ARID1B, a member of the human SWI/SNF chromatin remodeling complex, exhibits tumour-suppressor activities in pancreatic cancer cell lines

Abstract

Background: The human ATP-dependent SWItch/sucrose nonfermentable (SWI/SNF) complex functions as a primary chromatin remodeler during ontogeny, as well as in adult life. Several components of the complex have been suggested to function as important regulators of tumorigenesis in various cancers. In the current study, we have characterised a possible tumour suppressor role for the largest subunit of the complex, namely the AT-rich interaction domain 1B (ARID1B).

Methods: We performed Azacytidine and Trichostatin A treatments, followed by bisulphite sequencing to determine the possible DNA methylation-induced transcription repression of the gene in pancreatic cancer (PaCa) cell lines. Functional characterisation of effect of ARID1B ectopic expression in MiaPaCa2 PaCa cell line, which harboured ARID1B homozygous deletion, was carried out. Finally, we evaluated ARID1B protein expression in pancreatic tumour samples using immunohistochemistry on a tissue microarray.

Results: ARID1B was transcriptionally repressed due to promoter hypermethylation, and ectopic expression severely compromised the ability of MiaPaCa2 cells to form colonies in liquid culture and soft agar. In addition, ARID1B exhibited significantly reduced/loss of expression in PaCa tissue, especially in samples from advanced-stage tumours, when compared with normal pancreas.

Conclusion: The results therefore suggest a possible tumour-suppressor function for ARID1B in PaCa, thus adding to the growing list of SWI/SNF components with a similar function. Given the urgent need to design efficient targeted therapies for PaCa, our study assumes significance.

Figure 1

Evaluation of CpG methylation-induced transcriptional repression of ARID1B. Panels (A) and (B) show effect of Azacytidine and TSA, respectively, on several PaCa cell lines; fold increase in ARID1B transcript level in each cell line is plotted separately in response to varying concentrations (in μℳ indicated) of Azacytidine and TSA. The transcript level in absence of treatment in each cell line is normalised to 1.0. Error bars represent s.e.m.; each Azacytidine/TSA experiment was performed at least thrice. A, Azacytidine; T, Trichostatin (A). Panel (C) (top) shows position of the four ARID1B primer pairs used for PCR amplification of putative ARID1B promoter CpG island. Nucleotide position of the 5′ and 3′ ends of the four amplicons with respect to the translation initiation codon (ATG) are indicated. The bottom panel depicts the result of bisulphite sequencing-based evaluation of methylation status for the P3 region. Each row of boxes represents result for one cell line; each box represents one CpG dinucleotide and per cent of cytosine methylation is denoted by a colour code (white, <10% green, 10–33% orange, 34–66% red, >66%). The total number of clones analysed for each cell line is given at the end of each row of boxes. Methylation status in SW1990 was also evaluated, following Azacytidine (8 μℳ) treatment.

Figure 2

Functional characterisation of ARID1B permanent transfectants of MiaPaCa2. Panels (A) and (B) show result of MTT and crystal violet dye extraction growth assays performed for the two ARID1B (A3 and A8) and two vector (PC14 and PC15) clones of MiaPaCa2, respectively. Error bars represent s.e.m. of two (MTT assay) and three (crystal violet assay) independent experiments (performed in duplicate), respectively. Panels (C) and (E) show representative results of liquid and soft agar colony-formation assays, respectively. Panels (D) and (F) show graphical representation of result of liquid and soft agar colony-formation assays, respectively. Panel (G) shows quantitation of β-galactosidase staining in the same clones. Error bars represent s.e.m. of three independent liquid colony formation (in duplicate), soft agar and β-galactosidase staining (both in triplicate) experiments. P-value corresponds to unpaired Student's t-test.

Figure 3

Analysis of ARID1B and p53 expression using a pancreatic cancer tissue microarray. Panel (A) shows representative results for ARID1B for normal pancreas (left, strong nuclear stain) and pancreatic tumour (middle, moderate nuclear stain and right, negative nuclear stain). Representative results for p53 IHC performed on PaCa samples are shown in panel (B) (left, positive; right, negative). Panel (C) shows graphical representation of comparative analysis of ARID1B expression in pancreatic tumour and normal samples; samples were split into two categories based on their IHC scores (0–3 representing negative to weak staining and 4–7, indicating moderate to strong staining) calculated as described in MATERIALS AND METHODS. Panel (D) shows graphical representation of comparative analysis of ARID1B expression in early (T1–T2) and late (T3–T4) stage pancreatic tumour samples, based on IHC scores. Fisher's exact test P-values are shown.