Epigenetics Epigenetic inactivation of the candidate ...

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Epigenetic inactivation of the candidate tumor suppressor USP44 is a frequent and early event in colorectal neoplasia a

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Mathew A Sloane , Jason WH Wong , Dilmi Perera , Andrea C Nunez , John E Pimanda , b

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Nicholas J Hawkins , Oliver M Sieber , Michael J Bourke , Luke B Hesson & Robyn L Ward

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Adult Cancer Program; Lowy Cancer Research Centre and Prince of Wales Clinical School; Sydney, NSW Australia b

School of Medical Sciences; University of New South Wales; Sydney, NSW Australia

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Colorectal Cancer Genetics Laboratory; Systems Biology and Personalised Medicine Division; Walter and Eliza Hall Institute of Medial Research; Parkville, VIC Australia

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d

Faculty of Medicine, Dentistry and Health Sciences; Department of Medical Biology; University of Melbourne; Parkville, VIC Australia e

Department of Gastroenterology and Hepatology; Westmead Hospital; Sydney, NSW Australia Published online: 16 May 2014.

To cite this article: Mathew A Sloane, Jason WH Wong, Dilmi Perera, Andrea C Nunez, John E Pimanda, Nicholas J Hawkins, Oliver M Sieber, Michael J Bourke, Luke B Hesson & Robyn L Ward (2014) Epigenetic inactivation of the candidate tumor suppressor USP44 is a frequent and early event in colorectal neoplasia, Epigenetics, 9:8, 1092-1100, DOI: 10.4161/epi.29222 To link to this article: http://dx.doi.org/10.4161/epi.29222

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Research Paper Research Paper

Epigenetics 9:8, 1092–1100; August 2014; © 2014 Landes Bioscience

Epigenetic inactivation of the candidate tumor suppressor USP44 is a frequent and early event in colorectal neoplasia Mathew A Sloane1, Jason WH Wong1, Dilmi Perera1, Andrea C Nunez1, John E Pimanda1, Nicholas J Hawkins2, Oliver M Sieber3,4, Michael J Bourke5, Luke B Hesson1, and Robyn L Ward1,* Adult Cancer Program; Lowy Cancer Research Centre and Prince of Wales Clinical School; Sydney, NSW Australia; 2School of Medical Sciences; University of New South Wales; Sydney, NSW Australia; 3Colorectal Cancer Genetics Laboratory; Systems Biology and Personalised Medicine Division; Walter and Eliza Hall Institute of Medial Research; Parkville, VIC Australia; 4Faculty of Medicine, Dentistry and Health Sciences; Department of Medical Biology; University of Melbourne; Parkville, VIC Australia; 5Department of Gastroenterology and Hepatology; Westmead Hospital; Sydney, NSW Australia

Keywords: DNA methylation, epigenetic, CpG island, adenoma, colorectal cancer, aneuploidy, deubiquitinase

Downloaded by [UNSW Library] at 18:49 01 December 2014

Abbreviations: USP44, Ubiquitin Specific Protease 44; DUB, deubiquitinase; LOH, loss of heterozygosity; CNV, copy number variation; CRC, colorectal cancer; COBRA, Combined bisulfite restriction analysis; CGI, CpG island

In mouse models, loss of the candidate tumor suppressor gene Ubiquitin Specific Protease 44 (USP44) is associated with aneuploidy and cancer. USP44 is also transcriptionally silenced in human cancers. Here we investigated the molecular mechanism of USP44 silencing and whether this correlated with aneuploidy in colorectal adenomas. DNA methylation at the USP44 CpG island (CGI) promoter was measured using combined bisulfite restriction analysis (COBRA) in colorectal cancer (CRC) cell lines (n = 18), and with COBRA and bisulfite sequencing in colorectal adenomas (n = 89) and matched normal colonic mucosa (n = 51). The USP44 CGI was hypermethylated in all CRC cell lines, in most colorectal adenomas (79 of 89, 89%) but rarely in normal mucosa samples (3 of 51, 6%). USP44 expression was also compared between normal mucosa and paired hypermethylated adenomas in six patients using qRT-PCR. Hypermethylation of the USP44 CGI in adenomas was associated with a 1.8 to 5.5-fold reduction in expression compared with paired normal mucosa. Treatment of CRC cell lines with the DNA hypomethylating agent decitabine resulted in a 14 to 270-fold increase in USP44 expression. Whole genome SNP array data showed that gain or loss of individual chromosomes occurred in adenomas, but hypermethylation did not correlate with more aneuploidy. In summary, our data shows that USP44 is epigenetically inactivated in colorectal adenomas, but this alone is not sufficient to cause aneuploidy in colorectal neoplasia.

Introduction USP44 is a deubiquitinase (DUB) that plays an important role in the pathogenesis of cancer through its ability to regulate the mitotic spindle and the spindle checkpoint.1,2 In vitro studies have demonstrated that correct spindle geometry, interpole distance, and centrosome separation are regulated by direct binding of USP44 to the centriole protein centrin 2 (CETN2) at centrosomes,2 while USP44-mediated deubiquitination of CDC20, an inhibitor of the Anaphase Promoting Complex, maintains metaphase plate integrity, and prevents premature entry into anaphase and cytokinesis defects.1 Data suggesting a role for USP44 in tumorigenesis is derived from animal models and studies of human tissues. Usp44 knockout (Usp44−/−) mice have an increased incidence of spontaneous tumors and tumor burden.2 Aneuploidy, an abnormal number of chromosomes in a cell, either more or less than the diploid

number,3 occurs in tumors, splenocytes and mouse embryo fibroblasts (MEFs) derived from Usp44−/− mice.2 USP44 mRNA levels are significantly reduced in CRC and other cancers including B-ALL, breast, esophogeal, glioblastoma, kidney and testicular cancer.4 Reduced USP44 expression in human lung adenocarcinomas is also associated with more aggressive tumors and reduced overall survival.2 Taken together these data have led to the suggestion that USP44 is a bonafide tumor suppressor gene and that it functions to prevent aneuploidy from developing during cell division. However, the mechanistic basis of loss of USP44 expression in human tumors is unknown. Aneuploidy causes developmental abnormalities and is usually lethal. Paradoxically, it is common in cancer, indicating that it confers a selective survival advantage to cancer cells.3,5 Nonrandom structural rearrangements such as deletions or unbalanced translocations in chromosome 1p, and numerical changes in specific chromosomes, such as gains of chromosomes 7, 13,

*Correspondence to: Robyn L Ward; Email: [email protected] Submitted: 03/05/2014; Revised: 04/21/2014; Accepted: 05/13/2014; Published Online: 05/16/2014 http://dx.doi.org/10.4161/epi.29222 1092 Epigenetics

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©2014 Landes Bioscience. Do not distribute.

1



Figure 1. CGI methylation inactivates USP44 expression in CRC cell lines. (A) qRT-PCR showing the relative expression of USP44 in RKO and HEK293T cells. The methylation status of USP44 in each cell line is shown in the COBRA adjacent to the graph. +, TaqI digest; -, undigested control. Complete digestion of the PCR product in RKO cells indicates complete methylation. (B) A schematic showing the region analyzed for DNA methylation using COBRA within the USP44 CGI. (C) A COBRA assay showing USP44 is completely methylated in all tested CRC cell lines (n = 18). (D) qRT-PCR showing an increase in USP44 expression in four CRC cell lines after treatment with decitabine. Day 0, untreated; Day 3, 72 h exposure to decitabine; Day 5, 48 h post-exposure to decitabine. (E) Bisulfite sequencing of the USP44 promoter in untreated RKO cells and cells collected 2 d after a 72 h exposure to decitabine. Black circle, methylated CpG; white circle, unmethylated CpG; gray circle, inconclusive.

and X, and losses of 4, 8, 14, 15, 18, 21, and Y occur in CRC,6 with between 40 and 80% of CRCs displaying aneuploidy.7

In this study, we determined whether CGI promoter hypermethylation was responsible for loss of USP44 expression in colorectal neoplasia and whether this correlated with aneuploidy.

www.landesbioscience.com Epigenetics 1093

Location in GIT

Number of adenomas

Number of unmethylated

Number of hypermethylated

Appendix

1

0

1

Cecum

22

1

21

Ascending colon

19

3

16

Transverse colon

13

1

12

Descending colon

17

4

13

Rectum

17

1

16

Total

89

10

79


Results The USP44 CGI promoter is epigenetically inactivated in CRC cell lines To determine the relationship between USP44 expression and CGI methylation we first used qRT-PCR to identify a cell line expressing USP44 and a cell line in which USP44 was silent. USP44 was expressed in HEK293T cells but mRNA transcripts were undetectable in the CRC cell line, RKO (Fig. 1A). To assess DNA methylation levels at the USP44 CGI we designed a COBRA assay within the 5′ CGI promoter of the USP44 gene (NM_001042403) (Fig. 1B). The COBRA assay detected methylation at four CpG sites within the CGI. The promoter of USP44 was unmethylated in the USP44-expressing cell line, HEK293T, but was completely methylated in RKO cells (Fig. 1A). Using the same approach we found the CGI of USP44 was completely methylated in all CRC cell lines tested (n = 18), as well as in a range of other epithelial and hematological cancer cell lines including breast (SK-BR-3, MCF7, and MDA-MB-231), ovarian (OVCAR-3), glioma (U-251), leukemia (Kasumi-I and K-562) and lymphoma cell lines (U-937) (Fig. 1C). However, the CGI was not methylated in three ovarian cancer cell lines (TOV-11D, TOV-21G, and TOV-112D) or a prostate cancer cell line (DU145) (data not shown). To demonstrate that the CGI methylation observed in cell lines correlated with transcriptional silencing of USP44, we treated four completely methylated CRC cell lines (as determined by COBRA) with decitabine, a DNA methyltransferase inhibitor. Following a 72 h treatment, USP44 was re-expressed in RKO, SW620, HCT116 and DLD-1 cells. The increase in expression ranged from 14-fold in RKO cells to 270-fold in DLD1 cells (Fig. 1D). A previous study investigating the epigenetic resilencing of the MLH1 gene in RKO cells, found that maximum expression occurred 2 d after a 72 h decitabine exposure.8 We found that USP44 expression also continued to increase at this time point in RKO and SW620 cells (Fig. 1D). Bisulfite sequencing showed that this increased expression was also concomitant with more demethylated molecules compared with untreated cells (Fig. 1E). Taken together these results show that hypermethylation of the CGI promoter inactivates the expression of USP44 in these cell lines.

USP44 is methylated more frequently in colorectal adenomas than normal colorectal mucosa Cell lines are known to acquire epigenetic changes after longterm culture,9 so we next used the COBRA assay to assess the methylation status of the USP44 CGI in colorectal adenomas and matched normal colon mucosa samples. The CGI was hypermethylated in 89% of 89 adenomas (Table 1). Paired normal mucosa was available for 51 individuals in this study. In only 3 (6%) of the normal samples, all left-sided, was the CGI hypermethylated. One of the three matched adenomas had a hypermethylated CGI and the 2 other adenomas were unmethylated (Fig. 2A). The findings from a COBRA assay in one patient that showed hypermethylation in an adenoma but not in the matched normal, was confirmed using allelic bisulfite sequencing (Fig. 2B). USP44 hypermethylation correlates with loss of expression in adenomas To determine whether epigenetic inactivation of USP44 was potentially important in colorectal tumorigenesis we first assessed its expression in normal gastrointestinal tract (GIT) tissues. Using qRT-PCR we showed that USP44 was expressed throughout the GIT (Fig. 2C), however the levels of expression were higher in the stomach and small bowel compared with most of the lower GIT. Within the colon, the expression levels of USP44 were around 3-fold higher in the cecum compared with the rest of the colon. Next, we determined the levels of USP44 expression in six adenomas that showed hypermethylation and compared them with normal colorectal mucosa from the same patient. Methylation of matched normal DNA was tested in two patients and both were unmethylated. Hypermethylation in the adenomas was associated with a 1.8 to 5.5-fold reduction in USP44 expression compared with the paired normal mucosa (Fig. 2D). Although the correlation between CGI methylation and expression was imperfect, hypermethylation was always associated with reduced expression. To determine if genetic changes could also account for reduced expression we also determined the frequency of somatic mutations in USP44 using published data from the Cancer Genome Atlas.10 Non-synonymous or frameshift mutations in USP44 were

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Table 1. The methylation status of USP44 in colorectal adenomas in relation to location in the GIT. The methylation status of the USP44 CGI promoter was assessed in 89 colorectal adenomas and 51 normal tissue samples from throughout the GIT. COBRA was used to define the methylation level as unmethylated (no digestion or minor digestion of the COBRA PCR product producing a faint 289 and/or 332 bp fragment) or hypermethylated (at least six distinct digested bands or complete digestion of the COBRA PCR product)



Figure 2. USP44 is epigenetically inactivated in colorectal adenomas. (A) COBRA was used to assess the methylation status of the USP44 CGI promoter in adenomas (Ad) and matched normal colon mucosa (N). In this representative assay USP44 was classified as unmethylated in all normal samples and hypermethylated in all adenomas. USP44 was completely methylated in the adenoma from patient 2. (B) Bisulfite sequencing showing the methylation of the USP44 COBRA fragment in one patient. The COBRA assay on this patient showed the normal to be unmethylated and the adenoma sample to be hypermethylated. Black circle, methylated CpG; white circle, unmethylated CpG; gray circle, inconclusive. (C) qRT-PCR showing the relative expression of USP44 throughout the human GIT. USP44 expression in the ileocecum was normalized to one and expression in the other tissues expressed as a proportion of the ileocecum. Negative control, water-only PCR. (D) qRT-PCR showing the relative expression of USP44 in colorectal adenomas and matched normal colon mucosa in six patients. USP44 expression in the normal in each patient was normalized to one. The COBRA assay used to assess the methylation status of USP44 in each sample is shown below the graph. +, TaqI digest; -, undigested control. www.landesbioscience.com Epigenetics 1095

Discussion In this study we showed that the USP44 gene is frequently inactivated by CGI hypermethylation in colorectal adenomas. Despite reports that loss of USP44 causes aneuploidy,1,2 we found that hypermethylation did not correlate with aneuploidy in colorectal adenomas. We showed that USP44 is expressed throughout the GIT of healthy donors and patients with colorectal adenomas. However, in adenoma tissue the USP44 CGI promoter is frequently hypermethylated relative to normal colorectal mucosa from the same individual. Furthermore, hypermethylation correlated with reduced USP44 expression in adenomas, while treatment of methylated cell lines with a hypomethylating agent reactivated USP44 expression. These findings show that USP44 is transcriptionally silenced by CGI promoter methylation. Furthermore, because we focused specifically on adenomas, these data showed that USP44

hypermethylation is an early event in the development of colorectal neoplasia. USP44 hypermethylation in cell lines derived from five other cancer types also indicated that CGI hypermethylation inactivates USP44 expression in other cancers.4 USP44 was expressed throughout normal tissues of the GIT but mRNA was more abundant in the right sided colon. The site-specific differences in USP44 may be related to the different embryological origin of the proximal and distal colon.13 Indeed, the right and left side of the colon can be distinguished based on gene expression profile signatures,14,15 and 58 genes have been shown to be differentially expressed (more than 3-fold) between normal cecum and sigmoid/rectosigmoid mucosa.15 The functional significance of higher USP44 expression in the right sided colon however is unclear. In light of the known role of USP44 in preventing aneuploidy, our observation that USP44 was epigenetically silenced suggested a possible link with the high frequency of aneuploidy in colorectal neoplasia.16 Because the exact nature of possible aneuploidy following USP44 inactivation was unclear, we examined our whole genome copy number data in several ways. We first excluded serrated adenomas from the analysis to ensure our conclusions were not confounded by this type of lesion, which are less prone to chromosomal instability. Second we identified cases of whole genome duplication, and third, we identified the gain or loss of individual chromosomes. In addition, we considered that aneuploidy might only be related to higher levels of USP44 methylation and accordingly separated specimens showing no methylation or hypermethylation. Despite these measures, we found that USP44 hypermethylation alone did not correlate with aneuploidy in adenomas. Although hypermethylation served as a reliable surrogate for reduced USP44 expression, our results showed it did not correlate with complete silencing. Our bisulfite sequencing data showed that this was most likely due to the persistence of unmethylated promoter alleles. This suggested low levels of USP44 expression may be sufficient to prevent aneuploidy and that reduced levels alone are unlikely to impact the fidelity of chromosome segregation. However, previous studies have shown that deletion of Usp44 in mice causes aneuploidy and increased tumor incidence.2 This suggests that complete loss of USP44 function is required to promote aneuploidy, however we cannot exclude the possibility that reduced levels of USP44 expression, combined with other molecular changes, may result in vulnerability to mitotic defects. We combined SNP arrays and OncoSNP to analyze CNVs in our adenomas. SNP arrays have been extensively used to identify ploidy and large-scale genomic instability in cancers.12,17,18 In addition OncoSNP has been stringently validated as a tool to accurately measure ploidy and CNVs in heterogeneous tumor samples. OncoSNP is capable of detecting tumor-specific chromosome aberrations even when contaminating normal tissue constitutes at least half of a sample.12 The percentage of each adenoma genome with LOH and CNV was variable but low, irrespective of the level of USP44 methylation. This is consistent with a previous study of Usp44−/− MEFs, that found no chromosome breaks or gaps with Giemsa staining or SKY karyotyping.2 With the exception of one adenoma, close to normal average

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detected in 7 of 296 (216 colon, 80 rectal) samples. Thus somatic mutations in USP44 are present in less than 3% of CRCs. Hypermethylation of the USP44 CGI does not correlate with aneuploidy in colorectal adenomas Having established that USP44 frequently undergoes epigenetic inactivation, we next investigated if USP44 CGI hypermethylation was associated with LOH, CNV and changes in average ploidy in a subset of 28 adenomas (Table 2). These samples had previously been hybridized to Illumina Human610-Quad BeadChips SNP arrays,11 which allowed genomic aberrations to be detected with OncoSNP.12 USP44 CGI hypermethylation determined by COBRA was a reliable predictor of reduced expression (Fig. 1 and 2), so adenomas were divided into unmethylated (7 samples) and hypermethylated (21 samples) groups based on USP44 methylation status. LOH (Fig. 3A) and CNV (Fig. 3B) were variable but low in both groups, and there was no significant increase in LOH or CNV in hypermethylated adenomas (P > 0.05). With the exception of one hypermethylated sample that had an average ploidy of three, there was also no significant departure (P > 0.05) from diploidy in the hypermethylated group (Fig. 3C). The measurement of average ploidy has a limited ability to detect the gain or loss of individual chromosomes within cells when measuring average copy number across a population of cells. Therefore, using the SNP array data, we next determined if the gain or loss of each individual chromosome occurred more frequently in hypermethylated adenomas. Chromosome gain or loss was defined as greater than 2-fold copy number change encompassing > 80% of the length of a chromosome. Chromosome gain or loss was present in adenomas irrespective of USP44 methylation status (Fig. 3D). The average number of changes was 4.1 and 1.8 (4 – 10% of the genome) in the unmethylated and hypermethylated adenomas, respectively. In summary, SNP arrays detected aneuploidy in colorectal neoplasms irrespective of USP44 hypermethylation and increasing levels of methylation did not correlate with an increase in copy number alterations in adenomas.


56.3

60.2

80.2

81.0

M

M

M

F

M

M

F

F

F

F

45284

45073

7621

46132

45279

46158

46160

7614

45280

45282

76.8

45.8

73.6

F

F

M

F

M

M

M

F

M

F

M

M

M

M

400

45080

45283

45305

7606

8873

9637

46174

46150

46152

46155

46157

45105

45107

Tubulovillous

Tubulovillous

Tubular

Tubular

Tubular

Tubular

Villous

Tubulovillous

Tubulovillous

Tubular

Tubulovillous

Tubulovillous

Tubulovillous

Tubular

Tubulovillous

Villous

Tubular

Tubulovillous

Villous

Tubulovillous

Villous

Tubulovillous

Tubulovillous

Villous

Tubulovillous

Tubular

Tubulovillous

Villous

Classification

Cecum

Ascending

Cecum

Descending

Ascending

Descending

Transverse

Cecum

Rectum

Transverse

Rectum

Descending

Transverse

Descending

Descending

Rectum

Transverse

Transverse

Cecum

Descending

Ascending

Ascending

Cecum

Cecum

Descending

Ascending

Ascending

Rectum

Location

Hypermethylated

Hypermethylated

Unmethylated

Unmethylated

Unmethylated

Hypermethylated

Hypermethylated

Hypermethylated

Hypermethylated

Hypermethylated

Unmethylated

Hypermethylated

Hypermethylated

Hypermethylated

Hypermethylated

Hypermethylated

Hypermethylated

Hypermethylated

Hypermethylated

Unmethylated

Hypermethylated

Hypermethylated

Hypermethylated

Hypermethylated

Unmethylated

Unmethylated

Hypermethylated

Hypermethylated

USP44 methylation

0

3

3

0

1

0

0

12

5

0

2

0

1

0

0

0

2

5

0

0

0

0

0

9

9

14

0

1

CNVnumber of arms

0

188601463

246746504

0

125227369

0

0

750842177

202474122

0

57118452

0

32511889

0

0

0

153344757

141088077

0

0

0

0

0

555046121

301051944

917387864

0

26121695

CNV length (bp)

2.0

2.2

2.1

2.0

2.1

2.0

2.0

3.0

2.2

2.0

1.9

2.0

2.0

2.0

2.0

2.0

2.1

2.1

2.0

2.0

1.7

2.0

2.0

2.2

2.2

2.1

2.0

2.0

Average ploidy

0.00928

0.09593

0.16263

0.0414

0

0.01187

0.02283

0.69171

0.00009

0.04197

0.13102

0.00090

0.05032

0.02135

0.02272

0.00608

0.00167

0.00004

0.04127

0.04655

0.25095

0.00541

0.00012

0.02367

0.00108

0.08036

0.03294

0.03028

Proportion of genome with LOH


60

50

Severe Moderate

20

25

Severe

Moderate

30

35

Severe Moderate

20

35

Moderate

Moderate

40

35

Severe Moderate

25

30

60

40

20

80

24

40

50

50

17

30

20

45

15

20

50

30

Tumour max. size (mm)

Severe

Severe

Severe

Moderate

Moderate

Severe

Severe

Severe

Moderate

Severe

Moderate

Severe

Severe

Moderate

Moderate

Severe

Moderate

Severe

Dysplasia in adenoma

CNV, copy number variation; LOH, loss of heterozygosity

45.4

62.4

63.0

66.8

81.8

78.4

71.3

71.9

57.0

44.3

78.0

56.5

63.7

F

F

8859

46162

62.0

64.5

58.5

70.4

76.8

79.8

74.6

57.0

F

M

45071

Age

45278

Sex

ID number

Table 2. Clinicopathological features and whole genome copy number data for the adenomas analyzed with SNP arrays


0.00066

0.04495

0.14141

0.00001

0.00014

0

0.02216

0.00018

0.00001

0.00020

0.13102

0.00018

0.00902

0.00101

0.00243

0.00608

0.00003

0.00006

0.00424

0.00823

0.25103

0.00541

0.00005

0.02069

0.00010

0.03148

0.03297

0.00019

Proportion of genome deleted

0.00004

0.06835

0.09000

0.00008

0.03355

0.000001

0.00006

0.27517

0.06776

0.00064

0.03273

0

0.01361

0.00003

0

0.02323

0.05531

0.03550

0.00474

0

0

0

0

0.20721

0.05743

0.07143

0

0.01109

Proportion of genome amplified

ploidy (n = 2) occurred in all adenomas with a hypermethylated USP44 CGI. We acknowledge that SNP arrays may underestimate the frequency of whole genome duplication (2n vs. 4n), however given that previous studies have reported that this is rare in adenomas,19 the diploidy we observed is likely to be true rather than an array artifact. In summary, we concluded that the epigenetic inactivation of USP44 is not associated with whole genome duplication. After eliminating an association between whole genome duplication and USP44 methylation, we next investigated if USP44 methylation was associated with aneuploidy that involved the gain or loss of individual chromosomes, which has been observed in Usp44−/− cells and human tumors.2 Our approach was able to detect a small number of individual chromosome gains and losses in adenomas. Using this approach the number of aberrations detected was consistent with previous studies of adenomas. Hermsen et al. (2002) reported no chromosomal abnormalities in 29% of 66 non-progressed adenomas, and an average of 4.6 aberrations in the remaining 71% of samples that were randomly distributed over chromosomes. These consistencies support the veracity of our conclusions about aneuploidy based on SNP arrays. In conclusion we used a large cohort of patient samples, matched adenoma and normal tissue, and pharmacological

treatment of cell lines to demonstrate that USP44 is transcriptionally silenced by promoter hypermethylation and that this is a frequent and early event in colorectal neoplasia. Using SNP array data sets we found that aneuploidy was present in adenomas but was not significantly associated with hypermethylation. USP44 was also recently found to regulate total cellular levels of histone H2B ubiquitination,20 a histone modification involved in transcriptional regulation.21 It also negatively regulates the RNF8/ RNF168 pathway by deubiquitinating H2A at double strand DNA breaks.22 Further research into these functions is required to elucidate how the epigenetic inactivation of USP44 contributes to the progression of CRC.

Materials and Methods Patient samples Samples used in this study were collected from patients undergoing endoscopic (n = 46) or surgical removal (n = 43) of an advanced adenoma at Westmead or St Vincent’s hospitals, Sydney. These patients were part of the Australian Colonic Endoscopic resection study (ACE),23 or the MCO tumor collection study (ethics numbers 2009/6/4.6, 11194, H00/022, 00113).24 Matched normal mucosa sample was available from 51

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Figure 3. The association between USP44 CGI methylation and genomic aberrations in colorectal adenomas. Dotplots showing the association between (A) LOH, (B) CNV, (C) average ploidy and (D) the number of chromosome arm gains and losses in adenomas with an unmethylated (n = 7) or hypermethylated (n = 21) USP44 CGI. Horizontal bars, median value.

was performed according to the kit protocol. DNA was eluted in 50 μL of elution buffer. Two μL of bisulfite converted DNA was used as template in a primary PCR containing 1x reaction buffer, 2.5 mM MgCl2, 200 μM dNTPs, 0.5 μM of USP44-F and USP44-R, and 0.5 U of Platinum® Taq DNA polymerase (Invitrogen, 10966018). PCR conditions were 5 min at 95 °C, followed by 24 cycles of 95 °C for 30 s, 56 °C for 30 s, 72 °C 40 s, followed by a final extension at 72 °C for 5 min. One μL of the primary PCR was used in a secondary PCR using a nested forward primer (USP44-FN) and USP44-R. The secondary PCR was performed in a final volume of 50 μL, with 30 cycles, primer concentrations of 0.2 μM, and the same cycling temperatures as the primary PCR. PCRs were performed in a BIO-RAD C1000TM thermal cycler. Primer sequences were: USP44 -F: G A AGGT TAT YGGG A ATGGT T TTAGGAAGT USP4 4 -F N : G AT T T T TA AGT T T TA AGYG GT AGTAGGTGT USP44-R: ACAACCTCAA AATACCCRAA AATACTA Five microliters of PCR product was digested with 5 U of TaqI (Thermo Scientific, ER0671) at 65 °C for 3 h in a 25 μL reaction containing 1× TaqI buffer with BSA (Thermo Scientific, B28). The TaqI was inactivated at 80 °C for 20 min. Twenty microliters of the digest was visualized on a 3% agarose gel. A control digest without TaqI was included for each sample. Bisulfite PCR products were cloned by ligation into the pCR®2.1-TOPO® vector and transformation was performed using the TOPO TA Cloning kit (Life Technologies, K4560-01). Individual molecules were isolated from transformed colonies by colony PCR before sequencing using BigDye Terminator v3.1 and an ABI3730 genetic analyzer (ABI). LOH, CNV and average ploidy in adenomas Data for the 28 adenoma samples (Table 2) was acquired from SNP arrays performed at the Australian Genome Research Facility (AGRF) using Illumina Human610-Quad chips. The arrays acquired the genotype of 620 901 loci distributed across the genome, as described previously.11 Data was analyzed using OncoSNP (version 2.2)12 to identify LOH, CNV and average ploidy across the genome of each adenoma. To calculate the average ploidy for each sample, the ploidy state across each genome was averaged. To assess global genomic aberrations, the size of the output LOH and CNV regions from the OncoSNP analysis were measured as a fraction of the entire genome, to indicate the proportion of the genome with CNV or LOH in each adenoma. The average ploidy, proportion of the genome with CNV and LOH were compared between corresponding patients with an unmethylated (no digestion or minor digestion of the COBRA PCR product producing a faint 289 and/or 332 bp fragment) or hypermethylated (at least six distinct digested bands or complete digestion of the COBRA PCR product) USP44 CGI promoter. Statistical significance was evaluated using the Mann-Whitney test. Whole chromosome arm gain and loss in adenomas Adenoma SNP array data from Illumina Human610-Quad BeadChips (n = 28) were analyzed to assess the percentage of the genome with CNV and LOH. In order to assess aneuploidy, if




patients, 38 from surgical resections from the same location as the adenoma, and 13 from endoscopically resected rectal biopsy. The mean age of the patients was 68 y; age range, 17–89 y; number of females, 39; number of males, 50. The histology of the adenomas was 52 tubulovillous, 22 tubular, 10 villous and 5 sessile serrated. The location of lesions is shown in Table 1. The ascending colon included the hepatic flexure. The descending colon included the sigmoid colon and splenic flexure. Cell culture Cell lines were obtained from American Type Culture Collection (ATCC) and cultured in recommended medium supplemented with 10% (v/v) FBS, 100 U penicillin, 100 µg/ mL streptomycin and 2 mM glutamate (Life Technologies) and grown at 37 °C in 5% CO2. CRC cell lines (RKO, SW620, HCT116, and DLD-1) were treated every 24 h for a period of 72 h by replacing media supplemented with 2.5 µM decitabine (5-aza-2’deoxycytidine, Sigma A3656) freshly prepared in 50% filter sterilized acetic acid. RKO and SW620 cells were also cultured for an additional 48 h in media without decitabine. The media of untreated controls was supplemented with the same amount of 50% filter sterilized acetic acid only. Gene expression RNA was extracted from cell lines, normal colorectal mucosa and adenoma samples using the PureLink® RNA Micro Kit (Life Technologies, 12183016). cDNA was prepared using the SuperScript™III cDNA Synthesis Kit (Life Technologies, 18080051) according to the kit protocol. qRT-PCR was performed in triplicate with 40 ng of cDNA. For expression analysis in the adenoma and matched normal tissue from six patients, USP44 expression was normalized to one in the normal from each patient and mRNA levels in the adenoma were expressed as a ratio relative to the normal. USP44 expression in normal tissues of the GIT was measured using a panel of first strand cDNA preparations from poly A+ RNA. The cDNA from each tissue was pre-normalized against α-tubulin, β-actin, G3PDH and phospholipase A2 (Clonetech, 636746). qRT-PCR was performed in triplicate using 2 ng of cDNA. All qRT-PCRs were performed with 1 x iQ™ TMSYBR® Green supermix (Bio-Rad, 170–8880), and 0.5 μM of each primer (USP44 F: TGAGTACAAC TGGTTTGGAG GA; USP44 R: CAGCCATGTCT GGTTACTGAA A) in a Bio-Rad C1000TM thermal cycler. For expression analysis in the GIT, USP44 expression was normalized to one in the ileocecum where maximum expression was observed. Levels of mRNA in the other tissues in the GIT were then expressed as a ratio relative to the ileocecum. DNA methylation assays DNA was extracted from cell lines and tissues in 10 mM TRISHCl pH 7.8, 1 mM EDTA, 100 mM NaCl, 1% (w/v) SDS, treated with Proteinase K and purified by phenol-chloroform extraction and ethanol precipitation. Bisulfite conversion of genomic DNA was performed with the EZ DNA Methylation-GoldTM Kit (Zymo Research, D5006). One μg of genomic DNA was prepared in 20 μL of H2O and added to 130 μL of conversion reagent. The reaction was divided into three 50 μL aliquots and incubated at 98 °C for 10 min, followed by an overnight incubation at 53 °C. The three reactions were pooled and purification of converted DNA

the combined length of regions with CNV > 2 or < 2 was greater than 80% within a chromosome arm, this was classified as whole chromosome arm gain or loss, respectively. This was then correlated with the methylation status of the USP44 CGI promoter status in each sample. Methylation was measured using COBRA. Statistical significance in adenomas and the TCGA data set was evaluated using the Mann-Whitney test.

Acknowledgments

This work was supported by the NSW Cancer Council (RG 13-07) and Cancer Australia. OS is also supported by a NHMRC R.D. Wright Biomedical Career Development Fellowship, JWHW by a Cancer Institute NSW Early Career Fellowship and LBH by a Cancer Institute New South Wales Career Development Fellowship

Disclosure of Potential Conflicts of Interest


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Volume 9 Issue 8


The Authors state they have no conflict of interest