is found in several cancers and is associated with increased tumor aggressiveness. SHMT2 expression is an independent predictor of prognosis in glioma, breast and lung cancer [8,9,14,15]. The PI3K/Akt pathway regulates angiogenesis by stabilizing N-myc and is a commonly deregulated pathway in high-risk NB [11]. In addition, increased expression of SHMT2 in hepatocytes has been shown to lead to physiologic Akt activation via PI3K [12]. Given the common pathway, we hypothesized that SHMT2 plays a critical role in NB tumorigenesis and metastasis via the PI3K/Akt pathway.
In the present study, we found that increased SHMT2 gene expression was associated with MYCNamplification and a decrease in overall survival, in NB patients. These findings are similar to previous studies which have demonstrated an upregulation of SHMT2 in several cancers such as lymphoma, glioma, cholangiocarcinoma, breast cancer, gastric cancer, lung adenocarcinoma and colorectal cancer [6,7,14,16]. In addition, we found that SHMT2 mRNA and protein expression were increased in the aggressive MYCNamplified cell lines BE(2)-C and SK-N-DZ, compared to the non-MYCN-amplified cell lines, SK-N-AS and SK-N-SH. These findings are similar to a previous study which found that SHMT2 was upregulated in MYCN-amplified NB cells [10]. Several studies have demonstrated an association between SHMT2 expression and tumor aggressiveness, suggesting that SHMT2 is an independent predictor of prognosis in glioma, breast and lung cancer [8,9,14,15]. In addition, many related genes involved in serine metabolism have been associated with advanced stage disease and decreased overall survival in NB [17].
We have previously demonstrated that activation of Akt-2 specifically, is associated with NB tumor development, progression and metastasis [18]. A physiologic link between serine/glycine metabolism, SHMT2 and Akt has been shown in hepatocytes. However, to the best of our knowledge, this is not only the first investigation into the relationship between PI3K/Akt and SHMT2 in NB, but in all cancer studies [12]. In our study, we found that SHMT2 silencing decreased AKT-2 mRNA expression and pAkt-2 activity. In addition, SHMT2 silencing completely inhibited N-Myc protein expression at 24 hours and decreased MYCN mRNA expression by 500-fold in BE(2)-C cells, www.oncotarget.com suggesting SHMT2 regulates N-Myc via decreased activation of Akt-2. Our studies were performed on two additional cell lines, the non-MYCN amplified cell line, SK-N-SH, and the MYCN-amplified cell line, SK-N-DZ. Similar to BE(2)-C cells, the MYCN-amplified SK-N-DZ cells demonstrated decreased pAkt-2 and N-Myc protein expression with SHMT2 silencing and increased pAkt-2 and N-Myc protein expression with SHMT2 overexpression. However, there was no association between SHMT2 overexpression or silencing on pAkt-2 and N-Myc expression in the SK-N-SH cell line. Notably, the non-MYCN amplified cell line, SK-N-SH, which is known to have a single copy of MYCN [19], demonstrated N-Myc protein expression, but revealed minimal to no c-Myc protein expression. These findings are similar to previous studies which demonstrated a key role in the relationship between SHMT2 and MYC on cellular proliferation in other cancers, such as glioma [7,10]. Reprogrammed metabolism allows for pathologic cell survival. Some metabolic pathways are less important in solitary tumors but essential for metastasis [4]. In order to metastasize, cells must endure nutrient-poor and hypoxic conditions. SHMT2 has been shown to be upregulated in hypoxia and plays a key role in proliferation in areas of ischemia in other cancers, such as gliomas and breast cancer [6, 10,15]. The ability to adapt to hypoxic environments suggests NB cells with increased SHMT2 expression may be more readily able to metastasize given their ability to endure hypoxic environments. As expected, in the present study we found that SHMT2 silencing significantly impaired cellular proliferation, colony formation and cellular migration, while SHMT2 overexpression enhanced cellular proliferation, colony formation and cellular migration in vitro.
Although RT-qPCR and immunoblotting suggest the importance of SHMT2 in MYCN mRNA and N-Myc protein expression, SHMT2 silencing and overexpression impacted cellular function in both the MYCN-amplified cell line BE(2)-C and the non-MYCN-amplified cell line, SK-N-AS. This may be due to induction of c-Myc in the SK-N-AS cell line. In addition, the effects of cellular proliferation and colony formation with SHMT2 overexpression were more pronounced in the MYCNamplified cell line, BE(2)-C, likely due to an increased MYCN level at baseline compared to the non-MYCNamplified cell line, SK-N-AS. However, as previously discussed, SHMT2 silencing decreased colony formation and SHMT2 overexpression increased colony formation and c-Myc protein expression in the non-MYCN amplified cell line, SK-N-AS. Whereas, SHMT2 silencing and overexpression in the non-MYCN amplified cell line, SK-N-SH, which does not express c-Myc, did not significantly affect colony formation. Taken together, these results suggest SHMT2 affects cellular function via N-Myc or c-Myc protein expression, via pAkt-2, and that neither SHMT2 silencing or overexpression impacted cellular function in the non-MYCN amplified cell line, SK-N-SH. Moreover, while the effects of SHMT2 silencing on cellular proliferation, migration and colony formation may be related to decreased MYCN/N-Myc expression, different mechanisms such as induction of differentiation, may be related to SHMT2 and will be evaluated in future studies.
Given the effects of SHMT2 overexpression on cellular behavior, particularly colony formation and cellular migration, we sought to evaluate the role of SHMT2 in NB metastasis. We found increased SHMT2 mRNA and protein expression in the metastatic, highly aggressive cell line, LM2, compared to parental control. In addition, analysis of SHMT2 expression with relation to NB disease stage revealed increasing SHMT2 gene expression with increasing stage, with the highest SHMT2 expression in metastatic, stage 4 NB. These findings are concordant with a previous study which found increased SHMT2 expression in metastatic breast cancer tissue compared to primary tumor tissue [15]. In conclusion, SHMT2 expression is associated with poor overall survival and high-risk, MYCN-amplified NB. At the cellular level, SHMT2 silencing regulates N-Myc via decreased activation of Akt-2 and plays a key role in NB cellular proliferation, colony formation and cellular migration in vitro. Further studies are needed to evaluate the role of SHMT2 in NB in vivo. Elucidating the underlying pathophysiology and mechanisms of SHMT2 on reprogrammed metabolism is key to furthering our understanding of high-risk NB behavior and developing potential therapeutic targets in order to exploit the dependence of high-risk NB on altered serine metabolism. In addition, given SHMT2 is upregulated in several cancers, elucidating the mechanism underlying SHMT2 in NB will aid the development of potential therapies with broad applications.

Cell culture
The human NB cell lines BE(2)-C, SK-N-DZ, SK-N-SH and SK-N-AS were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in RPMI 1640 with 10% Fetal Bovine Serum (FBS) at 37°C in a humidified atmosphere consisting of 5% CO 2 and 95% air.

Plasmids, shRNA and transfections
Human NB cells were transfected with plasmids, shRNA, shCTL and pCo-SHMT2, using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions. Antibiotic selection was performed on stablytransfected shSHMT2 and shCTL cells with puromycin (10 mg/mL) at 0.5 µg/mL for SK-N-AS cells and 2.5 µg/ mL for BE(2)-C cells for two weeks. Antibiotic selection for stably-transfected pCo-SHMT2 cells was performed with blasticidin (10 mg/mL) at 16.0 µg/mL for SK-N-AS cells and 8.0 µg/mL for BE(2)-C cells. The antibiotic selection doses for the SK-N-AS cells and BE(2)-C cells were determined by performing antibiotic kill curves with increasing doses of puromycin and blasticidin.

SHMT2 gene expression analysis
The R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl, http://r2platform.com) and the Kocak -649 -custom -ag44kcwolf public NB database were used to create Kaplan-Meier survival curves in order to assess the relationship between SHMT2 expression and survival in NB patients. The R2 genomics database determines high versus low expression by performing a KaplanScan, a log rank test on increasing gene expression and defines a "cut-off" value of high versus low expression based on the value with the most significant expression value for performing survival analysis. Additional box and whisker plots of the relationship between SHMT2 expression and MYCN amplification, as well as NB disease stage, were also created.

Akt-2 inhibition
The Akt-2 inhibitor, CCT129830, was used to assess the effects of Akt-2 inhibition on downstream targets such as N-Myc, c-Myc and pGSK-3α/β [21]. BE(2)-C and SK-N-AS cells were plated at 0.25 × 10 6 and 0.5 × 10 6 cells/ well, respectively, in a 6-well plate. Cells were permitted to attach overnight and were then treated with control media or 10 μM of CCT129830, based on IC 50 dosing calculated in a previous study [22]. Lysate was collected after 24 hours of treatment and immunoblotting was performed.

Cell viability assay
To perform the cell viability assays, cells were seeded onto 96-well plates at equivalent densities (BE(2)-C cells at 500 cells/per well and SK-N-AS cells at 1000 cells/well) in RPMI culture medium with 10% FBS. Cell viability was measured using Cell Counting Kit-8 (CCK-8) colorimetric assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) at 24, 48, 72 and 96 hours after seeding.

Clonogenic assay
Cells were plated at clonal density (BE(2)-C cells at 500 cells/well and SK-N-SH/SK-N-AS cells at 1000 cells/well) on a 6-well plate, in triplicate. Cells were permitted to attach and grow for either a 7 or 14-day period. Colonies were stained with 0.05% crystal violet, photographed and counted.

Wound healing assay
Cellular migration was assessed using a wound healing assay. Cells were plated onto 6-well plate using