Genomic and proteomic characterization of ARID1A chromatin remodeller in ampullary tumors

Abstract

AT rich interactive domain 1A (ARID1A) is one of the most commonly mutated genes in a broad variety of tumors. The mechanisms that involve ARID1A in ampullary cancer progression remains elusive. Here, we evaluated the frequency of ARID1A and KRAS mutations in ampullary adenomas and adenocarcinomas and in duodenal adenocarcinomas from two cohorts of patients from Singapore and Romania, correlated with clinical and pathological tumor features, and assessed the functional role of ARID1A. In the ampullary adenocarcinomas, the frequency of KRAS and ARID1A mutations was 34.7% and 8.2% respectively, with a loss or reduction of ARID1A protein in 17.2% of the cases. ARID1A mutational status was significantly correlated with ARID1A protein expression level (P=0.023). There was a significant difference in frequency of ARID1A mutation between Romania and Singapore (2.7% versus 25%, P=0.04), suggestive of different etiologies. One somatic mutation was detected in the ampullary adenoma group. In vitro studies indicated the tumor suppressive role of ARID1A. Our results warrant further investigation of this chromatin remodeller as a potential early biomarker of the disease, as well as identification of therapeutic targets in ARID1A mutated ampullary cancers.

Keywords: ARID1A; Ampullary cancer; KRAS; Sanger sequencing.

Figure 1

Flow chart summarizing the patients groups, experiments and analysis performed within the study.

Figure 2

Analysis of ARID1A and KRAS non-synonymous somatic mutations. A: Plot summarizing samples with non-synonymous somatic mutations in ARID1A and KRAS based on tumor site and immunohistochemistry subtype classification. B: Graphic representation of ARID1A and KRAS somatic non-synonymous mutations (modified from cBio portal). C: Type of KRAS non-synonymous somatic mutations based on tumor site and percentage of KRAS mutations in the whole cohort. D: Survival curves of ampullary adenocarcinoma cohort on mutational status of ARID1A and KRAS (in the wild type group were included patients with somatic or germline synonymous mutations but non-synonymous germline mutations were excluded.

Figure 5

A: Immunohistochemistry of representative cases stained for CK20, CDX2, MUC1 and MUC2 as either negative or positive. B: Immunofluorescence of KRT19 in SNU-478 and SNU-869 cell lines (picture taken with 4× objective).

Figure 3

ARID1A genomic and proteomic characterization in representative cases with mutations. For each case, three panels are presented: upper left-sequencing chromatograms for normal and tumoral sample, bottom-left: immunohistochemistry staining, right-analysis of copy number alterations by ASCAT in tumoral sample (top panel-Log R ratio, middle panel: B allele frequency, bottom panel: allele specific copy numbers).

Figure 4

Functional studies in SNU-478 and SNU-869 cell lines. A: Relative proliferation curves, qPCR and western blot in SNU-478 cell line treated with non-targeting siRNA or Pool siRNA, siARID1A6 and siARID1A7. B: Relative proliferation curves, qPCR and Western blot in SNU-869 cell line treated with non-targeting siRNA or Pool siRNA, siARID1A6 and siARID1A7. C: Real time PCR analysis of EMT markers in SNU-478 and SNU-869 cell lines (in SNU-478, no amplification was detected for VIM and TWIST1 while CDH1 was reported to be hypermethylated (ref 34, 35).