Background: The non-steroidal anti-inflammatory drug (NSAID) sulindac has shown efficacy in preventing colorectal cancer. This potent anti-tumorigenic effect is mediated through multiple cellular pathways but is also accompanied by gastrointestinal side effects, such as colon inflammation. We have recently shown that sulindac can cause up-regulation of pro-inflammatory factors in the mouse colon mucosa. The aim of this study was to determine the signaling pathways that mediate the transcriptional activation of pro-inflammatory cytokines in colon cancer epithelial cells treated with sulindac sulfide.Results: We found that sulindac sulfide increased NF-κB signaling in HCT-15, HCT116, SW480 and SW620 cells, although the level of induction varied between cell lines. The drug caused a decrease in IκBα levels and an increase of p65(RelA) binding to the NF-κB DNA response element. It induced expression of IL-8, ICAM1 and A20, which was inhibited by the NF-κB inhibitor PDTC. Sulindac sulfide also induced activation of the AP-1 transcription factor, which co-operated with NF-κB in up-regulating IL-8. Up-regulation of NF-κB genes was most prominent in conditions where only a subset of cells was undergoing apoptosis. In TNFα stimulated conditions the drug treatment inhibited phosphorylation on IκBα (Ser 32) which is consistent with previous studies and indicates that sulindac sulfide can inhibit TNFα-induced NF-κB activation. Sulindac-induced upregulation of NF-κB target genes occurred early in the proximal colon of mice given a diet containing sulindac for one week.Conclusions: This study shows for the first time that sulindac sulfide can induce pro-inflammatory NF-κB and AP-1 signaling as well as apoptosis in the same experimental conditions. Therefore, these results provide insights into the effect of sulindac on pro-inflammatory signaling pathways, as well as contribute to a better understanding of the mechanism of sulindac-induced gastrointestinal side effects.

The mutated in colorectal cancer (MCC) is a multifunctional gene showing loss of expression in colorectal and liver cancers. MCC mutations can drive colon carcinogenesis in the mouse and in vitro experiments suggest that loss of MCC function promotes cancer through several important cellular pathways. In particular, the MCC protein is known to regulate beta‐catenin (β‐cat) signaling, but the mechanism is poorly understood. Here we show that the β‐cat repressor function of MCC is strongly impaired by the presence of a disease‐associated mutation. We also identify deleted in breast cancer 1 (DBC1) as a new MCC interacting partner and regulator of β‐cat signaling. RNA interference experiments show that DBC1 promotes β‐cat transcriptional activity and that the presence of DBC1 is required for MCC‐mediated β‐cat repression. In contrast to all other DBC1 interacting partners, MCC does not interact through the DBC1 Leucine Zipper domain but with a glutamic‐acid rich region located between the Nudix and EF‐hand domains. Furthermore, MCC overexpression relocalizes DBC1 from the nucleus to the cytoplasm and reduces β‐cat K49 acetylation. Treatment of cells with the SIRT1 inhibitor Nicotinamide reverses MCC‐induced deacetylation of β‐cat K49. These data suggest that the cytoplasmic MCC–DBC1 interaction sequesters DBC1 away from the nucleus, thereby removing a brake on DBC1 nuclear targets, such as SIRT1. This study provides new mechanistic insights into the DBC1–MCC axis as a new APC independent β‐cat inhibitory pathway.

MCC is a potential tumor suppressor gene, which is silenced by promoter hypermethylation in a subset of colorectal cancers. However, its functions have remained poorly understood. In the present study, we describe a novel function of MCC in the DNA damage response. Several novel phosphorylation sites were identified by mass spectrometry, including 2 highly conserved ATM/ATR consensus sites at serine 118 and serine 120. In addition, exposure to ultraviolet radiation (UV), but not phleomycin, caused PI3K-dependent phosphorylation of MCC and its nuclear localization. Re-expression of MCC in HCT15 colorectal cancer cells led to a G2/M arrest, and MCC knockdown impaired the induction of a G2/M arrest following UV radiation. Finally, mutation of S118/120 to alanine did not affect MCC nuclear shuttling following UV but did impair MCC G2/M checkpoint activity. Thus, these results suggest that MCC is a novel target of the DNA damage checkpoint and that MCC is required for the complete cell cycle arrest in the G2/M phase in response to UV.

Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC) protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions.