Bottom up proteomics reveals novel differentiation proteins in neuroblastoma cells treated with 13-cis retinoic acid

Abstract

Neuroblastoma, a cancer of the sympathetic nervous system, is the second most common pediatric cancer. A unique feature of neuroblastoma is remission in some patients due to spontaneous differentiation of metastatic tumors. 13-cis retinoic acid (13-cis RA) is currently used in the clinic to treat neuroblastoma due to its differentiation inducing effects. In this study, we used shotgun proteomics to identify proteins affected by 13-cis RA treatment in neuroblastoma SK-N-SH cells. Our results showed that 13-cis RA reduced proteins involved in extracellular matrix synthesis and organization and increased proteins involved in cell adhesion and neurofilament formation. These changes indicate that 13-cis RA induces tumor cell differentiation by decreasing extracellular matrix rigidity and increasing neurite overgrowth. Differentially-affected proteins identified in this study may be novel biomarkers of drug efficacy in the treatment of neuroblastoma. SIGNIFICANCE: As neuroblastoma can spontaneously differentiate, determining which proteins are involved in differentiation can guide development of novel treatments. 13-cis retinoic acid is currently used in the clinic as a differentiation inducer. Here we have established a proteome map of SK-N-SH cells treated with 13-cis retinoic acid. Bioinformatic analysis revealed the involvement of development, differentiation, extracellular matrix assembly, collagen biosynthesis, and neurofilament bundle association. This proteome map provides information as to which proteins are important for differentiation and identifies networks that can be targeted by drugs to treat neuroblastoma [1].

Keywords: CRABP2; Collagen biosynthesis; Differentiation; Extracellular matrix; ICAM1; NEFM; Neuroblastoma; PLAT; Proteomics; Retinoic acid.

Fig. 1.

13-cis RA inhibits proliferation and induces differentiation of SK-N-SH cells. A. Quantity of viable cells. SK-N-SH cells were treated with 10 μM 13-cis RA or vehicle control (DMSO) for up to 9 days. Relative quantity of viable cells were measured using a CellTiter-Blue assay. Quantities of treated cells relative to untreated cells are shown at each time point. Arrows represent where neurites are. Values represent the mean ± standard deviations of quadruplets over three different experiments. ** p < 0.01. B, C. Quantity of live and dead cells. Live and dead untreated and 13-cis RA treated SK-N-SH cells were quantified using CellTox assay. Quantities of treated cells relative to untreated cells are shown at each time point. Values represent the mean ± standard deviations of triplicates. D-F. 13-cis RA induces neurites in SK-N-SH cells over time. D. Phase contrast microscopy of untreated and 13-cis RA SK-N-SH cells over time. 10X magnification, bar 50 μm. E. % Neurites over 0-9 days. F. Length of neurites over 0-9 days. N- and S-type labeled (zero and one day).

Fig. 2.

Differentially altered proteins of SK-N-SH cells treated with 13-cis RA for three days. A heat map of significantly altered proteins using 1% FDR S0=1 was generated in Perseus. Green indicates decrease and red indicates increase (Z-score log2). Euclidean hierarchal clustering designates clusters.

Fig. 3.

Effect of 13-cis RA on protein and mRNA in SK-N-SH cells. A. Representative western blot of CRABP2, NEFM, ICAM in 10 μM of 13-cis RA-treated and control SK-N-SH cell proteins after up to 9 days of treatment. PLAT at 3 days of RA treatment. B. Quantitative analysis of relative levels of CRABP2, NEFM, PLAT, ICAM1 of three replicates of control and 13-cis RA treated SK-N-SH cells. * p< 0.05. C. Effect of 10 μM 13-cis RA on expression of mRNA levels of differentially expressed gene targets in SK-N-SH cells. SK-N-SH cells were treated with 10 μM 13-cis RA for 3 days. mRNA levels of CRABP2, PLAT, ICAM1, and NEFM were measured via quantitative RT-PCR with GAPD being used as a reference transcript. * p< 0.05, ** p< 0.01. D. Representative western blot of CRABP2, NEFM, PLAT in 10 μM of 13-cis RA treated and control IMR-32 and NB1690 cell proteins at 3 days of treatment. IMR-32 in triplicates and NB1690 in doubles. E. Quantitative analysis of relative levels of CRABP2, NEFM, PLAT of three replicates of control and 13-cis RA treated IMR-32 and two replicates of NB1690 cells. * p < 0.05.

Fig. 4.

Effect of 13-cis RA on expression of mRNA levels of differentially expressed collagens in SK-N-SH cells. mRNA levels of COL1A1, COL3A1, COL5A1, and COL18A1 were measured via quantitative RT-PCR with GAPD being used as a reference transcript. * p< 0.05

Fig. 5.

GOrilla web-server top GO biological process enrichments. 130 unranked DAP were matched to the entire data set. Top Panel: GOrilla performed DAG of top highly enriched BP. Bottom panel: A table with GO terms, description, p-value, q-value, enrichment value, and protein count.

Fig. 6.

GOrilla web-server top GO UDAP and DDAP biological process enrichments. Processes enriched for 63 unranked UDAP matched to the entire data set are shown on the left and processes enriched for 67 unranked DDAP matched to entire set are shown on the right.

Fig. 7.

GOrilla web-server top GO UDAP and DDAP molecular function enrichments. Processes enriched for 63 unranked UDAP matched to the entire data set are shown on the left and processes enriched for 67 unranked DDAP matched to entire set are shown on the right.

Fig. 8.

GOrilla web-server top GO UDAP and DDAP cellular component enrichments. Processes enriched for 63 unranked UDAP matched to the entire data set are shown on the left and processes enriched for 67 unranked DDAP matched to entire set are shown on the right.