Absent in melanoma 2 (AIM2) is a member of the interferon-inducible HIN-200 protein family. Recent findings point to a role of AIM2 function in both inflammation and cancer. In response to foreign cytoplasmic DNA, AIM2 forms an inflammasome, resulting in caspase activation in inflammatory cells. Moreover, AIM2 reduces breast cancer cell proliferation and mammary tumor growth in a mouse model and shows a high frequency of frameshift mutations in microsatellite unstable (MSI-H) gastric, endometrial and colorectal cancers. However, the consequences of AIM2 restoration in AIM2-deficient colon cancer cells have not yet been examined. Using different constructs for expression of AIM2 fusion proteins, we found that AIM2 restoration clearly suppressed cell proliferation and viability in HCT116 cells as well as in cell lines derived from other entities. In contrast to previous reports from breast cancer cells, our cell cycle analyses of colon cancer cells revealed that AIM2-mediated inhibition of cell proliferation is associated with accumulation of cells at late S-phase, resulting in G2/M arrest. The latter correlated well with upregulation of cyclin D3 and p21 Waf1/Cip1 as well as with inhibition of cdc2 activity through Tyr-15 phosphorylation. Furthermore, AIM2 restoration affected the adhesion of colorectal cancer cells to fibronectin and stimulated the invasion through extracellular matrix-coated membrane in transwell assays. Consistent with this phenotype, AIM2 induced the expression of invasion-associated genes such as VIM and MCAM, whereas ANXA10 and CDH1 were downregulated. Our data suggest that AIM2 mediates reduction of cell proliferation by cell cycle arrest, thereby conferring an invasive phenotype in colon cancer cells.

Tumor heterogeneity is a major obstacle for developing effective anticancer treatments. Recent studies have pointed to large stochastic genetic heterogeneity within cancer lesions, where no pattern seems to exist that would enable a more structured targeted therapy approach. Because to date no similar information is available at the protein (phenotype) level, we employed matrix assisted laser desorption ionization (MALDI) image-guided proteomics and explored the heterogeneity of extracellular and membrane subproteome in a unique collection of eight fresh human colorectal carcinoma (CRC) liver metastases. Monitoring the spatial distribution of over 1,000 proteins, we found unexpectedly that all liver metastasis lesions displayed a reproducible, zonally delineated pattern of functional and therapeutic biomarker heterogeneity. The peritumoral region featured elevated lipid metabolism and protein synthesis, the rim of the metastasis displayed increased cellular growth, movement, and drug metabolism, whereas the center of the lesion was characterized by elevated carbohydrate metabolism and DNA-repair activity. From the aspect of therapeutic targeting, zonal expression of known and novel biomarkers was evident, reinforcing the need to select several targets in order to achieve optimal coverage of the lesion. Finally, we highlight two novel antigens, LTBP2 and TGFBI, whose expression is a consistent feature of CRC liver metastasis. We demonstrate their in vivo antibody-based targeting and highlight their potential usefulness for clinical applications. Conclusion: The proteome heterogeneity of human CRC liver metastases has a distinct, organized pattern. This particular hallmark can now be used as part of the strategy for developing rational therapies based on multiple sets of targetable antigens. (HEPATOLOGY 2014;59:924-934) See Editorial on Page 757 D espite its great promise, clinically used targeted cancer therapy is unfortunately showing only limited success at a very high cost. 1 This failure is partly explained by the high level of genetic heterogeneity of malignant lesions and the fact that carcinogenesis is an evolutionally driven process that recapitulates Darwin's theory of selection of the fittest. 2 In such an unfavorable context, targeted therapy protocols are reaching only a subpopulation of tumor cells, leading to a punctual increase in the selective pressure, fueling resistance to therapy, and thus failing to cure the patient. 3 The notion of tumor heterogeneity and the apparent random distribution of tumor cells harboring different mutations are supported by recent genetic studies. [4][5][6] However, the lack of information at the protein level limits the understanding of how genetic Abbreviations: CRC, colorectal cancer; MALDI, matrix assisted laser desorption ionization; MS, mass spectrometry. From the

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The human gastrointestinal barrier comprises several layers which enable protection against the external environment. The mucosal epithelium, lamina propria, glycocalyx and secreted mucus each make a contribution to barrier protection. Glycocalyx and secreted mucins constitute a glycosylated environment which interacts with the enteric microflora. Turnover of the mucus layer and the creation of binding ligands for bacteria are significant factors in gut homeostasis. The gut microbiota is composed of many bacterial species, but improved technology has allowed detection of populations present at different stages of development and in disease. Interaction of the microflora with the gut occurs from birth onwards and enables maturation of gut angiogenesis and glycosylation as demonstrated in mouse models. Glycan legislation regulates the ongoing interaction between the microflora and the host mucosa. This accounts for host glycosylation mechanisms providing a dynamic response to fluctuations in the gut microflora. Evidence for glycan legislation is based on a surgical model where intact mucosa can be compared with and without contact to the faecal microflora. In addition, mucosal cell glycosylation is assessed using inhibitors of O-glycan synthesis. These inhibitors lead to growth arrest in cultured colorectal cancer cell lines through the induction of apoptosis and downregulation of proliferation.