ABSTRACT
BACKGROUND: The tumour suppressor gene 'mutated in colorectal cancer' (MCC) is silenced through promoter methylation in colorectal cancer and has been implicated as a regulator of the nuclear factor kappa B (NFκB) pathway. Therefore, we aimed to determine whether MCC modulates NFκB activation in colorectal cancer. MATERIALS AND METHODS: NFκB activation was assessed using luciferase reporter assays in colorectal cancer cells in vitro. MCC methylation was analysed in primary tumour specimens from patients with inflammatory bowel disease. RESULTS: Re-expression of MCC reduced NFκB-dependent transcription in tumour necrosis factor alpha (TNFα)- or lipopolysaccharide (LPS)-stimulated cells. Conversely, knockdown of MCC resulted in accumulation of the inhibitor of kappa B alpha (IκBα) protein, encoded by NFKBIA, a first response gene specifically and rapidly regulated by NFκB pathway activation. The MCC gene is methylated in up to 6/16 of inflammatory bowel disease-associated tissue specimens, and myosin-10 and valosin-containing protein were identified as MCC-interacting proteins. CONCLUSION: These findings suggest that MCC modulates NFκB pathway signalling indirectly in colorectal cancer cells.
Subject(s)
Colorectal Neoplasms/metabolism , DNA Methylation , Inflammatory Bowel Diseases/metabolism , NF-kappa B/metabolism , Tumor Suppressor Proteins/metabolism , Adenosine Triphosphatases/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis , Caspase 8/metabolism , Cell Cycle Proteins/metabolism , Colorectal Neoplasms/complications , Colorectal Neoplasms/genetics , Enzyme Activation , Female , Humans , I-kappa B Proteins/metabolism , Inflammatory Bowel Diseases/etiology , Inflammatory Bowel Diseases/pathology , Lipopolysaccharides/pharmacology , Luciferases/metabolism , Male , Middle Aged , Myosins/metabolism , NF-KappaB Inhibitor alpha , NF-kappa B/genetics , Promoter Regions, Genetic/genetics , RNA, Small Interfering/genetics , Signal Transduction , Spectrometry, Mass, Electrospray Ionization , Tumor Necrosis Factor-alpha/pharmacology , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics , Valosin Containing ProteinABSTRACT
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.
