Microarray analysis reveals ONC201 mediated differential mechanisms of CHOP gene regulation in metastatic and nonmetastatic colorectal cancer cells

The imipramine ONC201 has antiproliferative effects in several cancer cell types and activates integrated stress response pathway associated with the induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We investigated the signaling pathways through which ONC201/CHOP crosstalk is regulated in ONC201-treated nonmetastatic and metastatic cancer cell lines (Dukes' type B colorectal adenocarcinoma nonmetastatic SW480 and metastatic LS-174T cells, respectively). Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry, gene expression was assessed by Affymetrix microarray, signaling pathway perturbations were assessed in silico, and key regulatory proteins were validated by Western blotting. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment; Gene Ontology analysis of differentially expressed genes showed that cellular responsiveness to ONC201 treatment also differed substantially. In both ONC201-treated cell lines, CHOP expression was upregulated; however, its upstream regulatory mechanisms were perturbed. Although, PERK, ATF6 and IRE1 ER-stress pathways upregulated CHOP in both cell types, the Bak/Bax pathway regulated CHOP only LS-174T cells. Additionally, CHOP RNA splicing profiles varied between cell lines; these were further modified by ONC201 treatment. In conclusion, we delineated the signaling mechanisms by which CHOP expression is regulated in ONC201-treated non-metastatic and metastatic colorectal cell lines. The observed differences could be related to cellular plasticity and metabolic reprogramming, nevertheless, detailed mechanistic studies are required for further validations.

www.nature.com/scientificreports/ tained, independent of drug concentration. On the other hand, metastatic LS-174T cell proliferation/viability was gradually reduced in response to increasing concentrations of ONC201. These data suggest that ONC201 has a dose-dependent growth inhibitory effect on metastatic cells and only a moderate growth inhibitory effect on nonmetastatic cells.
To gain a better understanding of the cytotoxic effect of ONC201 on these cell lines, we used flow cytometry to evaluate cell proliferation stages in response to ONC201 treatment. As indicated in Fig. 1B, flow cytometric analyses were in agreement with cytotoxicity test results. Compared to nonmetastatic SW480 cells, which showed an 81% ± 1.5% survival rate, the viability of metastatic LS-174T cells was significantly reduced, i.e., 57.3% ± 1.8%. Relative to the vehicle-treated control, LS-174T cells treated with ONC201 showed a significant, tenfold increase in apoptotic cells and 3.3-fold increase in cell death. In contrast, nonmetastatic SW480 cells were more resistant to the drug treatment, with apoptotic and dead cells increasing by only 2.0-2.5-fold relative to the control (Fig. 1B). www.nature.com/scientificreports/ Pathway enrichment of differential microarray-based gene expression. The studied colorectal cancer cell lines exhibited a differential response to ONC201 treatment, suggesting a unique mechanism of action that may be related to the metastatic transformation of LS-174T cells. Gaining an understanding of these mechanisms will provide new insights into the effectiveness of ONC201 treatment. To this end, we performed microarray transcriptome profiling of RNA samples from LS-174T and SW480 cells with or without ONC201 treatment; we used Affymetrix expression console software for data analysis, applying a fold-change difference ≥ 2 and a p-values < 0.01 to determine which genes were differentially expressed between ONC201-and vehicletreated cells. This critical differentially expressed transcript filter is shown in volcano plots in Fig. 2A,B. In total, we detected 1,188 and 1,572 upregulated and downregulated gene transcripts, respectively, in ONC201-treated metastatic LS-174T cells relative to the expression in vehicle-treated cells ( Fig. 2A). In comparison, reduced numbers of differentially regulated transcripts were observed in nonmetastatic SW480 cells post-ONC201 treatment, i.e., only 519 and 379 gene transcripts were upregulated and downregulated, respectively, (Fig. 2B). Next, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on the differentially regulated transcripts. For both cell lines, the top signaling pathways that showed statistically significant regulation (p ≤ 0.001) in response to ONC201 treatment are listed in Supplemental Tables 1 and 2. Initially, these pathways were classified into major network mechanisms including oncogenesis, cell cycle, cellular metabolic pathways, DNA repair, micro-RNAs, and stress; the latter was affiliated only with ONC201-treated nonmetastatic SW480 cells (Supplemental Table 2). In comparison, the overall number of regulated signaling pathways and associated genes in ONC201-treated metastatic LS-174T cells was higher than that observed in treated nonmetastatic SW480 cells.
Detailed analysis of the total gene expression profile associated with each signaling pathway revealed remarkable diversity between the metastatic and nonmetastatic cancer cell lines. In drug-treated LS-174T cells, we observed a notable global downregulation of genes associated with oncogenesis, cell cycle, and DNA repair networks. Whereas, cell homeostasis networks, such as cellular metabolic pathways and micro-RNAs, showed a comparable number of upregulated or downregulated genes (Supplemental Table 1). Surprisingly, ONC201treated SW480 cells showed fewer regulated genes that were almost equivalently upregulated or downregulated, at least in part, for the studied networks. Notably, a large number of stress response network genes were upregulated only in the ONC201-treated nonmetastatic SW480 cells (Supplemental Table 2).

Meta-analysis of differentially regulated pathways and genes in metastatic versus nonmetastatic cells in response to ONC201 treatment.
In response to ONC201 treatment, observed differences between the two cell lines implied the existence of differentially regulated mechanisms. Accordingly, we performed a comparative meta-analysis of all the differentially expressed genes and their influence on signaling pathways. We used a computational method that considered the interplay between the gene products in the pathway in response to the drug treatment and scored a predicted functional perturbation for each protein (Supplemental Figure S1); the data were then further adjusted by Bonferroni corrections. This approach predicts functional results for the microarray data. For instance; the apoptosis map generated from extrinsic and intrinsic gene expression changes in response to ONC201 treatment does not explain the moderate apoptotic phenotype in nonmetastatic SW480 cells compared with the phenotype in metastatic LS-174T cells (Supplemental Figure S2A and S3A, respectively). On the other hand, the predicted functional perturbation changes in the apoptotic pathway clearly indicate that the observed phenotype in SW480 cells is due to a moderate induction of apoptotic genes (e.g., Casp7 and Casp9) that were not detected in the differential gene expression profile. This approach predicts the hidden functional effects of altered upstream regulatory genes (Supplemental Figure S1B and S3B). This map also shows that, upon ONC201 treatment, only the intrinsic apoptotic pathway is affected in nonmetastatic SW480 cells whereas both the extrinsic and intrinsic apoptotic pathway effectors are increased in metastatic LS-174T cells; hence, LS-174T cells show a higher apoptotic fraction (see Fig. 1).
Interestingly, when the computational methods were applied to both cell types, nine pathways were significantly perturbed in SW480 cells posttreatment with ONC201, whereas 44 signaling pathways were perturbed in the metastatic cell line LS-174T (Fig. 3A). Figure 3B shows the pathways that are commonly perturbed in both cell lines, while Table 1 details the 35 pathways that are perturbed only in LS-174T.
Detailed analysis of the p-values revealed that ONC201 treatment profoundly influenced genes associated with cell cycle signaling and ER-processing proteins, especially in metastatic LS-174T cells. Among the cell cycle regulatory genes, 36% and 65% were respectively perturbed in SW480 and LS-174T cells (Fig. 3B). Likewise, ONC201 administration altered 27% and 41% of genes regulating ER function in SW480 and LS-174T cells, respectively (Fig. 3B). Similarly, the other seven signaling pathways were also differentially regulated in either cell type in response to the drug treatment; of particular interest, genes associated with metabolic pathways, autophagy, and necroptosis may explain the observed phenotype shown in Fig. 1.
As previously mentioned, 35 signaling pathways were significantly perturbed in the genes differentially regulated in metastatic LS-174T cells but not nonmetastatic SW480 cells (Table 1). Of particular interest, changes to gene expression in the cellular senescence and colorectal cancer signaling pathways were most pronounced with notably low p-values. In addition, p53 signaling, DNA replication, and other metabolic signaling pathways were significantly decreased but to a lesser extent than the earlier described pathways.
We also performed comparative analysis of all data to identify genes associated with the differentially regulated pathways and subsequently ranked these genes in accordance with their p-values (Fig. 4A). Data analysis revealed that 2,404 and 3,902 genes were differentially regulated in the nonmetastatic SW480 and metastatic LS-174T cell lines, respectively. Of these, 2,218 were found to be commonly impaired in both cell types, albeit with varying p-values (Fig. 4A). The top 15 genes that were significantly upregulated in either cell line in response to ONC201 are listed in Fig. 4B. Notably, FAM129A, DDIT3/CHOP, and ASNS gene transcripts were significantly www.nature.com/scientificreports/ upregulated in both cell lines (p ≤ 0.001). FAM129A (also known as NIBAN1 or Niban apoptosis regulator 1) encodes a protein that is highly expressed in cancer. Since the cell lines are carcinogenic in nature, such a transcript should have been detected in our microarray data. DDIT3/CHOP is a transcription factor and a member of the C/EBP family. ASNS (asparagine synthetase) is involved in asparagine synthesis and facilitates progression through G1 phase of the cell cycle ( Fig. 4B).
Our initial results indicated that ONC201 induces apoptosis differently in both cell lines and since CHOP is a critical regulatory factor for this pathway therefore, we focused our study on the regulatory mechanisms associated with this gene. Mapping the differentially expressed gene transcripts detected in microarray data onto the map of the ER protein processing pathway 30,31 revealed similar, but nonidentical, regulatory mechanisms upstream of CHOP for each treated cell line (Fig. 5). In metastatic LS-174T cells, ONC201 treatment induced upregulation of CHOP transcripts through the upregulation of IRE1, ATF6, PERK, and Bak/Bax signaling networks (Fig. 5A). These regulatory mechanisms were also observed in ONC201-treated SW480 cells, except for Bak/Bax pathway expression (Fig. 5B). In addition, transcripts of downstream antiapoptotic BCL2 were significantly downregulated in metastatic LS-174T cells, relative to their expression in nonmetastatic SW480 cells, post-ONC201 treatment.

ONC201 treatment elicits apoptosis in LS-174T and SW480 human colorectal cancer cells.
The microarray gene expression data were validated by western blot analysis for selected proteins associated with the upregulation of CHOP. Indeed, treatment with ONC201 resulted in a significant increase in CHOP protein expression in both LS-174T (Fig. 6) and SW480 (Fig. 7) cells. The upstream CHOP regulatory proteins associated with different ER signaling pathways were also studied and showed similar patterns of expression to those observed in the microarray study. Specifically, ATF6 protein expression increased in response to ONC201 treatments in both cell lines, and the response was statistically significant when nonmetastatic SW480 cells were treated with 20-µM ONC201. In addition, Bax proteins were significantly increased in metastatic LS-174T cells, but not in nonmetastatic SW480 cells, post-ONC201 treatments (Figs. 5 and 7). PERK-regulated proteins (eIF2a, GADD34, and ATF4) were differentially expressed in response to ONC201 treatment in these cell lines. EIF2a protein expression was significantly increased in nonmetastatic SW480 cells (Fig. 7) while being significantly reduced in metastatic LS-174T cells at 20-µM ONC201 (Figs. 5). Low ONC201 concentration treatments were associated with slight reductions in ATF4 expression in both cell lines; however, at high ONC201 concentrations (i.e., 20 µM), significantly reduced ATF4 expression was observed in metastatic LS-174T cells only (Fig. 6). In contrast, GADD34 expression was significantly augmented in both cell lines in response to ONC201 treatment  Figures S4 and S5). Microarray meta-analysis indicated a significant reduction in BCL2 transcripts, particularly in ONC201-responsive metastatic LS-174T cells; this was confirmed at the protein level at which BCL2 protein was significantly downregulated post-ONC201 treatments (Fig. 6). Contrastingly, BCL2 protein expression was sustained in the presence or absence of ONC201 treatment in nonmetastatic SW480 cells (Fig. 7). Thus, microarray analysis data and protein expression levels of the studied signaling network markers were found to be alignment after ONC201 treatments.
Alternative splicing CHOP mRNA. Defects in RNA alternative splicing are a hallmark of cancerous cells.
Many RNA splicing regulators have been studied as tumor suppressors or are associated with drug resistance [32][33][34] . Exon splicing analysis of CHOP showed significant variation derived from metastatic LS-174T cells treated with vehicle versus those treated with ONC201: significantly reduced (up to 4.65-fold) splicing index signal levels were found in exon 2 in samples treated with the drug (Fig. 8A). To confirm these data experimentally, we performed quantitative RT-PCR and fractionated the products on a bioanalyzer. As shown in Fig. 8B, differential splicing patterns of CHOP mRNA were observed in metastatic LS-174T and nonmetastatic SW480 cells, and these patterns were further modified in response to ONC201 treatment. This is an additional indication of the multifaceted mechanisms of action of ONC201 as an anticancer drug.

Discussion
Using nonmetastatic SW480 and metastatic LS-174T colorectal cancer cell lines, we identified signaling pathways that were differently perturbed when cells were treated with ONC 201. In metastatic LS-174T cells, we identified differential mechanisms of CHOP regulation in response to ONC201 treatment that downregulate BCL2 specifically and induce apoptosis. Previous research has shown that ONC201 treatment interrupts ER homeostasis and induces the expression of three ER stress response signaling networks, namely PERK, ATF6, and IRE1, known to trigger unfolded protein response signaling 35,36 . Here, we report the prospective role of the ER stress Bak/Bax network in targeting CHOP regulation in the metastatic colorectal cell line LS-174T after ONC201 treatment. Additionally, our microarray data indicated that prospective crosstalk occurs between the IRE1 and Bak/Bax signaling pathways in LS-174T cells. Furthermore, the complexity of the CHOP regulatory mechanism in the metastatic cell line and the subsequent downregulation of BCL2 may explain the observed proliferation arrest and high apoptosis rates in ONC201-treated LS-174T cells relative to the response in nonmetastatic SW480 cells. Since BCL2, which was consistently downregulated in metastatic LS-174T cells, is inhibitory effector of the Bak/ Bax signaling pathway 37 , we cannot exclude the prospective feedback regulatory interplay among Bax/CHOP/ BCL2 as a regulatory factor in the response of LS-174T cells to ONC201 application. On the other hand, our meta-analysis indicated that the transcripts of BH3 proteins, Bak/Bax activators, are significantly upregulated, particularly in LS-174T cells; nevertheless, Bak/Bax autoactivation can occur, independently of the activator BH3s (i.e., BIM, BID, PUMA, and NOXA) following BCL2 downregulation 38 . Our observations are in accordance with previous studies that highlighted the role played by ONC201 in mediating the ER stress response in breast cancer cells 12,39 and high-grade central nervous system glioblastoma 40 . However, in these studies, the observed ER stress response was primarily due to ATF4 activation. Lev et al. 17 compared ONC201-sensitive HAPF-II www.nature.com/scientificreports/ against resistant PANC-1 pancreatic cell lines and reported a discrepancy in the ER stress response: ONC201 mediated upregulation of three ER stress response signaling molecules in PANC-1 cells, whereas ATF4 was the only protein to be upregulated in HPAF-II cells, in which substantial expression of IRE1 or ATF6 proteins was not detected. In nonmetastatic SW480 cells, ER homeostasis is restored by the upregulation of eIF2a, which explains the observed moderate effect of ONC201 treatment. Active eIF2a attenuates protein synthesis and reduces protein-processing workload on the stressed ER [41][42][43] . Taken together, these findings suggest that the cellular response to ONC201 treatment is cell type-dependent, but that the overall mechanisms are associated with ER stress and unfolded protein response signaling. Gene expression profiles in colorectal cancer cells revealed that ONC201 downregulates genes associated with energy metabolism. Specifically, ONC201 reduced the gene expression of citrate carrier (SLC25A1) and fumarate hydratase (FH) that regulate the mitochondrial metabolite carrier and substrate metabolism, respectively. SLC25A1 is involved in citrate mitochondria/cytoplasm translocations for cellular energy homeostasis 44 , whereas FH plays an important role in the Krebs cycle by providing FADH and NADH to the electron transport chain for ATP production 45 . Thus, ONC201 is involved in reducing metabolic pathways that may cause energy   46 observed a decrease in the ATP levels associated with low glycolysis and oxidative phosphorylation that caused energy stress to cancer cells. ONC201 can also reduce mitochondrial respiration in breast cancer cells, which may lead to energy stress (reducing ATP) and result in apoptosis 14 .
In our analysis, we identified several spliced variants of CHOP that were differentially expressed in ONC201treated metastatic and nonmetastatic colorectal cell lines. Notably, exon 2 was found to be the target for the CHOP splicing mechanism. The function of exon 2 in CHOP is unclear, although this exon encodes part of the 5'-untranslated region and in general, untranslated regions are considered to regulate the protein translation activity or mRNA expression 47,48 . CHOP belongs to the C/EBP family of transcription factors and functions as a dominant-negative inhibitor by forming heterodimers with other C/EBP members. Alternative splicing has also been reported for other family members; The C/EBP epsilon gene is regulated by an alternative translational initiation site and splicing mechanisms 49 , which generate four different isoforms with different functions 50,51 . In addition to alternative translational initiation, the expression of four alternative C/EBPε isoforms (p32, p30, p27, and p14) has been attributed to differential promoter usage and alternative splicing 51 . www.nature.com/scientificreports/ In summary, the efficacy and outcome of cancer treatment is dependent on the stage of the disease. Differences between nonmetastatic and metastatic cancer cells are associated with cellular plasticity and metabolic reprogramming 52 , which lead to differential responses to chemotherapy as observed here and in other studies. In the present study, we delineated the interactive signaling mechanisms and associated genes that differentially regulate CHOP expression in nonmetastatic and metastatic colorectal adenocarcinoma cells. In ONC201-treated metastatic LS174T cells, these mechanisms lead to increased expression of the core regulators, Bak and Bax, of the intrinsic apoptosis pathway.
We acknowledge that the current study has limitations; further mechanistic studies will be required to delineate the functional role of the signaling pathways up-and downstream of CHOP in both metastatic and nonmetastatic cell types. In particular, the crosstalk among Bak/Bcl2/CHOP must be assessed. In addition, further studies are required to determine the role of the observed CHOP alternative transcripts splicing between cell Preparation of protein extract and western blot analysis. Cells were harvested and lysed using modified RIPA buffer (50 mM Tris-HCl at pH 7.5, 150-mM NaCl, 1% Triton × 100, 1-mM EDTA, 0.5% sodium deoxycholate and 0.1% SDS). Cell lysates were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific GmbH, Driesch, Germany) and equal amounts of protein (30 μg) were resolved on 8%-12% polyacrylamide gels before using transferred to polyvinylidene fluoride membranes (EMD Millipore Corporation, Billerica, MA, USA) as previously described 53 . After blocking, membranes were blotted with the corresponding primary and horseradish peroxidase-linked secondary antibodies 54 . The primary antibodies used were as follows: β-actin (ab8224), eIF2a (ab169528), GADD34 (ab9869) and ATF4 (ab85049) (all from Abcam, USA). CHOP (2895S), Bax D2E11 (5023T), and BCL2 D55G8 (4223T) were purchased from Cell Signaling, USA; ATF6 (ALX-804-381-C100) was purchased from ANZO, USA. Finally, immunoblots were detected by chemiluminescence using the Chemidoc MP system (Bio-Rad, USA). ImageJ V1.49O software (https:// imagej. nih. www.nature.com/scientificreports/ gov/ ij/) was used to quantify the immunoblot signals as the mean value of the gray/white scale of all pixels in a band 55 .
MTT cytotoxicity assay. Cytotoxicity assays were performed as previously described 56   RNA extraction, microarray array , and PCR assays. Total RNA was isolated using the Trizol-Chloroform method as described by Al Madhoun et al. 57 . Isolated RNA was quantified and RNA integrity was assessed by microfluidic analysis using a Bioanalyser 2100 (Agilent Technologies, Santa Clara, CA, USA). For microarray assays, total RNA (100 ng) from each sample (in triplicate) was reverse transcribed as per the manufacturer's protocol (GeneChip WT PLUS Reagent, Thermo Fisher Scientific). Purified cDNA was fragmented, labeled, and hybridized onto a GeneChip Human Transcriptome Array 2.0 (Thermo Fisher Scientific GmbH) for 16 h at 45 °C and 60 rpm in an Affymetrix GeneChip Hybridization Oven 640. Chips were washed and stained using an Affymetrix GeneChip Fluidics Station 450 (Thermo Fisher Scientific) and scanned with an Affymetrix GeneChip Scanner 3000 7G (Thermo Fisher Scientific). CEL data files were analyzed using Affymetrix expression console software (version 1.0) provided by the manufacturer. For spliced isoform analysis, we followed similar procedures to those described by Al Al Madhoun et al. 32 ; cDNA was synthesized from 1 µg of RNA by reverse transcription using a QuantiTect Reverse Transcription Kit (Qiagen Inc., Hilden, Germany) as previously described 58 . Reverse-transcribed RNA was used as a template for CHOP spliced isoform quantitative PCR amplification using specific primers and a FastStart SYBR Green Kit (Roche Applied Sciences, Penzberg, Germany). Forward (5′-TAA GGC ACT GAG CGT ATC ATG-3′) and reverse (5′-CTG GAC AGT GTC CCG AAG GAG AAA -3′) primers were designed using Primer Bank 59 . PCR products were run on an Agilent 2100 Bioanalyzer system using a High Sensitivity DNA Electrophoresis Kit, as instructed by the manufacturers (Agilent Technologies) and then quantified using High Sensitivity DNA assay software.
Bioinformatics and meta-analysis of microarray data. Functional classification and enrichment analysis was performed based on GO annotation the KEGG database 30,60 . To detect pathways differentially perturbed in metastatic cancer cells relative to nonmetastatic cells following ONC201 treatment, we used a computational method to integrate differential gene expression into predefined pathways as described in Rivera et al. 61 . Briefly, we graphed P = (G, I), where P are pathways, G are their gene sets, and I are the interactions between these genes. For each cell line, we input the fold change of the treatment versus that of the control and determined which pathways were perturbed upon ONC201 treatment. The Liptak-Stouffer z-score was calculated as the perturbation of each subgraph. The most perturbed subpathway was then computed according to the algorithm of Rivera et al. 61 . We also applied Bonferroni corrections to the final computed p-value of the most perturbed pathways for more stringent results.

Statistical analysis.
All experiments and assays were conducted in technical duplicates or triplicates for three biological samples. Results were combined and statistical significance was determined using a two-tailed Student's t-test assuming equal variance. Test were performed in GraphPad Prism version 8.0. Data are presented as means ± standard error of the mean (SEM) as previously described 62 .

Data availability
All data generated and analyzed during this study are included in this article.