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. Author manuscript; available in PMC: 2023 Jun 1.
Published in final edited form as: J Pediatr Surg. 2022 Feb 14;57(6):1026–1032. doi: 10.1016/j.jpedsurg.2022.01.064

Serine-Threonine Kinase Receptor Associate Protein (STRAP) Confers an Aggressive Phenotype in Neuroblastoma via Regulation of Focal Adhesion Kinase (FAK)

Laura V Bownes a, Raoud Marayati a, Colin H Quinn a, Sara C Hutchins a, Jerry E Stewart a, Joshua C Anderson b, Christopher D Willey b, Pran K Datta c, Elizabeth A Beierle a
PMCID: PMC9119921  NIHMSID: NIHMS1780274  PMID: 35272839

Abstract

Background:

Serine-threonine kinase receptor associated protein (STRAP), a scaffolding protein, is upregulated in many solid tumors. As such, we hypothesized that STRAP may be overexpressed in neuroblastoma tumors and may play a role in neuroblastoma tumor progression.

Methods:

We examined two publicly available neuroblastoma patient databases, GSE49710 (n=498) and GSE49711 (n=498), to investigate STRAP expression in human specimens. SK-N-AS and SK-N-BE(2) human neuroblastoma cell lines were stably transfected with STRAP overexpression (OE) plasmid, and their resulting phenotype studied. PamChip® kinomic peptide microarray evaluated the effects of STRAP overexpression on kinase activation.

Results:

In human specimens, higher STRAP expression correlated with high-risk disease, unfavorable histology, and decreased overall neuroblastoma patient survival. STRAP OE in neuroblastoma cell lines led to increased proliferation, growth, supported a stem-like phenotype and activated downstream FAK targets. When FAK was targeted with the small molecule FAK inhibitor, PF-573,228, STRAP OE neuroblastoma cells had significantly decreased growth compared to control empty vector cells.

Conclusion:

Increased STRAP expression in neuroblastoma was associated with unfavorable tumor characteristics. STRAP OE resulted in increased kinomic activity of FAK. These findings suggest that the poorer outcomes in neuroblastoma tumors associated with STRAP overexpression may be secondary to FAK activation.

Keywords: serine-threonine kinase receptor-associated protein, STRAP, focal adhesion kinase, FAK, neuroblastoma

1. Introduction

Neuroblastoma, a tumor of neural crest origin, is the most common extra-cranial solid tumor in the pediatric population and is considered high risk based on several factors, including MYCN amplification, patient age, tumor differentiation, and imaging characteristics [1]. Current management for children with high-risk disease results in significant life-altering toxicities even if life is extended. Unfortunately, only about half of children with high-risk disease survive beyond 5 years [1, 2], making it imperative to continue to investigate potential therapeutic targets for this tumor.

Serine-threonine kinase receptor associate protein (STRAP) is a WD40 domain protein that functions as a scaffold to facilitate protein-to-protein interactions [3, 4]. STRAP is important in regulating numerous cellular processes, including cell cycle [5, 6], apoptosis [7], and signal transduction [8–13]. STRAP knockdown in colorectal cancer was associated with a decrease in cancer cell stemness [3]. In addition, STRAP knockout led to decreased anchorage independent growth and stemness, and an increase in differentiation markers in the hepatoblastoma cell line, HuH6 [11]. Because of these findings, and its involvement in several oncogenic pathways [5, 9–11, 14], STRAP has become target for investigation as a potential cancer therapeutic.

We have previously shown that CRISPR-Cas9 mediated STRAP knockout decreased the malignant phenotype of neuroblastoma in vitro and in vivo, providing evidence for STRAP’s role in maintenance of neuroblastoma [6]. In the current study, we sought to determine the effects of STRAP overexpression on human neuroblastoma cells to further investigate its role in neuroblastoma tumorigenicity and its potential as a therapeutic target.

2. Materials and methods

2.1. Patient database

R2 (http://r2.amc.nl), a publicly available genomic analysis platform, was utilized to interrogate two primary neuroblastoma patient databases available from GEO [https://www.ncbi.nlm.nih.gov/geo/], GSE49710 (n=498) and GSE49711 (n=498) containing microarray data and RNA-Seq to investigate STRAP expression and whether there is a correlation between STRAP and FAK in patient samples.

2.2. Cells and cell culture

The human neuroblastoma cell lines SK-N-AS (AS, CRL-2137, and MYCN non-amplified) and SK-N-BE(2) (BE, CRL-2271, and MYCN amplified) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained under standard culture conditions at 37 °C and 5% CO2. AS cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 30–2601, ATCC) containing 10% fetal bovine serum (FBS, Hyclone, Suwanee, GA, USA), 4 mM L-glutamine (Thermo Fisher Scientific Inc., Waltham, MA, USA), 1 μM non-essential amino acids, and 1 μg/mL penicillin/streptomycin (Gibco, Carlsbad, CA, USA) (AS media). BE cells were maintained in a 1:1 mixture of minimum Eagle medium and Ham F-12 medium (30–2004, ATCC) with 10% FBS (Hyclone), 1 μM non-essential amino acids, 2 mM l-glutamine (Thermo Fisher Scientific Inc.), and 1/μg/mL penicillin/streptomycin (Gibco) (BE media). Stable lines of STRAP knockout (KO) were established as previously described in AS and BE cells [6]. Cells transfected with either empty vector (EV) or STRAP over expression (OE) plasmids were maintained in AS or BE media with the addition of G418 (600 μg/mL EMD Millipore, Burlington, MA) for antibiotic plasmid selection. Cell lines were verified within the last 12 months using short tandem repeat analysis (University of Alabama at Birmingham (UAB) Genomics Core, Birmingham, AL, USA) and tested for and deemed free of mycoplasma infection.

2.3. Reagents and antibodies

Trypan blue stain was obtained from Life Technologies Corporation (Grand Island, NY, USA). Primary antibodies used for Western blotting included: polyclonal rabbit anti-STRAP (18277–1-AP) from Proteintech (Rosemont, IL, USA) and monoclonal mouse anti-β-actin (A1978) and anti-FLAG (F1804) from Sigma Aldrich (St. Louis, MO). The anti-phospho-FAK (Tyr397, D20B1, #8556), anti-FAK [(D5O7U) XP® (#71433)], and anti-vinculin (E1E9V, #13901) rabbit monoclonal antibodies were from Cell Signaling (Danvers, MA). Antibodies were used according to manufacturers’ suggestions.

2.4. STRAP overexpression

The STRAP overexpression vector, pcDNA3-STRAP-Flag, was a generous gift from Dr. Pran Datta and was generated as described [13]. The plasmid was sequenced for verification (UAB Genomics Core). Empty vector (pcDNA3-Flag, EV) served as a control. Transfection was accomplished using FuGENE® HD Transfection Reagent (Promega, Madison, WI) per manufacturer’s protocol. Briefly, cells were plated 24 hours prior to transfection. The appropriate plasmid was incubated for 15 minutes at room temperature in OptiMEM™ media (Thermo Fisher Scientific, Inc.) with FuGENE® HD Transfection Reagent (Promega) in a 3:1 ratio of transfection reagent to plasmid DNA (7 μg DNA per 1 × 105 cells) and added to the cells while swirling the flask. Cells were transfected 72 hours prior to use in experiments, and G418 was added for antibiotic selection. Cells were labeled and referred to as SK-N-AS empty vector (AS EV), SK-N-AS STRAP overexpression (AS STRAP OE), SK-N-BE(2) empty vector (BE EV) or SK-N-BE(2) STRAP overexpression (BE STRAP OE).

2.5. Immunoblotting

Cell lysis, gel electrophoresis and transfer were performed as previously described [6]. To confirm the expected size of target proteins, Precision Plus Protein Kaleidoscope Standards (161–0375, Bio-Rad, Hercules, CA) molecular weight markers were used. Luminata Classico or Luminata Crescendo (EMD Millipore) substrates were used to visualize immunoblots by enhanced chemiluminescence (ECL) of horseradish peroxidase (HPR)-conjugated secondary antibodies. Vinculin or β-actin served as internal protein loading controls.

2.6. Proliferation

Proliferation was examined using the CellTiter 96® Aqueous One Solution Cell Proliferation assay (Promega). Cells (5 × 103) were plated onto 96-well plates. After 24 hours, CellTiter 96® dye (10 μL) was added to each well and the absorbance was measured at 490 nm using a microplate reader (Epoch Microplate Spectrophotometer, BioTek Instruments, Winooski, VT). Proliferation experiments were completed with at least three biologic replicates, and data reported as fold change ± standard error of the mean (SEM).

2.7. Cell growth

Cells (5 × 104) were plated in 12-well plates. To measure cell growth over time, cells were lifted, and live cells counted after staining with trypan blue at 1, 2, and 3 days. Experiments were completed with at least three biologic replicates, and data reported as cell count ± SEM.

2.8. Cell cycle

AS EV and AS STRAP OE cells (5 × 105) were plated in 6-well plates and maintained in AS and G418 media with decreased FBS (4%). After 24 hours, cells were washed with phosphate buffered saline (PBS) and fixed with 100% ethanol at 4 °C for at least 30 min. After a second wash with PBS, cells were stained with propidium iodide (Invitrogen, Waltham, MA), 0.1% TritonX (Active Motif, Carlsbad, CA), and RNAse A (0.1 mg/mL, Qiagen, Germantown, MD) and cell cycle data were obtained using the FACSCalibur™ Flow Cytometer (BD Biosciences) and analyzed using the FlowJo software (FlowJo, LLC, Ashland, OR, USA). Experiments were completed with at least three biologic replicates, and data reported as percent of cells per phase ± SEM.

2.9. Extreme limiting dilution analysis

AS EV or AS STRAP OE cells were plated in conditioned media in non-adherent 96-well plates with a decreasing number of cells in each row of 12 wells (5000 to 10 cells). After one week, each well was assessed for tumorsphere formation. The number of wells containing spheres was counted and analyzed using an extreme limiting dilution analysis (ELDA) software [15].

2.10. CD133 expression

AS EV or AS STRAP OE cells (1 × 106) were labeled with allophycocyanin (APC) conjugated human CD133/1 (AC133)-APC (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s instructions. Unlabeled cells served as negative controls. The percent of cells positive for APC was determined via flow cytometry using the FACSCalibur™ Flow Cytometer (BD Biosciences) and analyzed using the FlowJo software (FlowJo, LLC). Experiments were completed with at least three biologic replicates, and data reported as fold change in percent CD133 positive cells ± SEM.

2.11. Real-time PCR (qPCR)

iScript cDNA Synthesis kit (Bio-Rad) was used to synthesize cDNA with 1 μg of RNA used in a 20 μL reaction. The reverse transcription products were stored at −20 °C until further use. SsoAdvanced™ SYBR® Green Supermix (Bio-Rad) was utilized according to the manufacturer’s protocol for quantitative real-time PCR (qPCR). Primers specific for Octamer-binding transcription factor 4 (Oct4), homeobox protein Nanog, sex determining region Y-box 2 (Sox2) and β-actin were utilized (Thermo Fisher Scientific Inc.). qPCR was performed with 10 ng cDNA in 20 μL reaction volume as previously described [6]. β-actin was utilized as an internal control. Gene expression was calculated using the ΔΔCT method [16] and reported as mean fold change in mRNA abundance ± SEM.

2.12. Kinome assay

Kinomic profiling of AS EV and AS STRAP OE cells was completed as previously described [17]. Briefly, 2–15 μg of protein lysates (M-Per lysis buffer, Pierce Scientific, Waltham, MA) containing 1:100 Halt’s protease and phosphatase inhibitors (78420, 78415, respectively, Pierce) were examined on the PamStation® 12 platform (PamGene, ‘s-Hertogenbosch, Netherlands) and results analyzed within the UAB Kinome Core (https://www.uab.edu/medicine/radonc/research/labs-core-facilities/kinome-core). After protein quantification (BCA protein determination, Pierce Scientific, Thermo Fisher Scientific, Inc.), total soluble protein lysates were loaded onto the appropriate PamChip® [PTK (tyrosine kinome, article code 86312) or STK (serine/threonine kinome, article code 87102)] in kinase buffer [17, 18]. This platform utilizes a high throughput peptide microarray system analyzing 144 individual tyrosine phosphorylatable peptides, or 144 serine and threonine phosphorylatable peptides imprinted and immobilized in a 3D format to assess kinomic activity. As such, molecular profiles of samples were measured by kinase activity against 288 phosphorylatable peptides (phosphopeptides) containing a distinct 12–15 amino acid sequence (comprising over 560 phosphorylatable tyrosine, serine, or threonine residues in total). Fluorescein (FITC) conjugated phosphospecific antibodies (PamGene) were used for visualization during and after lysates were pumped through the array. Capture of peptide phosphorylation signal was via a computer-controlled CCD camera. Kinomic profiling was analyzed using software including Evolve2 (PamGene) for array processing and image capture, and BioNavigator v6.0 (PamGene) for raw data transformation into kinetic (initial velocity) and steady state (postwash) values. Peptide spot intensity (brightness) was captured across multiple exposure times (10–200 ms) and the slope was taken, multiplied by 100, log2 transformed, and used as signal. Resulting heat map is shown in Supplemental Figure 1. The peptide lists within the clusters were then analyzed for upstream kinase prediction (UpKin PamApp; PTK v6.0, STK v6.0) using scoring from Kinexus Phosphonet (phosphonet.ca) to generate kinase statistics and specificities as previously described [17, 18].

2.13. Statistical analysis

All experiments were performed with a minimum of three biologic replicates. Data were reported as mean ± SEM of separate experiments. Student’s t test or analysis of variance (ANOVA) were used where appropriate. Statistical significance was defined as p ≤ 0.05.

3. Results

3.1. STRAP is associated with poor neuroblastoma prognosis

High STRAP expression in colorectal cancer [3] was associated with worse patient prognosis leading us to examine STRAP expression in neuroblastoma patient samples. Using the R2 online genetic database application (http://r2.amc.nl), we examined GSE49711, a database consisting of 498 primary neuroblastoma patient samples investigated with RNA-Seq and found that increased STRAP expression was significantly associated with unfavorable histology (Figure 1 A), high-risk disease (Figure 1 B), disease progression (Figure 1 C), worse event free (Figure 1 D) and overall (Figure 1 E) survival, and worse disease specific survival (Figure 1 F). All these data correlated increased STRAP with poor neuroblastoma prognosis.

Figure 1: STRAP expression is associated with poor patient prognosis.

Figure 1:

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Using R2, a publicly available genomic analysis platform, we examined the neuroblastoma patient database, GSE49711 (n=498) consisting of RNA-Seq data and investigated STRAP expression in human patient tumors. Increased STRAP mRNA was significantly associated with (A) unfavorable tumor histology, (B) high-risk disease, (C) disease progression, (D) decreased event free survival, (E) decreased overall survival, and (F) increased risk of death from disease.

3.2. STRAP overexpression resulted in increased proliferation

STRAP has been shown to promote tumorigenicity in several malignancies [5, 19]. To study the effects of STRAP overexpression in neuroblastoma, we developed cell lines with stable transfection of STRAP. Immunoblotting showed increased STRAP protein in the STRAP OE cells compared to wild type and EV cells (Figure 2 A, D). The presence of FLAG protein confirmed the successful transfection of EV and STRAP OE plasmids into AS and BE neuroblastoma cells (Figure 2 A, D). STRAP OE significantly increased AS and BE cell proliferation (Figure 2 B, E) when compared to control EV cells. STRAP OE in AS and BE cell lines resulted in significantly increased cell growth over time (Figure 2 C, F).

Figure 2: Overexpression (OE) of STRAP increased proliferation and growth in SK-N-AS and SK-N-BE(2) cells.

Figure 2:

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SK-N-AS and SK-N-BE(2) neuroblastoma cells were stably transfected with pcDNA3-Flag (empty vector, EV) or pcDNA3-STRAP-Flag (STRAP overexpression, OE) plasmids. Immunoblotting confirmed the expression of Flag protein, a marker of successful transfection, and overexpression of STRAP protein in STRAP OE cells compared to control EV or wild type (WT) cells (A, D). (B, E) Proliferation was compared between EV and STRAP OE cells using CellTiter 96® assay. Cells (5 × 103) were plated onto 96-well plates. After 24 hours, CellTiter 96® dye (10 μL) was added to each well and the absorbance was measured at 490 nm using a microplate reader. At 24 hours, AS STRAP OE (B) and BE STRAP OE cells (E) demonstrated significantly increased proliferation compared to control EV cells. (C, F) EV and STRAP OE cells (5 × 104) were plated in 12-well plates. To measure cell growth over time, cells were lifted, stained with trypan blue and live cells counted at 24, 48, and 72 hours. AS STRAP OE (C) and BE STRAP OE (F) cells had significantly increased cell growth over time compared to respective control EV cells. Data reported as mean fold change ± SEM. Experiments were repeated with at least three biologic replicates. *p≤0.05, **p≤0.01, ***p≤0.001.

3.3. STRAP overexpression led to cell cycle progression and stemness

Since STRAP OE increased cell growth more in the AS (46%, Figure 2 C) than the BE (29%, Figure 2 F) cell lines, we chose to focus on the AS cell line for the remainder of the experiments. We first investigated the effect of STRAP overexpression on cell cycle progression. Compared to control AS EV cells, AS STRAP OE cells had significantly less percentage of cells in G1 phase (28 ± 2% vs 33 ± 2%, AS STRAP OE vs AS EV, p≤0.05) and higher percentage of cells in G2 phase (18 ± 2% vs 23 ± 3%, AS STRAP OE vs AS EV, p≤0.05), indicating increased progression through the cell cycle (Figure 3 A). Representative histograms from a single experiment are demonstrated in Figure 3 B, and aggregate cell cycle results are presented in tabular form in Figure 3 B.

Figure 3: STRAP OE resulted in progression of the cell cycle and increased stemness.

Figure 3:

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(A) AS EV and AS STRAP OE cells were plated and serum starved for 24 hours to synchronize the cells. Cell cycle was analyzed using flow cytometry. AS STRAP OE cells had significantly decreased percentage of cells in G1 phase and increased percentage in G2 phase compared to AS EV cells, indicating increased progression through the cell cycle. (B) Representative histograms from a single cell cycle experiment are shown for AS EV and AS STRAP OE cells. (C) Cumulative cell cycle data are presented in tabular form and represent data from three biologic replicates. (D) Tumorsphere formation was examined using an extreme limiting dilution assay. AS EV and AS STRAP OE cells were plated at increasing cell concentrations per row ranging from 10 to 5000 cells per well in a 96 well plate. After 1 week, tumorspheres were counted. AS STRAP OE cells had an increased ability to form tumorspheres at lower cell numbers, indicating increased cell stemness. (E) AS EV and AS STRAP OE cells were stained with CD133 antibody, and percent CD133 expression was analyzed by flow cytometry. AS STRAP OE cells had significantly increased percentage of CD133 positive cells compared to EV cells. (F) Abundance of mRNA for stemness markers, Oct4, Nanog, and Sox2, was evaluated using real-time PCR. AS STRAP OE cells had significantly increased abundance of mRNA of stemness markers compared to AS EV cells. Data reported as mean ± SEM. Experiments were repeated with at least three biologic replicates. *p≤0.05, **p≤0.01, ***p≤0.001.

STRAP supports cancer stem cells in other malignancies [3, 11], prompting us to examine whether STRAP affected neuroblastoma stemness. We utilized extreme limiting dilution assay (ELDA), CD133 expression, and real-time PCR for stemness markers, Oct4, Nanog [19], and Sox2 [20] to evaluate stemness. We found that AS STRAP OE cells had increased ability to form tumorspheres at lower cell concentrations (p≤0.001, Figure 3 C). The percentage of cells expressing CD133 on their surface was significantly increased in AS STRAP OE (Figure 3 D), as was the abundance of mRNA for the stemness markers Oct4, Nanog and Sox2 (Figure 3 E) compared to EV cells. These data indicated that STRAP supports the stem cell phenotype in neuroblastoma.

3.4. STRAP overexpression is associated with increased FAK activation

We utilized a kinome assay to investigate a potential mechanism responsible for the changes in the phenotype seen with STRAP overexpression. Kinome studies showed that downstream targets of FAK had increased phosphorylation in the AS STRAP OE cells compared to AS EV cells (Figure 4 A, Supplemental Figure 1), suggesting that FAK was activated with STRAP OE. We had previously designed a CRISPR-Cas9 STRAP knockout (KO) SK-N-AS cell line [6]. We used immunoblotting and examined the phosphorylation of FAK in STRAP KO cells at the tyrosine 397 (Y397) site, the autophosphorylation site for FAK activation. We found that STRAP KO resulted in decreased FAK expression, and therefore phosphorylation, compared to AS WT cells (Figure 4 B), further indicating that STRAP may play a role in regulating FAK expression. We employed a small molecule inhibitor of FAK, PF-573,228 (PF), and found that the AS STRAP OE cells, those found to have increased FAK activation in the kinome studies, had significantly decreased proliferation (46.7 ± 1.1% vs 67.7 ± 1.2% at 15 μM, p≤0.001, AS STRAP OE vs EV, Figure 4 C) after FAK inhibition compared to EV cells (no FAK activation on kinome studies). Similar results were seen with viability (51.2 ± 1.1% vs 67.8 ± 1.7% at 15 μM, p≤0.001, AS STRAP OE vs EV, Figure 4 D). Using R2 online tool (http://r2.amc.nl), we queried the GSE49710 database, consisting of RNA-Seq and microarray data, and found a significant correlation between STRAP and FAK expression in neuroblastoma (R=0.662, p≤0.001, Supplemental Figure 2). These findings suggest that STRAP overexpression may be promoting a malignant phenotype in neuroblastoma through FAK activation.

Figure 4: STRAP is associated with FAK activation.

Figure 4:

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(A) AS EV and AS STRAP OE cells were examined using PamChip® kinomic peptide microarray, and the resulting phosphorylation curves shown (far right column). AS STRAP OE cells had an increase in the phosphorylation of FAK downstream targets compared to AS EV cells, suggesting an increase in FAK activity in the OE cells. (B) Immunoblotting was used to detect target proteins. CRISPR-Cas9 was used to generate STRAP knockout (KO) cells. The AS STRAP KO cells had decreased FAK phosphorylation compared to the control AS wildtype (WT) cells. (C, D) AS EV and AS STRAP OE cells were treated with increasing concentrations (0, 7.5, 15 μM) of the small molecule FAK inhibitor, PF-573,228 (PF), for 24 hours. STRAP OE cells had decreased (C) proliferation and (D) viability compared to EV. Respective IC50 was 19.4 ± 3.4 μM vs 11.8 ± 1.4μM (EV vs OE, p=0.03) and LD50 18.6 ± 3.1 μM vs 13.0 ± 1.0 μM (EV vs OE, p=0.05). Data reported as mean fold change ± SEM. Experiments were repeated with at least three biologic replicates. **p≤0.01, ***p≤0.001.

4. Discussion

Our lab previously demonstrated the tumor promoting effects of STRAP in neuroblastoma by inhibiting STRAP with genetic knockdown and KO [6]. The current study serves to corroborate and validate the importance of STRAP in neuroblastoma by focusing on the overexpression of STRAP and the resulting effects on tumor phenotype. STRAP is overexpressed and has tumor promoting effects in numerous malignancies, including colorectal [5], lung [5], osteosarcoma [21], and breast [22] cancer. Jin et. al investigated STRAP in colorectal cancer patient samples using the TCGA dataset and found that high STRAP mRNA expression was significantly associated with worse survival [3]. In osteosarcoma, Pruksakorn et. al found immunohistochemical staining and immunoblotting for STRAP protein in patient tumor specimens was increased in tumor tissue compared to healing bone [21]. We examined the association between STRAP expression and tumor histology, disease risk, and survival in patient neuroblastoma samples using the R2 database application and found high STRAP mRNA expression was significantly associated with poor prognosis. These findings support the continued investigation of STRAP’s contribution to neuroblastoma tumorigenicity.

FAK, a 124 kDa non-receptor protein tyrosine kinase, promotes tumor progression and metastasis in breast [23, 24], colon cancer [23], and neuroblastoma [25, 26]. FAK is activated upon phosphorylation of the Y397 autophosphorylation site [27]. Investigators have shown that scaffolding proteins function to activate FAK [27]. In breast cancer, Kiely et al. showed that the association of the scaffolding protein, RACK1, with FAK is required for Y397 phosphorylation in response to insulin-like growth factor I [27], and RACK1 knockdown resulted in decreased FAK phosphorylation [27]. Since STRAP is a scaffolding protein, and in cancer, FAK is regulated by other scaffolding proteins [28–30], we investigated the potential relation between STRAP and FAK in neuroblastoma after a kinome assay revealed that FAK activation was increased in STRAP OE cells. This assay demonstrated an increase in the phosphorylation of FAK targets, CDK2, PAXI, and RET. When STRAP was knocked out with CRISPR-Cas9, FAK phosphorylation was decreased without a change in protein expression. These data implied a relation between STRAP and FAK. These findings were further strengthened when data from the GSE49710 database revealed a correlation between STRAP and FAK in patient samples (Supplemental Figure 2). These findings provide evidence that STRAP may exert its effects on neuroblastoma through the FAK pathway.

Stem cell-like cancer cells have garnered interest as potential therapeutic targets. These subsets of cells are thought to be responsible for neuroblastoma disease relapse and therapeutic resistance [28]. Other investigators have studied the role of STRAP in promoting cancer cell stemness and its association with various oncogenic pathways [3, 11]. Using qPCR, Wang et. al found that the loss of STRAP in hepatoblastoma resulted in a reduction in the abundance of mRNA of stemness markers [11]. In the present study, our findings mirrored those of Wang in that we demonstrated that STRAP OE led to an increase in stemness as demonstrated by increased tumorsphere formation, increased cell surface expression of CD133, and increased abundance of mRNA of stemness markers.

In the studies presented, we utilized long-term passage cell lines which pose some limitations. Long-term, or conventional cancer cell lines, have adapted over time through multiple passages in contrived culture conditions to effectively grow in vitro, often resulting in a lack of predictive value when testing therapeutics prior to advancing to clinical trials [29, 30]. Patient-derived xenografts (PDXs) better retain the histologic and genetic features of the original patient tumor [38–40] and have become an exciting avenue for preclinical therapeutic studies since they overcome some of the limitations seen with conventional cancer cell lines grown in standard 2D culture [29–31]. There are, however, some limitations to PDXs [32]. Human PDX tumors must be maintained in immunosuppressed mice, negating the ability to evaluate the contribution of the immune system to the therapeutic in question. Further, the tumor microenvironment (TME) is largely murine, again, making the study of the effects of the therapeutics on the TME difficult. Finally, pediatric tumors are notorious in their heterogeneity [33], harboring various subclones of cells with different genotypes, so establishing a PDX based upon one clone may not completely recapitulate the human condition or provide an adequate model for testing therapeutics.

5. Conclusion

In this study, we investigated the overexpression of STRAP in neuroblastoma and showed that this scaffolding protein supports the malignant phenotype, likely through its effects FAK. In addition, we found that increased STRAP expression was associated with poor patient prognosis. Based on the results of the current study, STRAP warrants further examination as a potential therapeutic target and potential prognostic marker for neuroblastoma.

Supplementary Material

1

Supplemental Figure 1: Kinomic profiling AS EV and AS STRAP OE cells. PamChip® kinomic peptide microarray was performed. The heat map represents the kinomic signature data. The peptides are colored by log difference from mean per cell type per peptide. Clustering is unsupervised using a geometric means-distance method. The peptides are clustered by row, and samples are clustered by column. FAK downstream targets are denoted by the arrow. Red color indicates increased phosphorylation or activity, and blue represents decreased phosphorylation.

2

Supplemental Figure 2: STRAP and FAK correlation in patient tumors. Using R2, we examined the neuroblastoma patient database, GSE49710 (n=498), consisting of RNA-Seq and microarray data, and found a significant correlation (R = 0.662, p≤0.001) between STRAP and FAK expression in patient tumors.

Acknowledgements

The authors wish to thank Pran Datta’s laboratory for their collaboration and for providing plasmids. We would like to thank Anita Hjemeland’s laboratory for their assistance with the qPCR. Furthermore, we would like to thank Dr. Christopher Willey’s laboratory and the UAB Kinome Core for assistance with kinomic studies as well as Sagar Hanumanthu and UAB Comprehensive Flow Cytometry Core for his assistance with flow cytometry.

Funding:

This work was partially funded by institutional grants from the National Institutes of Health including T32 CA229102 (LV Bownes, R Marayati), 5T32GM008361: Medical Scientist Training Program (CH Quinn), P30 AR048311 and P30 AI27667 to the Flow Cytometry Core at the University of Alabama at Birmingham. Further funding was provided by the Veterans Affairs Merit Review Award (1I01BX005143), UAB U54 Pilot Project (CA 118948) (PK Datta). The funding sources had no role in study design, analysis, or interpretation.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental Figure 1: Kinomic profiling AS EV and AS STRAP OE cells. PamChip® kinomic peptide microarray was performed. The heat map represents the kinomic signature data. The peptides are colored by log difference from mean per cell type per peptide. Clustering is unsupervised using a geometric means-distance method. The peptides are clustered by row, and samples are clustered by column. FAK downstream targets are denoted by the arrow. Red color indicates increased phosphorylation or activity, and blue represents decreased phosphorylation.

NIHMS1780274-supplement-1.tiff (627.1KB, tiff)
2

Supplemental Figure 2: STRAP and FAK correlation in patient tumors. Using R2, we examined the neuroblastoma patient database, GSE49710 (n=498), consisting of RNA-Seq and microarray data, and found a significant correlation (R = 0.662, p≤0.001) between STRAP and FAK expression in patient tumors.

NIHMS1780274-supplement-2.tiff (124.2KB, tiff)

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