Quantitative Proteomic Analysis of 2D and 3D Cultured Colorectal Cancer Cells: Profiling of Tankyrase Inhibitor XAV939-Induced Proteome

Recently there has been a growing interest in three-dimensional (3D) cell culture systems for drug discovery and development. These 3D culture systems better represent the in vivo cellular environment compared to two-dimensional (2D) cell culture, thereby providing more physiologically reliable information on drug screening and testing. Here we present the quantitative profiling of a drug-induced proteome in 2D- and 3D-cultured colorectal cancer SW480 cells using 2D nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS) integrated with isobaric tags for relative and absolute quantitation (iTRAQ). We identified a total of 4854 shared proteins between 2D- and 3D-cultured SW480 cells and 136/247 differentially expressed proteins (up/down-regulated in 3D compared to 2D). These up/down-regulated proteins were mainly involved in energy metabolism, cell growth, and cell-cell interactions. We also investigated the XAV939 (tankyrase inhibitor)-induced proteome to reveal factors involved in the 3D culture-selective growth inhibitory effect of XAV939 on SW480 cells. We identified novel XAV939-induced proteins, including gelsolin (a possible tumor suppressor) and lactate dehydrogenase A (a key enzyme of glycolysis), which were differentially expressed between 2D- and 3D-cultured SW480 cells. These results provide a promising informative protein dataset to determine the effect of XAV939 on the expression levels of proteins involved in SW480 cell growth.


Results and Discussion
Quantitative Proteomic Analysis of 2D-and 3D-cultured SW480 Cells Treated with XAV939.
We investigated the effects of XAV939 on the cell growth of 2D-and 3D-cultured human CRC SW480 cells. APC-mutant SW480 cells, which are constitutively active in the Wnt/β-catenin signaling pathway, were grown in 2D and 3D cultures and treated with various concentrations of XAV939 ranging from 0 to 20 μM. XAV939 did not show any noticeable anti-proliferation effects on 2D-cultured SW480 cells, whereas it suppressed the growth of the 3D-cultured cells in a dose-dependent manner (Fig. 1A). Compared to the untreated 3D-cultured cells, SW480 cells grown in 3D culture showed 48 ± 12% cell survival in the presence of 20 μM XAV939. However, SW480 cells were completely resistant to the same concentration of XAV939 in 2D culture.
The XAV939-mediated inhibition of tankyrase induces the stabilization of AXIN2 (axin-2, a negative regulator of Wnt/β-catenin signaling) and the reduction of CTNNB1 (β-catenin) protein levels. Therefore, we next examined the expression levels of AXIN2 and CTNNB1 in 2D-and 3D-cultured SW480 cells after treatment with XAV939. XAV939 treatment led to the up-regulation of AXIN2 and the down-regulation of CTNNB1 in both 2D-and 3D-cultured SW480 cells (Fig. 1B). The stabilization of AXIN2 by XAV939 treatment was more effective in 2D than 3D-cultured SW480 cells, but CTNNB1 was similarly down-regulated in 2D-and 3D-cultured SW480 cells. These results indicate that XAV939 effectively impairs the Wnt/β-catenin signaling in both 2D-and 3D-cultured SW480 cells.
The quantitative proteomic analysis was carried out using iTRAQ labeling coupled with online 2D-nLC-MS/ MS to gain insight into the global changes between 2D-and 3D-cultured SW480 cells and to reveal factors involved in the 3D culture-specific growth inhibitory effects of XAV939 on SW480 cells (Fig. 2). We compared the proteomes of 2D-and 3D-cultured SW480 cells treated with either 20 μM of XAV939 or DMSO as a control. Proteins extracted from these four samples were tryptically digested and labeled with different isobaric tags: 113 tag for 2D-cultured cells; 114 tag for 2D-cultured cells treated with 20 μM of XAV939; 115 tag for 3D-cultured SCIeNTIfIC RePoRts | (2018) 8:13255 | DOI: 10.1038/s41598-018-31564-6 cells; and 116 tag for 3D-cultured cells treated with 20 μM of XAV939. The labeled peptides were equally pooled, followed by online 2D-nLC-MS/MS analysis (see Materials and Methods). The quantitative analysis relied on measuring the relative intensities of iTRAQ reporter ions with different masses (m/z 113, 114, 115, and 116) produced during the fragmentation of precursor ions in MS/MS experiments. We calculated iTRAQ 115/113 ratios for the comparison of 2D-and 3D-cultured cells and iTRAQ 116/115 versus 114/113 ratios for the comparison of XAV939-induced proteomic changes between 2D-and 3D-cultured cells. A total of 4854 proteins were quantified with confidence corresponding to peptide and protein FDR < 0.01 and with at least two unique peptides per protein (Table S1 in Supplementary Information). Both quantitative datasets for iTRAQ ratios 115/113 and 116/115 versus 114/113 followed a normal distribution ( Fig. S1 in Supplementary Information).

Comparison of Proteomic Differences between 2D-and 3D-Cultured SW480 Cells.
To compare the proteomes of 2D-and 3D-cultured SW480 cells, statistically significant differences in protein abundance were determined based on the fold change with a cut-off of 1.6 and a t-test p-value threshold of 0.05 (red dots in Fig. 3A). We identified 136 up-regulated proteins and 247 down-regulated proteins in 3D compared to 2D culture (Tables S2 and S3 in Supplementary Information). To validate the global proteomic data, the expression levels of several selected proteins were confirmed using western blot analysis. LDHA (lactate dehydrogenase A), PGK1 (phosphoglycerate kinase 1), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) were highly expressed in 3D than 2D-cultured cells, whereas the expression levels of NPM1 (nucleophosmin), NCL (nucleolin), and DBN1 (drebrin) were lower in 3D-cultured cells (Fig. 3B). These results are consistent with previous iTRAQ-based quantitative analyses. Figure 3C shows representative MS/MS spectra for tryptic peptides VIISAPSADAPMFVMGVNHEK (m/z 941.18 with 943 charge) and TLVLSNLSYSATEETLQEVFEK (m/z 1037.23 with 103 charge), which are derived from GAPDH and NCL, respectively.
We also analyzed the GO and KEGG pathway enrichment to characterize functionally the significantly changed proteins between 2D-and 3D-cultured SW480 cells. The differentially expressed proteins were classified according to their biological processes and molecular functions (Fig. 4). The 12 GO terms of biological processes including glycolysis, metabolic processes, and amino acid biosynthesis were enriched in 3D-up-regulated proteins. The 14 GO terms were enriched by proteins down-regulated in 3D culture, and these proteins were involved in DNA/RNA processing and cellular component organization. For the category of molecular functions, up-regulated proteins were related to enzyme activities such as catalytic activity, transferase, and oxidoreductase activity, while DNA/ RNA binding proteins were enriched in proteins down-regulated in 3D culture. The results from the KEGG pathway analysis are shown in Table 1. The 3D-up-regulated pathways included metabolic pathways and glycolysis/ gluconeogenesis, whereas oxidative phosphorylation was down-regulated in 3D culture. These results indicate that 3D-cultured cells tend to depend on glycolysis rather than mitochondrial oxidative respiration for energy production, which possibly results from oxygen or nutrient gradients that occur in 3D microenvironments. In agreement with these results, up-regulation of the HIF-1 (hypoxia-inducible factor-1) signaling pathway was also observed in 3D culture. In addition to metabolic changes, the gap junction pathway including cytoskeletal proteins (α-and β-tubulin) and MAPK (mitogen-activated protein kinase) signaling proteins (MEK1 and ERK1), which contributes to intercellular communications, was down-regulated in 3D culture. The down-regulation of proteins involved in DNA/RNA processing and cellular component biogenesis/organization is correlated with the relatively slower growth of 3D-cultured cells in comparison to those in 2D-cultured cells.
Our results are mostly in agreement with those reported by Yue et al. in that proteins involved in the metabolic pathway, amino acid biosynthesis/metabolism, HIF-1 signaling pathway, and DNA replication were significantly different between 2D and 3D cultures of CRC HT29 cells. However, the changes in abundance of proteins related to energy metabolism were not consistent with this previous report 16 . Although the glycolysis/gluconeogenesis pathway was highly enriched in the 3D-up-regulated proteins in both results, proteins associated with oxidative phosphorylation were down-regulated in this study (ATP5D, ATP5J, COX17, COX6B1, NDUFA8, NDUFAB1, UQCRB, and UQCRH), but up-regulated in the results of Yue et al. (COX6C, COX7A2, NDUFA13, NDUFA8, PPA2, SDHA, SDHB, SDHC, UQCR10, UQCRB, UQCRC1, etc.). He et al. also reported global proteomic comparisons of 2D-and 3D-cultured cells using glioma U25l cells, which also showed the enrichment of glycolysis-associated terms in 3D-up-regulated proteins 15 . This proteomic study identified seven oxidative phosphorylation-associated proteins that were down-regulated in 3D culture (AHCY, ATP5A1, ATP5F1, COX4I1, UQCRC1, UQCRC2, and PPA1). Taken together, these results suggest that metabolic reprogramming to increase glycolysis commonly occurred in 3D culture, but the regulation of mitochondrial oxidative phosphorylation (either activation or inactivation) could be influenced by the types of cells, 3D cell culture systems, spheroid sizes, or other factors.
Of these 24 proteins, four proteins were previously proposed as TNKS protein interactors (GSK3A/B and TAX1BP1) 35,36 or a Wnt/β-catenin signaling-target protein (HPGD) in 2D culture-based assays 37,38 , while the  other proteins are novel XAV939-induced proteins. We are particularly interested in GSN and LDHA because XAV939-induced changes in their expression levels occurred only in 3D-cultured cells. We then verified the iTRAQ data of GSN and LDHA using western blot analysis (Fig. 6A,B). Representative MS/MS spectra for tryptic peptides of GSN and LDHA are shown in Fig. 6C. GSN (gelsolin) is an actin-binding protein that regulates cytoskeleton dynamics and various cellular signalings, such as differentiation, proliferation, invasion, and apoptosis 39 . GSN has been shown to have dual roles as both a tumor promoter and suppressor, depending on the type of cancer cells [40][41][42] . Upon treatment of XAV939, we observed the up-regulation of GSN in 3D, but not in 2D culture (Fig. 6A, 2D, 1; 2D_XAV939, 0.99-fold; 3D, 1.66-fold; 3D_XAV939, 2.15-fold). This is interesting in the context of previous results showing that the overexpression of GSN inhibited the proliferation and invasion of renal cancer cells and promoted apoptosis in cardiac myocyte through the GSN/HIF-1α/DNase І pathway 42,43 . A possible explanation is that the up-regulation of GSN led to the growth inhibition of CRC cells in 3D culture upon treatment with XAV939. Also, the up-regulation of GSN was also observed in 3D compared with 2D culture, which was proposed in a previous report to be the result of the hypoxic conditions 44 .
LDHA belongs to the lactate dehydrogenase family and converts pyruvate to lactate in the final step of the glycolytic pathway 45 . The enhanced glycolytic activity has long been known as a hallmark of cancer because tumor cells rely on aerobic glycolysis for the production of energy, which is called the Warburg effect 45,46 . Therefore, the inhibition of LDHA suppresses tumor progression by alteration of energy metabolism and induction of oxidative stress 45 . We observed the up-regulation of glycolysis ( Fig. 4A and Table 1) and LDHA levels (Fig. 3B) in 3D compared to 2D culture. These results indicate that the 3D culture system used in this study more closely represented the metabolic states of tumors in vivo than that of 2D culture. Interestingly, LDHA was significantly down-regulated in 3D-cultured cells in the presence of XAV939, whereas 2D-cultured cells showed no significant differences in LDHA expression (Fig. 6A, 2D, 1; 2D_XAV939, 1.03-fold; 3D, 3.05-fold; 3D_XAV939, 2.32-fold). These results suggest that XAV939 treatment induced the reduction of glycolysis via the down-regulation of LDHA in 3D culture, resulting in suppression of cell growth. Our results are consistent with a previous study in which a Wnt inhibition-mediated metabolic change to reduce glycolysis suppressed the growth of CRC cells in a xenograft tumor model 47 . In addition to LDHA, ALDH1A3 (aldehyde dehydrogenase 1 family member A3), which participates in the glycolytic pathway, also showed similar expression patterns in 2D-and 3D-cultured cells after treatment with XAV939 (Fig. 5C, 2D, 1; 2D_XAV939, 0.91-fold; 3D, 1.79-fold; 3D_XAV939, 1.23-fold).
Our quantitative profiling of XAV939-target proteins based on 2D and 3D cell culture could be useful for the discovery and development of new drugs for CRC. Nevertheless, it remains unclear why XAV939-target proteins are differentially expressed in 2D-and 3D-cultured cells. One conceivable mechanism is functional changes in β-catenin signaling that depend on the cellular microenvironments, hypoxia, and normoxia conditions 48 . In normoxia, β-catenin directly interacts with TCF-4 (T-cell factor-4) and enhances TCF-4-mediated transcription. In hypoxic conditions, however, the interaction of β-catenin and HIF-1 is predominant over the β-catenin-TCF-4 complex. Therefore, the HIF-1-dependent transcription is improved, but the TCF-4 transcriptional activity is reduced. Hypoxia conditions are one notable characteristic of 3D culture, while 2D-cultured cells are commonly grown under normoxic conditions. It is therefore reasonable to argue that the expression of TCF-4/HIF-1-target genes could be regulated differentially in 2D versus 3D culture conditions when β-catenin protein levels are decreased by XAV939. The fact that LDHA is one of the HIF-1-target genes supports this suggestion 46 . Further investigations are required to fully understand these mechanisms.  Table 2. Significantly changed proteins between 2D-and 3D-cultured SW480 cells upon treatment with XAV939. Notes: GO terms determined using PANTHER analysis tool. 3D_XAV939: 3D culture treated with XAV939, 3D:3D culture, 2D_XAV939: 2D culture treated with XAV939, 2D: 2D culture. Abbreviations: GO, Gene ontology.

Conclusion
Herein, we presented a comparative proteomic analysis of 2D-and 3D-cultured SW480 cells with a focus on the XAV939-induced changes. We observed the up-regulation of glycolytic proteins and down-regulation of proteins associated with cell growth in 3D-cultured SW480 cells compared to 2D-cultured SW480 cells. This supports the fact that 3D cell culture models more appropriately represent cancer phenotypes in vivo than 2D cell culture models. Compared to previous proteomic studies of 3D-cultured cells, it is noticeable that mitochondrial oxidative phosphorylation was either activated or inactivated depending on the 3D cell culture models. This was possibly affected by the type of cells, 3D cell culture systems, spheroid sizes, or other factors. In addition, we showed that XAV939 suppresses the growth of SW480 cells in 3D culture, but not in 2D culture, even though Wnt/β-catenin signaling was successfully impaired by XAV939 in both 2D-and 3D-cultured SW480 cells. We identified the novel XAV939-responsive proteins involved in the growth inhibitory effects of XAV939 in 3D-cultured SW480 cells including gelsolin and lactate dehydrogenase A. These results provide novel insight into the mechanisms of XAV939-induced growth inhibition of 3D-cultured SW480 cells.   The fluorescence is proportional to the number of cells and was read using a Spectramax ® i3 (Molecular Devices) with a 485/520-nm filter. For iTRAQ analysis, the dried peptides were dissolved in 30 μl of 0.5 M triethylammonium bicarbonate (TEAB) buffer. The iTRAQ reagents were used to label individual peptide samples as follows: 2D-cultured cells, 113 tag; 2D-cultured cells treated with 20 μM XAV939, 114 tag; 3D-cultured cells, 115 tag; 3D-cultured cells treated with 20 μM XAV939, 116 tag. After incubation for 2 h at room temperature, the reaction was stopped by addition of 1% formic acid (v/v). The four iTRAQ-labeled samples were combined in one tube and desalted on 10-mg OASIS HLB cartridges. The resulting peptides were dried in a SpeedVac and reconstituted with 0.1% formic acid (FA) in water for online 2D-nLC-MS/MS analysis. Two independent biological replicates were performed, and each sample was analyzed in duplicate online 2D-nLC-MS/MS runs.
The peptide samples were loaded onto the SCX resins of the biphasic trap column and eluted with 12-step salt gradients (0, 15, 20, 22.5, 25, 27.5, 30, 40, 50, 100, 200 mM, and 1 M ammonium bicarbonate buffer containing 0.1% FA). After each salt fractionation, eluted peptides were bounded on RP resins, followed directly by RP gradient elution with mobile phase A (0.1% FA in water) and mobile phase B (2% water and 0.1% FA in acetonitrile). Electrospray ionization (ESI) was performed at a spray voltage of 2.5 kV. MS precursor ions were analyzed in positive mode with a scan range of 300 to 1800 m/z. MS spectra were acquired with a resolution of 70,000, AGC target of 3 × 10 6 , and maximum injection time of 80 ms. The twelve most intense precursor ions were selected for data-dependent MS/MS scans with exclusions of singly charged ions. Selected precursor ions were fragmented by high-energy collision dissociation (HCD) with NCE 27. For the MS/MS scans, the first fixed mass was set at 100 m/z, and the resolution was 35,000. Dynamic exclusion was allowed for 30 s.