ABSTRACT
Colorectal cancer (CRC) is the second most common malignant neoplasia in women and men worldwide. The B-cell lymphoma 2 (Bcl-2) protein family is mainly known for its pivotal role in the regulation of the mitochondrial death pathway. Anti-apoptotic Bcl-2 proteins may provide survival benefits and induce therapy resistance in cancer cells. Among anti-apoptotic Bcl-2 proteins, we found solely Bcl-xL strongly upregulated in human CRC specimens. In order to study protein function in the context of tumor initiation and progression in vivo, we generated a mouse model lacking Bcl-xL in intestinal epithelial cells (Bcl-xL(IEC-KO)). If challenged in an inflammation-driven tumor model, Bcl-xL(IEC-KO) mice showed a significantly reduced tumor burden with lower tumor numbers per animal and decreased tumor sizes. Analysis of cell death events by immunohistochemistry and immunoblotting revealed a striking increase of apoptosis in Bcl-xL-negative tumors. qRT-PCR and immunohistochemistry excluded changes in proliferative capacity and immune cell infiltration as reasons for the reduced tumor load and thereby identify apoptosis as key mechanism. Human CRC tissue was cultured ex vivo and treated with the small molecule compound ABT-737, which inhibits Bcl-xL and Bcl-2. Under ABT-737 treatment, the amount of apoptotic tumor cells significantly increased compared with controls, whereas proliferation levels remained unaltered. In summary, our findings identify Bcl-xL as a driver in colorectal tumorigenesis and cancer progression, making it a valuable target for clinical application.
Subject(s)
Colorectal Neoplasms/genetics , Oncogenes , bcl-X Protein/genetics , Animals , Apoptosis/drug effects , Apoptosis/genetics , Biphenyl Compounds/pharmacology , Carcinogenesis/drug effects , Carcinogenesis/genetics , Carcinogenesis/pathology , Cell Culture Techniques , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/pathology , Disease Models, Animal , Enterocytes/drug effects , Enterocytes/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Inflammation/genetics , Inflammation/pathology , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/metabolism , Mice , Mice, Knockout , Nitrophenols/pharmacology , Organ Specificity , Phenotype , Piperazines/pharmacology , Sulfonamides/pharmacology , Up-Regulation/drug effects , Up-Regulation/genetics , bcl-X Protein/metabolismABSTRACT
BACKGROUND: Metabolic and transcriptomic differences between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) compartments, particularly in the context of obesity, may play a role in colorectal carcinogenesis. We investigated the differential functions of their metabolic compositions. OBJECTIVES: Biochemical differences between adipose tissues (VAT compared with SAT) in patients with colorectal carcinoma (CRC) were investigated by using mass spectrometry metabolomics and gene expression profiling. Metabolite compositions were compared between VAT, SAT, and serum metabolites. The relation between patients' tumor stage and metabolic profiles was assessed. DESIGN: Presurgery blood and paired VAT and SAT samples during tumor surgery were obtained from 59 CRC patients (tumor stages I-IV) of the ColoCare cohort. Gas chromatography time-of-flight mass spectrometry and liquid chromatography quadrupole time-of-flight mass spectrometry were used to measure 1065 metabolites in adipose tissue (333 identified compounds) and 1810 metabolites in serum (467 identified compounds). Adipose tissue gene expression was measured by using Illumina's HumanHT-12 Expression BeadChips. RESULTS: Compared with SAT, VAT displayed elevated markers of inflammatory lipid metabolism, free arachidonic acid, phospholipases (PLA2G10), and prostaglandin synthesis-related enzymes (PTGD/PTGS2S). Plasmalogen concentrations were lower in VAT than in SAT, which was supported by lower gene expression of FAR1, the rate-limiting enzyme for ether-lipid synthesis in VAT. Serum sphingomyelin concentrations were inversely correlated (P = 0.0001) with SAT adipose triglycerides. Logistic regression identified lipids in patients' adipose tissues, which were associated with CRC tumor stage. CONCLUSIONS: As one of the first studies, we comprehensively assessed differences in metabolic, lipidomic, and transcriptomic profiles between paired human VAT and SAT and their association with CRC tumor stage. We identified markers of inflammation in VAT, which supports prior evidence regarding the role of visceral adiposity and cancer.
Subject(s)
Carcinoma/physiopathology , Colorectal Neoplasms/physiopathology , Gene Expression Regulation, Neoplastic , Intra-Abdominal Fat/metabolism , Panniculitis/etiology , Paraneoplastic Syndromes/etiology , Subcutaneous Fat, Abdominal/metabolism , Aged , Biomarkers/blood , Biomarkers/metabolism , Carcinoma/metabolism , Carcinoma/pathology , Carcinoma/surgery , Cohort Studies , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Colorectal Neoplasms/surgery , Female , Gene Expression Profiling , Humans , Intra-Abdominal Fat/immunology , Intra-Abdominal Fat/pathology , Lipid Metabolism , Male , Metabolomics/methods , Middle Aged , Neoplasm Staging , Oligonucleotide Array Sequence Analysis , Panniculitis/immunology , Panniculitis/metabolism , Panniculitis/pathology , Paraneoplastic Syndromes/immunology , Paraneoplastic Syndromes/metabolism , Paraneoplastic Syndromes/pathology , Prospective Studies , Subcutaneous Fat, Abdominal/immunology , Subcutaneous Fat, Abdominal/pathologyABSTRACT
Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.
