Pharmacogenomic profiling and pathway analyses identify MAPK-dependent migration as an acute response to SN38 in p53 null and mutant colorectal cancer cells

Abstract

The topoisomerase I inhibitor irinotecan is used to treat advanced colorectal cancer and has been shown to have p53-independent anti-cancer activity. The aim of this study was to identify the p53-independent signalling mechanisms activated by irinotecan. Transcriptional profiling of isogenic HCT116 p53 wild-type and p53 null cells was carried out following treatment with the active metabolite of irinotecan, SN38. Unsupervised analysis methods demonstrated that p53 status had a highly significant impact on gene expression changes in response to SN38. Pathway analysis indicated that pathways involved in cell motility (adherens junction, focal adhesion, MAPK and regulation of the actin cytoskeleton) were significantly activated in p53 null cells, but not p53 wild-type cells, following SN38 treatment. In functional assays, SN38 treatment increased the migratory potential of p53 null and mutant colorectal cancer cell lines, but not p53 wild-type lines. Moreover, p53 null SN38-resistant cells were found to migrate at a faster rate than parental drug-sensitive p53 null cells, whereas p53 wild-type SN38-resistant cells failed to migrate. Notably, co-treatment with inhibitors of the MAPK pathway inhibited the increased migration observed following SN38 treatment in p53 null and mutant cells. Thus, in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells, and inhibiting MAPK blocks this potentially pro-metastatic adaptive response to this anti-cancer drug.

INTRODUCTION

In advanced colorectal cancer, response rates remain disappointingly low, at 40-50% for combined 5-FU-based regimes (1, 2). The main reason for the lack of response is acute or acquired drug resistance. The identification and manipulation of drug resistance mechanisms in colorectal cancer could significantly enhance patient response to therapy.

The p53 tumour suppressor gene is the most frequently mutated gene in all human cancers and has been demonstrated to be mutated in at least 50% of CRC tumours (3). A number of studies have reported that p53 is a predictive marker of sensitivity to chemotherapy (4-8), however, other studies have reported conflicting findings (9, 10). Previously, we reported that CRC cells display similar levels of sensitivity to irinotecan irrespective of p53 status; this is not the case for either 5-FU or oxaliplatin both of which have significantly reduced cytotoxic effects in p53 null cells (11). Ravi et al. demonstrated that the combination of irinotecan and TRAIL eliminates hepatic metastasis in both p53 wild-type and null CRC cells in vivo. They further demonstrated that the synergy displayed between irinotecan and TRAIL was mediated via a p53-independent mechanism that involved the inhibition of JAK-STAT3/5 signalling (8). Indeed, work carried out in our laboratory demonstrated that irinotecan, but not 5-FU or oxaliplatin, up-regulated Fas cell surface expression via a novel p53-independent mechanism that involved the activation of STAT1 followed by enhanced Fas cell surface trafficking (11). Bhonde et al used expression profiling to identify the genes that were associated with induction of apoptosis in p53 mutant cells following SN38 treatment. They identified a significant number of mitotic genes that were differentially regulated between p53 wild-type and p53 mutant cells and further demonstrated that suppression of the mitotic gene, hMps1, reduced the apoptosis induced following SN38 treatment in p53 mutant cells (12).

The aim of the current study was to carry out transcriptional profiling of isogenic p53 wild-type and p53 null CRC cells following treatment with SN38 (the active metabolite of irinotecan) to identify the signalling pathways activated by this agent in these genetic backgrounds. Bioinformatics analyses revealed that, in response to SN38, a number of pathways involved in promoting cell migration were activated in the p53 null setting, but not in the p53 wild-type setting. Importantly, cell migration assays revealed that SN38 treatment increased the migratory potential of p53 null and mutant colorectal cancer cells, but not p53 wild-type cells. Of note, inhibitors of MAPK signalling, such as the MEK inhibitor AZD6244, attenuated SN38-induced migration in p53 null and mutant colorectal cancer cells. These results suggest that in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells. Moreover, inhibiting MAPK blocks this potentially pro-metastatic adaptive response in p53 mutant/null cells.

MATERIALS AND METHODS

Cell Lines and Cell Culture

All CRC cells were grown as previously described (13). Following receipt, cells were grown up, and as soon as surplus cells became available, they were frozen as a seed stock. All cells were passaged for a maximum of 2 months, after which new seed stocks were thawed for experimental use. All cell lines were tested for mycoplasma contamination at least every month. RKO (2001) cells were obtained from the American Type Culture Collection [ATCC; authentication by short tandem repeat (STR) profiling/karyotyping/isoenzyme analysis] and maintained in Dulbecco’s modified Eagle’s medium (DMEM). The p53 wild-type HCT116 human colorectal adenocarcinoma cell line and a matched isogenic p53 null cell line (kindly provided by B. Vogelstein, Johns Hopkins University School of Medicine, Baltimore) were maintained in McCoy’s medium (GIBCO Invitrogen, Paisley, UK). . LoVo (2004) cells were obtained from the European Collection of Cell Cultures (ECACC), maintained in DMEM. HCC2998 cells were obtained from the National Cancer Institute-Frederick Cancer DCT Tumour repository (10/2008; authentication: SNP arrays, oligonucleotide-base HLA typing, karyotyping and STR (5/2007)) and maintained in Roswell Park Memorial Institute 1640 (RPMI). DLD-1 cells were kindly provided by Senji Shirasawa in 8/2008, were maintained in DMEM. HCT116, HT29, LoVo, DLD-1 and RKO cell lines were validated by STR profiling by LGC Standards (14) in May 2011. The p53 wild-type and p53 null SN38-resistant HCT116 sub-lines were generated in our laboratory as previously described (6). All medium was supplemented with 10% dialysed FCS, 50μg/mL penicillin-streptomycin, 2mM L-glutamine, and 1mM sodium pyruvate (all medium and supplements from Invitrogen Life Technologies Corp., Paisley, United Kingdom). All cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Reagents

The following reagents were used: SN38 (Abatra Technology Co, LTD, China) (Figure 1A), U0126 (Cell Signalling Technology, Inc.), AZD6244 (Astra Zeneca, UK) (15).

Figure 1.

A. The structure of SN38, the active metabolite of irinotecan. B. Principal Component Analysis (PCA) plot and C. Condition tree displaying the separation between HCT116 p53 wild-type and p53 null cells following treatment with 5nM (IC₅₀) SN38 for 6, 12 and 24h. The combined p53 wild-type and p53 null genelist was used in each case. D. The SN38-inducible genelist for the p53 wild-type setting and the SN38 inducible genelist for p53 null setting were used as the starting genelists for the Venn diagram functionality to display the number of genes altered following SN38 treatment in p53 wild-type cells only, p53 null cells only or the genes that are commonly regulated in both cell lines.

Microarray analysis

HCT116 p53wild-type and p53 null cells were treated with 5nM SN38 for 0, 6, 12, and 24 hours. Total RNA was isolated from three independent experiments using the RNA STAT-60™ Total RNA isolation reagent (Tel-Test, Inc., Texas, USA) according to the manufacturer’s instructions. Five micrograms of total RNA was sent to Almac Diagnostics (Craigavon, UK) for cDNA synthesis, cRNA synthesis, fragmentation and hybridisation onto Affymetrix HG U133 Plus2.0 microarrays. Detailed experimental protocols and raw expression data are available at http://www.ebi.ac.uk/arrayexpress/ (Acession numbers E-MEXP-1171 and E-MEXP-1194).

Data analysis

Microarray data analysis was performed using GeneSpring v7.3 (Agilent Technologies UK Limited, Stockport, UK) as previously described (16). Firstly, the HCT116 p53 wild-type and p53 null arrays were assessed as a single experiment. Following standard normalisation and filtering, a 4800 drug inducible genelist was created using a 1.5-fold cut-off for each gene relative to their 0 hour control, with genes meeting this criterion in at least one time point retained. This genelist was used as the input for Principal Component Analysis (PCA) and Hierarchical clustering analysis. For PCA, the genelist was utilised for all samples, with mean centering and scaling. For hierarchical clustering, the genelist was analysed using Pearson’s correlation as the similarity measure and the average linkage clustering algorithm. Secondly, constitutive gene expression in p53 wild-type and p53 null cells was examined. Normalisations and filtering were carried out, and those genes that were altered ≥1.5-fold were retained as the ‘basal’ genelist and used in pathway analysis. Finally, the drug-inducible data from each cell line were analysed separately to identify SN38-inducible genelists for p53 wild-type and null cells. A Venn diagram was used to identify genes that were common and unique to each experimental setting; these common and unique genelists were retained for pathway analysis.

Pathway Analysis

Pathway analysis was conducted using KEGG and GenMAPP pathways. Pathway analysis was carried out on the ‘basal’ and ‘drug-inducible’ genelists described above. Pathway statistical significance was assessed using hypergeometric statistics. For both basal and SN38 inducible analyses, pathways were retained that contained ≥10 genes per pathway. Pathway analysis from the p53 wild-type experiments were compared to the p53 null experiments, and common pathways were retained and further analysed for differential expression either basally or following SN38 treatment.

Real-time reverse transcription-PCR analysis

Total RNA was isolated as described above. Reverse transcription was carried out as previously described (16) using 2μg of RNA using a Moloney murine leukemia virus–based reverse transcriptase kit (Invitrogen) according to the manufacturer’s instructions. All amplifications were primed by pairs of chemically synthesized 18- to 22-mer oligonucleotides designed using freely available primer design software (17).

Western Blotting

Western blots were performed as previously described (11). Anti–phospho-extracellular signal-regulated kinase 1/2 (pErk1/2; Thr²⁰²/Tyr²⁰⁴, Cell Signalling, Beverly, MA) mouse monoclonal antibody was used in conjunction with a horseradish peroxidase–conjugated anti-mouse secondary antibody (Amersham) and anti-Erk1/2 (K-23, Santa Cruz) rabbit polyclonal antibody was used in conjunction with a horseradish peroxidase–conjugated anti-rabbit secondary antibody (Amersham).

Cell Migration assay

Cell migration was assessed using the CIM-plate 16 and the Xcelligence system (Roche Applied Sciences), according to the manufacturer’s instructions.

Statistical Analyses

Pearson’s product moment correlation (r) and the coefficient of determination (r²) were calculated using MS excel. The significance of the correlation was determined using a two-tailed test of significance (18). The unpaired two-tailed t-test was used to determine statistically significant differences between treatment effects. Significance was defined as p<0.05.

RESULTS

p53 dependence of SN38-induced gene expression

Transcriptional profiling was performed to compare the acute changes in mRNA expression induced by SN38 in isogenic p53 wild-type and p53 null HCT116 CRC cell lines. The results were combined into a single data set that contained 4800 genes that changed by ≥1.5-fold in at least one treatment condition. These 4800 genes were used as the starting point for Principal Component Analysis (PCA) and hierarchical clustering. The PCA revealed that the greatest variability in gene expression was due to p53 status, with the p53 wild-type and p53 null treatment groups clustering separately (Figure 1B). Secondly, within these groups, the drug-treated samples were more closely grouped than the non-treated control samples. Hierarchical clustering also demonstrated two main groups: one branch that contained all of the p53 wild-type samples and one that contained all the p53 null samples (Figure 1C). Within each branch, the 12h and 24h samples were more closely related than the 6h sample, and the least closely correlated sample was the control sample. Thus, both PCA and hierarchical clustering analyses segregated the samples according to p53 status and drug treatment; therefore the signalling pathways activated by SN38 in p53 null cells are significantly different from those activated in p53 wild-type cells.

To further compare the mechanism of action of SN38 in p53 wild-type and p53 null cells, we analysed each data set independently and identified the unique and common genes in the two lists. Following normalisation and filtering, it was found that 3296 genes changed ≥1.5-fold in at least one time point in p53 wild-type cells, while 2738 genes changed ≥1.5-fold in at least one time point in p53 null cells. Of these genes, only 752 were common to both p53 wild-type and null samples (Figure 1D and Supplementary Table 1), whereas 2,544 genes (77.2%) were unique to p53 wild-type (Figure 1D and Supplementary Table 2) and 1,986 (72.5%) genes were unique to p53 null cells (Figure 1D and Supplementary Table 3). From this analysis, it is again clear that the downstream effects of SN38 are significantly different in p53 wild-type and null cells. The constitutive differences in gene expression between the p53 wild-type and null cells were also assessed, with a significant number of genes (2102) found to be constitutively altered ≥1.5-fold between the two cell lines (Supplementary Table 4).

Q-PCR validation of microarray results

Q-PCR analysis was carried out to validate the microarray datasets. Validation of the p53 wild-type dataset following SN38 treatment has previously been reported (16). For the comparison of constitutive gene expression in the p53 wild-type and p53 null cells, the results for selected genes are displayed in Supplementary Table 5. Overall, when the Q-PCR data was compared to the microarray data, the Pearson’s correlation product moment (r) was 0.789 (r²=0.6225 and p=0.0002) (Figure 2A). The results of the Q-PCR analyses for the genes induced by SN38 treatment in the p53 null cells are presented in Supplementary Table 6. For the selected genes, Pearson’s correlation demonstrated an r value of 0.73, with r²=0.5383 (p=0.000015) (Figure 2B). Overall, statistically significant correlations (P<0.05) were observed for 24 of the 26 (~92%) genes (data not shown). In the analysis of the p53 wild-type cells following SN38 treatment, 25 genes were analysed by Q-PCR and demonstrated excellent correlation with the microarray results, with an r value of 0.80, and r²=0.61 (p=0.000001) (16). Altogether, these results indicate a strong overall concordance of gene expression trends between the microarray analysis and real-time RT-PCR analysis and demonstrate the robustness of the microarray datasets for further analysis.

Figure 2.

A. Correlation between average fold-changes in gene expression levels for 16 target genes that are basally altered between the HCT116 p53 wild-type and p53 null cells. B. Correlation between average fold-changes in gene expression levels for 26 target genes that are altered in the HCT116 p53 null cells following treatment with 5nM SN38. The correlation coefficients were determined by comparing the DNA microarray fold-changes and Q-PCR fold-changes following treatment of HCT116 p53 null parental cells with 5nM SN38 for 6, 12 and 24h. All data are displayed as log₂. C. Real-time Q-PCR validation of the results of the pathway analyses. A number of upstream and downstream genes were selected from the focal adhesion pathway and analysed by Q-PCR 24h following treatment with 5nM SN38 in the HCT116 p53 wild-type and HCT116 p53 null cell lines. All graphs are the result of three independent experiments and values are expressed as a fold-change compared to the 0h control sample.

Pathway analysis of SN38-induced signalling in p53 wild-type and null settings

Pathway analysis was carried out to identify those signalling pathways constitutively altered between p53 wild-type and p53 null cells and those altered following SN38 treatment in the p53 wild-type and p53 null settings. Eleven pathways containing ≥10 genes were found to be differentially active in p53 wild-type and p53 null cells (p<0.05) (Supplementary table 7). The majority of the identified pathways were down-regulated in the p53 null cells compared to the p53 wild-type cells. Further analyses demonstrated that a total of 32 pathways were altered following SN38 treatment in either p53 wild-type, p53 null or both settings (Table 1). Of the 32 pathways, 16 contained genes that were altered in both the p53 wild-type and null settings (Table 1, highlighted in bold). Of these 16 pathways, 5 were differentially regulated in p53 wild-type and p53 null cells following SN38 treatment (Table 2). The pathways that displayed differential regulation were adherens junction, focal adhesion, JAK-STAT signalling, MAPK signalling and regulation of the actin cytoskeleton, and in all cases, the pathways were up-regulated in p53 null samples following SN38 treatment compared to p53 wild-type samples. In addition, all of these pathways were constitutively lower in the p53 null samples compared to p53 wild-type samples (Supplementary table 7).

Table 1.

Results from KEGG pathway analysis of HCT116 p53wild-type and p53 null cells following treatment with 5nM SN38. The table displays the number of genes that are altered for a given pathway, based on genes that are uniquely altered in p53 wild-type cells, uniquely altered in p53 null cells or commonly altered in both p53 wild-type and p53 null cells following SN38 treatment. The pathways that are highlighted in bold are those that contain genes that are altered following SN38 treatment in both p53 wild-type and p53 null cells.

Pathways	Number of unique genes: p53wt	Number of unique genes: p53null	Number of common genes
Adherens junction	24	16	5
Apoptosis	14	14	9
Arginine and proline metabolism		11
Benzoate degradation via CoA ligation	10	12
Butanoate metabolism		13
Cell cycle	25	14	10
Cytokine-cytokine receptor interaction	12	13	12
ECM-receptor interaction	10	11	13
Fatty acid metabolism		18
Focal adhesion	29	25	13
Fructose and mannose metabolism	12
Glycosphingolipid metabolism	10
Glutamate metabolism		15
Glycolysis Gluconeogenesis		10
Inositol phosphate metabolism	14	12
Jak-STAT signaling pathway	12	21	7
Lysine degradation		13
MAPK signaling pathway	42	27	12
Nicotinate and nicotinamide metabolism	12
Oxidative phosphorylation	13
Phosphatidylinositol signaling system	18	19	6
Proteasome	14
Purine metabolism	17	14
Regulation of actin cytoskeleton	30	36	11
Ribosome		15
TGF-beta signaling pathway	14	10	7
Tight junction	18	20
Toll-like receptor signaling pathway		16
Tryptophan metabolism		12
Ubiquitin mediated proteolysis	10		6
Valine, leucine and isoleucine degradation		13
Wnt signaling pathway	37	18	7

Table 2.

Results from KEGG pathway analysis of HCT116 p53wild-type and p53 null cells following treatment with 5nM SN38. The table shows those pathways that are altered following SN38 treatment in both p53 wild-type and p53 null samples and also in which direction the specific pathways are regulated (up ↑ or down ↓ ) in response to SN38.

Pathway	p53 wt	p53 null
Adherens junction	↓	↑
Apoptosis	↓	↓
Benzoate degradation	↓	↓
Cell cycle	↓	↓
Cytokine-cytokine receptor interactions	↓ ↑	↑
ECM receptor interactions	↓ ↑	↑
Focal adhesion	↓	↑
Inositol phosphate metabolism	↑	↑
JAK-STAT signalling	↓	↑
MAPK signalling	↓	↑
PI3K signalling	↑	↑
Purine metabolism	↓	↓
Regulation of actin cytoskeleton	↓	↑
TGF-β signalling	↓	↓
Tight junction	↓	↓↑
Wnt signalling	↓	↓ ↑

To validate the pathway analysis results, we selected 15 members of the focal adhesion pathway and analysed their mRNA expression in p53 wild-type and p53 null cells following SN38 treatment. The focal adhesion pathway was selected, as this pathway included differentially expressed genes that were also members of the actin signalling (ITGB4, ITGA6, ITGA3, MYLK, ROCK1, PIK3R1, PIK3CD, ACTN1, CALM3, MAPK1, ACT, FAK, B-Raf, PAK1 and PAK2) and MAPK signalling (MAPK1, GRB2, PRKCA, B-Raf, RAP1A, MAPK8, c-Jun, PAK1 and PAK2) pathways. Genes from various points in the pathway were chosen, including receptors (INTGA6 and INTGA3), members of cell motility signalling (Actn1, Actn4, ZYX, VASP, CPN and Actb) and cell proliferation genes (CCND3, MAPK1, DUSP5, DUSP6, PRKCA, MAPK8 and Jun). Analysis of these genes demonstrated that ZYX Actb, CCND3, MAPK1 (ERK2), MAPK8, Jun, INTGA3 and DUSP5 were down regulated in p53 wild-type cells, but were highly up-regulated in p53 null cells following SN38 treatment (Figure 2C). INTGA6, Actn1, Actn4, VASP, CPN, PRKCA and DUSP6 were up-regulated in both p53 wild-type and p53 null cells following SN38 treatment, however, the level of up-regulation was significantly greater in the p53 null setting. Overall, these results demonstrate differential activation of the focal adhesion pathway in p53 wild-type and null cells and thereby confirm the results of the pathway analysis.

Differential cell migration following SN38 treatment in p53 wild-type and null cells

The results of the pathway analyses indicated that adherens junctions, ECM receptor interaction, focal adhesion, actin cytoskeleton, MAPK signalling and JAK-STAT signalling were all constitutively down-regulated in p53 null cells compared to their p53 wild-type counterparts, which suggested that the p53 null cells may migrate more slowly than the p53 wild-type cells. In addition, it was noted that following SN38 treatment, all of these pathways were up-regulated in p53 null cells compared to p53 wild-type cells, suggesting that SN38 may increase cell migration in the p53 null setting. To test these hypotheses, we measured migration of p53 wild-type and null isogenic cells in the presence and absence of SN38. Both cell lines started to migrate after ~8 hours, and active migration continued to ~28 hours, therefore all measurements of cell migration, cell index and slope of the curve were measured between 8 and 28 hours. As predicted by the pathway analyses, we found that constitutively, the p53 null cells migrated more slowly than the p53 wild-type cells, as the p53 null cells displayed a reduced slope of the curve (0.115 h⁻¹) compared to the p53 wild-type cells (0.278 h⁻¹) (Figures 3A, p<0.001).

Figure 3.

Migration assay results measured using the Xcelligence system. The level of cell migration was assessed by increases in the slope (1/hr) of the curve between 8h and 28h (active migration). A. The migratory rate of the HCT116 p53 wild-type cells are displayed in the white bars and the HCT116 p53 null in the grey bars. B. Migration rate analysis of HCT116 p53 wild-type and p53 null cells following treatment with 25nM SN38. C. Migration analysis of HCT116 p53 wild-type and p53 null parental (drug-sensitive) and respective SN38-resistant daughter cell line. In all cases statistical significance was assessed using an unpaired two-tailed t test (**** denotes p<0.001).

Importantly, following treatment with SN38, the migration of p53 null cells significantly increased (Figure 3B, p<0.001). In contrast, the p53 wild-type cells did not display any increase in cell migration following SN38 treatment (Figure 3B). To assess whether these findings were unique to SN38, we carried out migration experiments following treatment with the two other chemotherapies commonly used in the treatment of colorectal cancer, 5-FU and oxaliplatin. Treatment with 5-FU induced cell migration in both p53 wild-type and null cells to a similar extent, whereas treatment with oxaliplatin significantly reduced migration of p53 wild-type cells, but did not significantly alter migration of p53 null cells (data not shown). These results suggest that in this panel of clinically relevant chemotherapeutic agents, SN38 is unique in its ability to differentially induce migration based on p53 status.

We also compared the migratory potential of p53 wild-type and p53 null SN38-resistant daughter cell lines (6). The p53 wild-type SN38 resistant daughter cell line displayed highly reduced migratory ability compared to its parental SN38-sensitive cell line (Figure 3C, p<0.001). Most notably however, the p53 null SN38-resistant daughter cell line migrated at a significantly faster rate than the p53 null SN38-sensitive parental line (Figure 3C, p<0.001). These results further strengthen the hypothesis that increased migratory potential is an important adaptive response to SN38 treatment in p53 null CRC cells.

Inhibition of MAPK signalling attenuates SN38-induced cell migration

As the pathway analyses of the microarray data (Tables 1 and 2) and the molecular analyses (Figure 2) indicated differential MAPK signalling in p53 wild-type and p53 null cells following SN38 treatment and given the role of this pathway in cell migration (19), we next assessed whether MAPK signalling was involved in promoting the migration of p53 null cells following SN38 treatment. Western blot analyses demonstrated that ERK1/2 activation was inhibited following treatment with the MEK inhibitor, AZD6244 (Figure 4A). Importantly, it was found that the increased migration of HCT116 p53 null cells following SN38 treatment was significantly reduced following co-treatment with the MEK inhibitors U0126 and AZD6244 (Figure 4A, p<0.001 and data not shown). To determine whether these observations were cell line-dependent, we assessed migration in 4 other CRC cell lines: DLD1 and HCC2998 (both p53 mutant); and LoVo and RKO (both p53 wild-type). These analyses demonstrated that cell migration was significantly induced following SN38 treatment in p53 mutant cells (DLD1, p<0.001 and HCC2998, p<0.001) and that this was inhibited following co-treatment with AZD6244 (Figure 4B). In agreement with results from the HCT116 model, SN38 treatment did not induce cell migration in the p53 wild-type LoVo and RKO cell lines (Figure 4C). These results provide further evidence that in the absence of functional p53, SN38 enhances MAPK-dependent migration of CRC cells.

Figure 4.

Migration assay results measured using the Xcelligence. The level of cell migration was assessed by increases in the slope (1/hr) of the curve during active migration. All graphs display fold changes in the slope (1/hr) compared to the untreated control sample. A. The migration of HCT116 p53 null cells was assessed following treatment with 25nM SN38 in the presence and absence of 0.5μM AZD6244. Western blot analysis confirmed inhibition of ERK activation (pERK) by this concentration of AZD6244. B. The migration of p53 mutant DLD1 and HCC2998 cells was assessed following treatment with 30nM and 10nM SN38 respectively in the presence and absence of 1μM and 0.5μM AZD6244 respectively. C. The migration of p53 wild-type LoVo and RKO cells was assessed following treatment with 20 nM and 10nM SN38 respectively, in the presence and absence of 1μM and 0.1μM AZD6244 respectively. In all cases statistical significance was assessed using an unpaired two-tailed t test (**** denotes p<0.001).

DISCUSSION

p53 is one of the most commonly mutated genes in cancer and at least 50% of CRC tumours have dysfunctional p53 (3, 20, 21). Several studies have evaluated the role p53 plays in determining sensitivity to irinotecan therapy, with some suggesting a predictive capability and others reporting no correlation (6-10). Indeed, studies from our own laboratory have demonstrated that there was no significant difference in the sensitivity of p53 wild-type and p53 null CRC cells to irinotecan (6); this was not the case for either 5-FU or oxaliplatin, the other two chemotherapeutic agents used to treat CRC, both of which had reduced activity in the p53 null setting. Previous studies have demonstrated that p53 wild-type CRC cells induce a prolonged G2/M arrest in response to irinotecan, whereas p53 null CRC cells undergo a more short-term arrest followed by apoptosis. (22) We previously reported that although p53 wild-type and p53 null CRC cells are similarly sensitive to irinotecan, the molecular signalling pathways activated in the two settings can be significantly different. (11) Therefore the aim of the current study was to understand p53-independent signalling in response to irinotecan in CRC.

Following transcriptional profiling of SN38-treated p53 wild-type and null HCT116 cells, PCA and hierarchical clustering analysis demonstrated that there was a clear separation between the p53 wild-type and p53 null samples. Furthermore, it was found that only ~25% of the genes significantly altered by SN38 treatment in each cell line were similarly altered in the other cell line. These findings indicate that distinct genes/pathways are activated in response to SN38 in the different p53 backgrounds. The microarray results were validated by Q-PCR, and the level of correlation was found to be highly significant and similar to those of other previously published studies (16, 23-25).

Using KEGG and GenMAPP pathway analysis programmes, we identified adherens junction, focal adhesion, JAK-STAT signalling, MAPK signalling and actin cytoskeleton as the key pathways that were differentially regulated following SN38 treatment in p53 wild-type and p53 null cells. Previous work from our laboratory investigated the role p53 and STAT1 play in regulating Fas-mediated apoptosis in response to chemotherapy in CRC cells (11). In that study, we found that irinotecan treatment resulted in STAT1 activation in p53 null cells, but not p53 wild-type cells, and this resulted in up-regulation of Fas receptor expression at the cell membrane. The microarray data and pathway analyses presented in this study also indicated that the JAK-STAT pathway was activated in p53 null cells following exposure to SN38. Further work is ongoing to investigate the roles of JAK1/2 and STAT3 in colorectal cancer in regulating the response of colorectal cancer cells to chemotherapy in clinically relevant genetic contexts (manuscript submitted).

In this study, we focussed on the role of the focal adhesion pathway in regulating the response of colorectal cancer cells to SN38. This pathway contains components of MAPK signalling and regulators of the actin cytoskeleton, which pathway analyses also identified as being differentially regulated between p53 wild-type and null cells following SN38 treatment. Several studies have demonstrated a link between focal adhesion kinase (FAK) and p53; specifically, p53 binds to the FAK promoter and regulates its activity. In addition, several studies have demonstrated that FAK is up-regulated in breast and CRC tumours which contain p53 mutations compared to those expressing wild-type p53 (26-28). Real-time RT-PCR results demonstrated that following SN38 treatment, a number of key components of the focal adhesion pathway were differentially regulated in p53 wild-type and p53 null cells. Some of the most marked differences between p53 wild-type and p53 null cells were observed for those focal adhesion pathway genes that are also components of the MAPK signalling pathway.

As several pathways that regulate cell migration were identified as being differentially regulated in p53 wild-type and p53 null cells, we carried out a number of cell migration experiments. Constitutively, p53 null cells displayed a lower level of cell migration compared to the p53 wild-type cells, which is in agreement with the results of the pathway analyses. Importantly, following SN38 treatment, the p53 null cells displayed a significantly faster rate of migration compared to the p53 wild-type cells, which again is in agreement with the transcriptional profiling and pathway analysis results. In addition, we also found that a stably SN38-resistant p53 null daughter cell line migrated at a faster rate than the parental p53 null cell line; in contrast, the corresponding p53 wild-type SN38-resistant cell line failed to migrate at all. We speculated that the differences between the migratory potential of the p53 wild-type and null HCT116 isogenic cell lines was likely due to the differences we observed in the focal adhesion signalling pathway, which was found to be constitutively lower in the p53 null setting but which, following SN38 treatment, was up-regulated in p53 null cells, but not their p53 wild-type counterparts. In support of this, the increased migratory potential observed in p53 null cells following SN38 treatment was completely abrogated following co-treatment with MEK inhibitors. This suggests that following SN38 treatment in the p53 null setting, activation of the MAPK signalling pathway leads to enhanced migratory potential, which can be reversed by MEK inhibition. One of the limitations of the current study is that all of the initial findings have been carried out in one isogenic cell line model system; it would be of benefit to confirm these findings in additional p53 wt and null isogenic cell line pairs. In confirmation of our results in the isogenic HCT116 cell line models, we also found that SN38 treatment increased the migratory potential of the p53 mutant DLD1 and HCC2998 cell lines in a MEK-dependent manner, whereas SN38 had no effect or actually decreased the migration of the p53 wild-type LoVo and RKO cell lines respectively. A recent study by Muller et al., demonstrated that mutant p53 can promote cell migration in vitro and in vivo through recycling of integrins to the membrane (29). We also found differential expression of integrins following SN38 treatment in the isogenic HCT116 cell line models, with a higher expression of INTGA6 and INTGA3 in p53 null compared to p53 wild-type cells. In terms of SN38 resistance, MAPK signalling pathway plays a key role in inflammatory response, differentiation, cell cycle arrest, apoptosis, senescence and survival (30), with many of the substrates of ERKs involved in promoting cell proliferation and survival. Previously, a number of studies have highlighted the importance of p38 MAPK signalling in regulating resistance to irinotecan therapy. Specifically, p38 has been demonstrated to be activated in SN38-resistant colorectal cancer cells (31). In addition, pharmacological inhibition of p38α and β was found to sensitise SN38-resistant CRC cells to treatment with SN38 (32).

In conclusion, we have used microarray profiling to identify a number of functionally relevant pathways that are differentially regulated following SN38 treatment in p53 wild-type and p53 null cells. Notably, following SN38 treatment, the focal adhesion pathway is activated in p53 null cells but down-regulated in p53 wild-type cells. Furthermore, we report that a component of the focal adhesion pathway, MAPK, is involved in mediating the enhanced migratory potential of p53 null cells following SN38 treatment. We have also shown that there are differential migration rates in parental and SN38-resistant daughter cells that differ depending on p53 status. In addition, we have demonstrated that the increased migratory potential observed in p53null/mutant cells is a unique feature of SN38 treatment as the other two treatments used in the management of advanced colorectal cancer, 5-FU and oxaliplatin do not differentially effect CRC cell migration based on p53 status. Thus, targeting MAPK signalling may prove a viable strategy for enhancing the therapeutic potential of irinotecan in p53 null or mutant CRC by decreasing drug-induced cell migration. Finally, in terms of personalised therapy, p53 status would represent an important prognostic biomarker for patients who receive irinotecan therapy. This will require further retrospective and prospective clinical testing Moreover; further analyses are needed to assess the effectiveness of irinotecan-based therapy in patients with p53null/mutant tumours.