Abstract
BackgroundActivin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers.MethodsFifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH) was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2′-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria.ResultsOf 51 MSS colon tumors, 7(14%) lost ACVR2, 2 (4%) ACVR1, and 5(10%) pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05).ConclusionsOnly a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer.
Highlights
Colon cancers with high frequency microsatellite instability (MSI-H) are associated with mutations in several genes with coding repetitive sequences, such as transforming growth factor b receptor 2 (TGFBR2) and activin type 2 receptor (ACVR2) [1,2,3]
We found a subset of microsatellite stable (MSS) colon cancers that lost Activin receptor 2 (ACVR2) expression [2], akin to TGFBR2 loss found in MSS colon cancers [4]
We found that loss of ACVR2 expression occurs in a subset of MSS tumors, which is often associated with retained pSMAD2, the downstream effector of both TGFb and activin signaling
Summary
Colon cancers with high frequency microsatellite instability (MSI-H) are associated with mutations in several genes with coding repetitive sequences, such as transforming growth factor b receptor 2 (TGFBR2) and activin type 2 receptor (ACVR2) [1,2,3]. Similar to TGFb, activin utilizes two cell surface receptors, activin receptor 1 (ACVR1) and activin receptor 2 (ACVR2), followed by SMAD activation. Another type 2 receptor, ACVR2B, cannot substitute for the functions and signaling of ACVR2 [9]. Activin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. We examined activin signaling disruption in microsatellite stable (MSS) colon cancers
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