CHD5, A Tumor Suppressor Gene Deleted From 1p36.31 in Neuroblastomas

Tomoyuki Fujita; Jun Igarashi; Erin R. Okawa; Takahiro Gotoh; Jayanthi Manne; Venkatadri Kolla; Jessica Kim; Huaqing Zhao; Bruce R. Pawel; Wendy B. London; John M. Maris; Peter S. White; Garrett M. Brodeur

Disclosures

J Natl Cancer Inst. 2008;100(13):940-949. 

In This Article

Results

CHD5 expression is restricted to neural tissues, and expression was very low or absent in all the neuroblastoma cell lines examined.[22] We previously examined CHD5 for inactivating mutations in 30 neuroblastoma cell lines but found only one missense mutation.[12] Thus, CHD5 is not homozygously inactivated by deletion or mutation with substantial frequency, so homozygous inactivation would require an epigenetic mechanism. Therefore, we investigated whether CHD5 expression was silenced by methylation in these lines.

We determined the methylation status of the CHD5 promoter in four cell lines—two with no CHD5expression (both with 1p deletion: NLF, IMR5) and two with low CHD5 expression (both without 1p deletion: SK-N-SH, S-KN-FI)—and in human fetal brain, which has high CHD5 expression. There were strong sites of methylation in NLF and IMR5 (60%–100%) between base pairs –780 to –450 and a secondary site between –400 and –340 bp (Figure 1, A and B). The distal site showed much lower or no methylation in the two cell lines with low CHD5 expression (Figure 1, C and D) or in human fetal brain (data not shown). We grew these lines in increasing concentrations of 5-aza-2-deoxycytidine for up to 7 days and measured normalized CHD5 expression using quantitative RT-PCR. A dose- and time-dependent increase in expression was observed in all lines that was most dramatic (one to two orders of magnitude) in the two cell lines with 1p deletion and no CHD5 expression, compared with only two- to fourfold in the cell lines with readily detectable CHD5 expression (data not shown). Thus, there was a strong association between methylation of the distal promoter site and CHD5 expression, although no well-established regulatory motifs were apparent. Moreover, these data suggest that this site contains an important regulatory domain and they support promoter methylation as a possible mechanism for epigenetic silencing of CHD5 transcription.

Methylation of the CHD5 promoter and 5' coding region in four neuroblastoma cell lines. AB) CHD5 promoter methylation in NLF (A) and IMR5 (B) cells, both of which have hemizygous 1p deletions and virtually no CHD5 expression. CD) CHD5 promoter methylation in SK-N-SH (C) and SK-N-FI cells (D), which lack 1p deletion and have low expression of CHD5. Methylation at a given GC dinucleotide, as determined by methylation-specific sequencing, is shown as a percentage of sequences analyzed (10 per site). Methylation was determined as the number of clones with methylation of the given region divided by 10 and is expressed as a percentage.

We stably transfected CHD5 sense and antisense vectors into two neuroblastoma cell lines with 1p deletion and MYCN amplification (NLF and IMR5) and two with neither aberration (SK-N-SH and SK-N-FI). We examined the morphology, growth, differentiation, and apoptosis rates of these cell lines, but no major differences were observed (data not shown). We repeated these experiments under low serum conditions but again observed no major differences.

Next, we plated cells from CHD5-transfected and CHD5-AS–transfected lines into soft agar. After 3 weeks of culture, NLF-CHD5-AS clones formed statistically significantly more colonies (mean = 74 large colonies per plate, 95% confidence interval [CI] = 62 to 86 colonies) than NLF-CHD5 clones (mean = 43 colonies, 95% CI = 35 to 51 colonies (P < .001) (Figure 2, A). Similarly, the IMR5-CHD5-AS control cells formed more colonies (mean = 39 colonies, 95% CI = 17 to 60 colonies) than the CHD5-transfected cells (mean = 11 colonies, 95% CI = 2 to 20 colonies) (P = .01) (Figure 2, B). However, there was no difference between the number of colonies formed by CHD5-transfected and CHD5-AS–transfected clones of SK-N-SH (P = .16) and SK-N-FI (P = .1) (Figure 2, C and D). Thus, CHD5 expression caused a statistically significant reduction in colony formation in the CHD5-expressing clones relative to the CHD5-AS clones, but only in cell lines with 1p deletion.

Effect of altered CHD5 expression on clonogenicity in neuroblastoma cell lines. Plasmids containing CHD5 or antisense CHD5 (CHD5-AS) were stably transfected into NLF and IMR5 neuroblastoma cell lines, both of which have hemizygous 1p deletion and MYCN amplification, and into SK-N-SH and SK-N-FI neuroblastoma cell lines, which have neither. Transfected cells were plated on soft agar, and colonies were counted 3 weeks later. A) NLF cells. CHD5 vs CHD5-AS, P < .001. B) IMR5 cells. CHD5 vs CHD5-AS, P = .001. C) SK-N-SH cells. CHD5 vs CHD5-AS, P = .16. D) SK-N-FI cells. CHD5 vs CHD5-AS, P = .10. Means and 95% confidence intervals (error bars) are shown. P values (two-sided) were calculated using the Student t test. Data are representative of three independent experiments performed in quadruplicate.

Next, we injected neuroblastoma cells stably expressing CHD5 or CHD5-AS into the flanks of immunosuppressed nu/nu mice. Tumors became palpable within 1–2 weeks, but there was dramatic growth inhibition of CHD5-expressing clones for both NLF and IMR5 neuroblastoma cell lines compared with parental cell lines or CHD5-AS controls (Figure 3, A and B). The mean tumor volume for NLF-CHD5-AS at 5 weeks after injection was 1.65 cm3 (CI = 0.83 to 2.46 cm3), compared with 0.36 cm3 (CI = 0.17 to 0.44 cm3) for NLF-CHD5 (P = .002). The mean tumor size for IMR5-CHD5-AS at 5 weeks after injection was 1.15 cm3 (CI = 0.43 to 1.87 cm3), compared with 0.28 cm3 (CI = 0.18 to 0.38 cm3) for IMR5-CHD5 (P = .01). Both studies were repeated with similar results (data not shown). There was no statistically significant difference in growth rates between CHD5- and CHD5-AS–transfected SK-N-SH and SK-N-FI tumors (Figure 3, C and D), again demonstrating the specificity of tumor growth suppression for neuroblastoma lines with 1p deletion.

Effect of CHD5 expression on tumor growth in neuroblastoma cell lines. NLF, IMR5, SK-N-SH, and SK-N-FI cells (107 each) stably transfected with plasmids containing CHD5- or CHD5-AS were injected into the flanks of nu/nu mice (n = 10 mice per group), and tumor growth was measured as volume weekly over 5 weeks. A) NLF CHD5-AS vs NLF CHD5, P = .002. B) IMR5 CHD5-AS vs IMR5 CHD5, P = .01. C) SK-N-SH CHD5-AS vs SK-N-SH CHD5, P = .07. D) SK-N-FI CHD5 vs SK-N-FI CHD5-AS, P = .26. Means and 95% confidence intervals (error bars) are shown. All P values (two-sided, comparisons at the 5-week time points) were calculated using a two-sample Student t test. Data are representative of two independent experiments.

We also observed an effect on histology for the CHD5-transfected tumors. The control tumors for NLF and IMR5 were undifferentiated with scant cytoplasm, as is typical of neuroblastomas (Figure 4, A). The NLF-CHD5 tumors had areas of differentiation and necrosis, whereas the IMR5-CHD5–transfected tumors had a more elongated morphology (Figure 4, A). We examined the mRNA expression of CHD5 in xenograft tumors derived from parental NLF and IMR5 cells, as well as the CHD5-transfected and CHD5-AS–transfected clones, by real-time RT-PCR. CHD5 mRNA expression was higher in the CHD5-transfected clones than their respective parental lines (Figure 4, B), and expression in CHD5-AS cells was intermediate. We also examined CHD5 protein expression in the NLF and IMR5 lines. The CHD5 protein (250–260 kD) was detected only in the lines that were transfected with the CHD5-sense construct (Figure 4, C). In summary, there was clear suppression of both clonogenicity and xenograft tumor growth of CHD5-transfected clones, but only in tumors with 1p deletion.

Histology and CHD5 expression in xenografts derived from NLF and IMR5 cells transfected with either CHD5 sense or CHD5 antisense constructs. A) Histology of NLF and IMR5 xenograft tumors (see Figure 3) after 5 weeks of growth. NLF-CHD5-AS tumors were composed of undifferentiated cells with scant cytoplasm (top left), whereas NLF-CHD5 tumors showed areas of necrosis (arrowheads) and differentiation (arrows; top right). Cells in the IMR5-CHD5-AS tumors were undifferentiated (bottom left), whereas cells in the IMR5-CHD5 tumors had a more elongated appearance (bottom right). Bar = 20 µm. B) Relative expression of CHD5, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was determined by real-time reverse transcription–polymerase chain reaction in parental NLF and IMR5 cell lines, as well as the CHD5 sense and CHD5 antisense transfected lines used for the xenograft experiments. The normalized values indicated by each bar graph represent the mean of three measurements. Replicate measurements were within 10% of the mean for each bar shown. C) Expression of CHD5 protein, as detected by immunoblotting, is shown for the NLF and IMR5 parental lines and the corresponding sense- and antisense-transfected cells used in these experiments.

We analyzed microarray expression data from 101 neuroblastomas representing well-defined risk groups (low, intermediate, high, and ultrahigh)[23] to assess clinical associations of candidate tumor suppressor gene expression from the SRD.[10,12] Only 12 of the 23 genes from the SRD were represented on the microarray chip, but these included CHD5, three other leading tumor suppressor gene candidates (TNFRSF25, CAMTA1, and AJAP1),[12] and eight other genes. We compared expression patterns (high vs low) of all genes with clinical and biologic variables currently used in risk stratification of neuroblastoma patients, including patient age and INSS stage, as well as features of the tumor (MYCN amplification, 1p deletion, 11q deletion, tumor cell DNA content/ploidy, Shimada histopathology). These features were also combined into risk groups, as described previously.[23,29]

CHD5 expression had the strongest association with all eight clinical and biologic features ( Table 1 ). Expression of ICMT, THAP3, CAMTA1, ACOT7, KCNAB2, RPL22, and KLHL21 had weaker associations and/or associations with fewer variables (two to seven variables). Expression of TNFRSF25 was associated with only one variable, and expression of the remaining three genes (AJAP1, NPHP4, HES2) showed no statistically significant association with any of the variables. High CHD5 expression was strongly associated with favorable clinical and biologic features: younger age (either <12 or <18 months), lower stage (1, 2, 4S vs 3 and 4), non-amplified MYCN, hyperdiploidy, favorable histopathology, and normal 11q ( Table 1 ) (P < .001 for all). CHD5 expression was also statistically significantly lower in tumors with 1p deletion, and expression was inversely proportional to risk group (Figure 5, A and B; P < .001). Together, these data strongly suggest that CHD5 is the tumor suppressor gene deleted from the 1p36 SRD.

Association of CHD5 expression with risk factors and outcome in primary neuroblastomas. A) Normalized CHD5 expression in 101 primary neuroblastomas stratified based on the presence (n = 26) or absence (n = 75) of 1p deletion (two-sample t test, P < .001). B) Association of normalized CHD5 expression with the risk group (low, intermediate, high, and ultrahigh) as defined above and in.[23] Briefly, for this study, low-risk patients were defined as infants (<1 year of age) with stage 1 or 2 disease by the International Neuroblastoma Staging (INSS) system[24] and favorable biologic features. Intermediate-risk patients were almost all patients with INSS stage 3 disease who were older than 1 year. High-risk patients were defined as those with INSS stage 3 or 4 (only two had stage 3) disease who were older than 1 year. High-risk patients were divided into two subsets: those without MYCN amplification (high risk) and those with MYCN amplification (ultrahigh risk). Twenty-eight were low-risk, 21 were intermediate-risk, 32 were high-risk (without MYCN amplification), and 20 were ultrahigh risk (with MYCN amplification) patients. Survival data were available for 99 of the 101 patients. The association of CHD5 expression with risk group was assessed (analysis of variance, P < .001). The box stretches from the 25th to 75th percentile. The median is shown with a line across the box. The whiskers extend to the highest and lowest observed values that are lower than and higher than 1.5 times the interquartile range from the third and first quartile values. The solid circles are outlying values beyond the whisker edges. C) Association of CHD5 expression with event-free survival in univariate analysis (log-rank, P < .001). D) Association of CHD5 expression with overall survival in univariate analysis (log-rank, P < .001). HR = hazard ratio; CI = confidence interval. All P values are two-sided.

We compared event-free survival and overall survival for 99 of the 101 neuroblastoma patients based on the expression of candidate tumor suppressor genes, with or without adjustment for 1p deletion and MYCN amplification. CHD5 was the only gene whose expression was strongly associated with event-free survival in both univariate and multivariable analyses ( Table 1 ). Expression of the KCNAB2, CAMTA1, THAP3, and TNFRSF25 genes was weakly associated with outcome by log-rank and/or Cox regression, but only in univariate analyses. High chd5 expression was strongly associated with favorable event-free survival and overall survival (event-free survival: HR = 6.7, 95% CI = 2.8 to 16.1, and overall survival: HR = 7.1, 95% CI = 2.5 to 20.8; both P < .001; Figure 5, C). Cox regression analysis showed that, after adjustment for CHD5 expression, 1p deletion was no longer statistically significantly associated with survival. Conversely, after including 1p deletion or MYCN amplification separately or jointly in the models, CHD5 expression remained statistically significantly associated with event-free survival (P = .027) ( Table 1 ). These results demonstrate that CHD5 expression is strongly associated with outcome in neuroblastomas, CHD5 is the likely target of 1p deletions in these tumors, and it may have a direct role in the biology and behavior of neuroblastomas.

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