MCC inhibits beta-catenin transcriptional activity by sequestering DBC1 in the cytoplasm.

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 (ß-cat) signaling, but the mechanism is poorly understood. Here we show that the ß-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 ß-cat signaling. RNA interference experiments show that DBC1 promotes ß-cat transcriptional activity and that the presence of DBC1 is required for MCC-mediated ß-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 ß-cat K49 acetylation. Treatment of cells with the SIRT1 inhibitor Nicotinamide reverses MCC-induced deacetylation of ß-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 ß-cat inhibitory pathway.

The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress.

Mutations in BRCA1 are associated with a high risk of breast and ovarian cancer. BRCA1 participates in the DNA damage response and acts as a ubiquitin ligase. However, its regulation remains poorly understood. Here we report that BRCA1 is modified by small ubiquitin-like modifier (SUMO) in response to genotoxic stress, and co-localizes at sites of DNA damage with SUMO1, SUMO2/3 and the SUMO-conjugating enzyme Ubc9. PIAS SUMO E3 ligases co-localize with and modulate SUMO modification of BRCA1, and are required for BRCA1 ubiquitin ligase activity in cells. In vitro SUMO modification of the BRCA1/BARD1 heterodimer greatly increases its ligase activity, identifying it as a SUMO-regulated ubiquitin ligase (SRUbL). Further, PIAS SUMO ligases are required for complete accumulation of double-stranded DNA (dsDNA) damage-repair proteins subsequent to RNF8 accrual, and for proficient double-strand break repair. These data demonstrate that the SUMOylation pathway plays a significant role in mammalian DNA damage response.

Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform.

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.

Loss of special AT-rich sequence-binding protein 1 (SATB1) predicts poor survival in patients with colorectal cancer.

AIM: Special AT-rich sequence-binding protein 1 (SATB1) is a cell type-specific matrix attachment region binding protein, functioning as a global genome organizer. This study aims to investigate the expression pattern and the prognostic value of SATB1 in colorectal cancer. METHODS AND RESULTS: Prospectively collected data were obtained and tissue microarrays were constructed from a cohort of 352 patients. SATB1 protein expression was evaluated by immunohistochemistry and scored by two independent investigators. SATB1 expression was predominantly nuclear in both normal and cancer tissues. Loss of SATB1 nuclear expression was seen in 22% of colorectal cancers compared to 1.5% of adjacent normal colorectal tissue, and was associated with worse overall survival (P = 0.02) independent of age and stage of disease (HR 2.48 with 95% CI 1.31-4.70). Loss of SATB1 expression was more evident in younger patients (P = 0.03), tumours with mucinous or signet ring histology (P = 0.0001) and poor differentiation (P = 0.005). SATB1 expression was associated with a survival advantage in patients with Dukes C tumours who received adjuvant chemotherapy. CONCLUSION: Loss of SATB1 nuclear expression correlates with poor survival and a less favourable response to adjuvant chemotherapy in colorectal cancer. The value of SATB1 in individualized colorectal cancer therapy warrants further evaluation.

The PDZ-binding motif of MCC is phosphorylated at position -1 and controls lamellipodia formation in colon epithelial cells.

In this study, we describe a new post-translational modification at position -1 of the PDZ-binding motif in the mutated in colorectal cancer (MCC) protein and its role in lamellipodia formation. Serine 828 at position -1 of this motif is phosphorylated, which is predicted to increase MCC binding affinity with the polarity protein Scrib. We show that endogenous MCC localizes at the active migratory edge of cells, where it interacts with Scrib and the non-muscle motor protein Myosin-IIB. Expression of MCC harboring a phosphomimetic mutation MCC-S828D strongly impaired lamellipodia formation and resulted in accumulation of Myosin-IIB in the membrane cortex fraction. We propose that MCC regulates lamellipodia formation by binding to Scrib and its downstream partner Myosin-IIB in a multiprotein complex. Importantly, we propose that the function of this complex is under the regulation of a newly described phosphorylation of the PDZ-binding motif at position -1.

Sulindac activates NF-κB signaling in colon cancer cells.

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.

Is chromosome radiosensitivity and apoptotic response to irradiation correlated with cancer susceptibility?

PURPOSE: Individuals who have been treated for breast cancer have been reported to have increased lymphocyte chromosomal sensitivity to ionizing radiation and a significantly lower apoptotic response to irradiation compared to controls. We set out to test these findings using a substantial number of cases sampled before treatment (which could alter the parameters measured), compared to age-matched controls with normal mammograms. MATERIAL AND METHODS: We used the G2 chromosome breakage, and apoptotic response assays of peripheral blood lymphocytes to ionizing radiation to compare 211 unselected newly diagnosed and untreated breast cancer patients, with 170 age, sex and ethnically matched controls. RESULTS: We found no significant differences between breast cancer patients and their matched controls in the G2 assay or apoptotic response. However, there was some evidence that both cases and controls with a strong family history of breast cancer had higher radiosensitivity than those without. CONCLUSIONS: This is the largest and best controlled study of its kind, but it has not replicated previous reports of differences between chromosome breakage or apoptotic response in breast cancer cases vs. controls. However there was a suggestion of increased radiosensitivity in patients with a strong family history, which may indicate a heritable cancer susceptibility trait, warranting further study.

HIF1α deficiency reduces inflammation in a mouse model of proximal colon cancer.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates the adaptation of cells to hypoxic microenvironments, for example inside solid tumours. Stabilisation of HIF1α can also occur in normoxic conditions in inflamed tissue or as a result of inactivating mutations in negative regulators of HIF1α. Aberrant overexpression of HIF1α in many different cancers has led to intensive efforts to develop HIF1α-targeted therapies. However, the role of HIF1α is still poorly understood in chronic inflammation that predisposes the colon to carcinogenesis. We have previously reported that the transcription of HIF1α is upregulated and that the protein is stabilised in inflammatory lesions that are caused by the non-steroidal anti-inflammatory drug (NSAID) sulindac in the mouse proximal colon. Here, we exploited this side effect of long-term sulindac administration to analyse the role of HIF1α in colon inflammation using mice with a Villin-Cre-induced deletion of Hif1α exon 2 in the intestinal epithelium (Hif1α(ΔIEC)). We also analysed the effect of sulindac sulfide on the aryl hydrocarbon receptor (AHR) pathway in vitro in colon cancer cells. Most sulindac-treated mice developed visible lesions, resembling the appearance of flat adenomas in the human colon, surrounded by macroscopically normal mucosa. Hif1α(ΔIEC) mice still developed lesions but they were smaller than in the Hif1α-floxed siblings (Hif1α(F/F)). Microscopically, Hif1α(ΔIEC) mice had significantly less severe colon inflammation than Hif1α(F/F) mice. Molecular analysis showed reduced MIF expression and increased E-cadherin mRNA expression in the colon of sulindac-treated Hif1α(ΔIEC) mice. However, immunohistochemistry analysis revealed a defect of E-cadherin protein expression in sulindac-treated Hif1α(ΔIEC) mice. Sulindac sulfide treatment in vitro upregulated Hif1α, c-JUN and IL8 expression through the AHR pathway. Taken together, HIF1α expression augments inflammation in the proximal colon of sulindac-treated mice, and AHR activation by sulindac might lead to the reduction of E-cadherin protein levels through the mitogen-activated protein kinase (MAPK) pathway.

Mutated in colorectal cancer protein modulates the NFκB pathway.

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.

Neuropeptide Y1 receptor in immune cells regulates inflammation and insulin resistance associated with diet-induced obesity.

Recruitment of activated immune cells into white adipose tissue (WAT) is linked to the development of insulin resistance and obesity, but the mechanism behind this is unclear. Here, we demonstrate that Y1 receptor signaling in immune cells controls inflammation and insulin resistance in obesity. Selective deletion of Y1 receptors in the hematopoietic compartment of mice leads to insulin resistance and inflammation in WAT under high fat-fed conditions. This is accompanied by decreased mRNA expression of the anti-inflammatory marker adiponectin in WAT and an increase of the proinflammatory monocyte chemoattractant protein-1 (MCP-1). In vitro, activated Y1-deficient intraperitoneal macrophages display an increased inflammatory response, with exacerbated secretion of MCP-1 and tumor necrosis factor, whereas addition of neuropeptide Y to wild-type macrophages attenuates the release of these cytokines, this effect being blocked by Y1 but not Y2 receptor antagonism. Importantly, treatment of adipocytes with the supernatant of activated Y1-deficient macrophages causes insulin resistance, as demonstrated by decreased insulin-induced phosphorylation of the insulin receptor and Akt as well as decreased expression of insulin receptor substrate 1. Thus, Y1 signaling in hematopoietic-derived cells such as macrophages is critical for the control of inflammation and insulin resistance in obesity.

Loss of heterozygosity of the Mutated in Colorectal Cancer gene is not associated with promoter methylation in non-small cell lung cancer.

'Mutated in Colorectal Cancer' (MCC) is emerging as a multifunctional protein that affects several cellular processes and pathways. Although the MCC gene is rarely mutated in colorectal cancer, it is frequently silenced through promoter methylation. Previous studies have reported loss of heterozygosity (LOH) of the closely linked MCC and APC loci in both colorectal and lung cancers. APC promoter methylation is a marker of poor survival in non-small cell lung cancer (NSCLC). However, MCC methylation has not been previously studied in lung cancer. Therefore, we wanted to determine if MCC is silenced through promoter methylation in lung cancer and whether this methylation is associated with LOH of the MCC locus or methylation of the APC gene. Three polymorphic markers for the APC/MCC locus were analysed for LOH in 64 NSCLC specimens and matching normal tissues. Promoter methylation of both genes was determined using methylation specific PCR in primary tumours. LOH of the three markers was found in 41-49% of the specimens. LOH within the MCC locus was less common in adenocarcinoma (ADC) (29%) than in squamous cell carcinoma (SCC) (72%; P=0.006) or large cell carcinoma (LCC) (75%; P=0.014). However, this LOH was not accompanied by MCC promoter methylation, which was found in only two cancers (3%). In contrast, 39% of the specimens showed APC methylation, which was more common in ADC (58%) than in SCC (13%). Western blotting revealed that MCC was expressed in a subset of lung tissue specimens but there was marked variation between patients rather than between cancer and matching non-cancer tissue specimens. In conclusion, we have shown that promoter methylation of the APC gene does not extend to the neighbouring MCC gene in lung cancer, but LOH is found at both loci. The variable levels of MCC expression were not associated with promoter methylation and may be regulated through other cellular mechanisms.

Loss of special AT-rich binding protein 1 expression is a marker of poor survival in lung cancer.

INTRODUCTION: Lung cancer is the leading cause of cancer-related mortality and requires more effective molecular markers of prognosis and therapeutic responsiveness. Special AT-rich binding protein 1 (SATB1) is a global genome organizer that recruits chromatin remodeling proteins to epigenetically regulate hundreds of genes in a tissue-specific manner. Initial studies suggest that SATB1 overexpression is a predictor of poor prognosis in breast cancer, but the prognostic significance of SATB1 expression has not been evaluated in lung cancer. METHODS: A cohort of 257 lung cancers was evaluated by immunohistochemistry. Epigenetic silencing of SATB1 was examined in cell lines by 5-Aza 2-deoxycytidine and trichostatin A treatment, and chromatin immunoprecipitation. RESULTS: Significant loss of SATB1 expression was found in squamous preinvasive lesions (p < 0.04) and in non-small cell lung cancers (p < 0.001) compared with matched normal bronchial epithelium. Loss of SATB1 independently predicted poor cancer-specific survival in squamous cell carcinomas (SCCs; hazard ratio: 2.06, 95% confidence interval: 1.2-3.7, p = 0.016). Treatment of lung cancer cell lines with the histone deacetylase inhibitor trichostatin A resulted in up-regulation of SATB1. SATB1 was associated with a decrease in the active chromatin mark acetylated histone H3K9 and an increase in the repressive polycomb mark trimethylated H3K27 in a SCC cell line relative to a normal bronchial epithelial cell line. CONCLUSIONS: This is the first study showing that SATB1 expression is lost in early preinvasive squamous lesions and that loss of SATB1 is associated with poor prognosis in lung SCC. We hypothesize that the SATB1 gene is epigenetically silenced through histone modifications.

The "Mutated in Colorectal Cancer" Protein Is a Novel Target of the UV-Induced DNA Damage Checkpoint.

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.

Lymphocyte radiosensitivity in BRCA1 and BRCA2 mutation carriers and implications for breast cancer susceptibility.

There is conflicting evidence as to whether individuals who are heterozygous for germ-line BRCA1 or BRCA2 mutations have an altered phenotypic cellular response to irradiation. To investigate this, chromosome breakage and apoptotic response were measured after irradiation in peripheral blood lymphocytes from 26 BRCA1 and 18 BRCA2 mutation carriers without diagnosed breast cancer, and 38 unaffected age, ethnically and sex-matched controls. To assess the role of BRCA1 and BRCA2 in homologous recombination, an S phase enrichment chromosome breakage assay was used. BrdUrd incorporation studies allowed verification of the correct experimental settings. We found that BRCA1 mutation carriers without cancer had increased chromosome breaks as well as breaks and gaps per cell post irradiation using the classical G2 assay (p = 0.01 and 0.004, respectively) and the S phase enrichment assay (p = 0.01 and 0.01, respectively) compared to age-matched unaffected controls. BRCA2 mutation carriers without cancer had increased breaks as well as breaks and gaps per cell post irradiation using the S phase enrichment assay (p = 0.045 and 0.012, respectively). No difference was detected using the G2 assay (p = 0.88 and 0.40 respectively). BRCA1 and BRCA2 mutation carriers had normal cell cycle kinetics and apoptotic response to irradiation compared to age-matched controls. Our results show a demonstrable impairment in irradiation induced DNA repair in women with heterozygous germline BRCA1 and BRCA2 mutations prior to being diagnosed with breast cancer.

Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility.

The N-terminus of the Breast Cancer-1 predisposition protein (BRCA1) associates with the BRCA1-associated RING domain-1 protein (BARD1) to form a heterodimer, which exhibits ubiquitin ligase activity that is abrogated by known cancer-associated BRCA1 missense mutations. The majority of missense substitutions identified in patients with a personal or a family history of disease have not been followed in pedigrees, nor there is a functional understanding of their impact. We have examined, by extensive missense substitution, the interaction of BRCA1 with components that contribute to its ubiquitin ligase activity, BARD1 and the E2 ubiquitin-conjugating enzyme, UbcH5a. Selection from a randomly generated library of BRCA1 missense mutations for variants that inhibit the interaction with these components identified substitutions in residues found altered in patient DNA, indicating a correlation between loss of component-binding and propensity to disease development. We further show that the BRCA1:E2 interaction is sensitive to substitutions in all structural elements of the BRCA1 N-terminus, whereas the BARD1 interaction is sensitive to a subset of BRCA1 substitutions, which also inhibit E2-binding. Patient variants that inhibit the BRCA1:E2 interaction show loss of ubiquitin ligase activity and correlate with disease susceptibility and theoretical predictions of pathogenicity. These data link the loss of ubiquitin ligase activity, through loss of E2-binding, to the majority of non-polymorphic patient variants described within the N-terminus of BRCA1 and illustrate the likely significant role of BRCA1 ubiquitin ligase activity in tumour suppression.