RESUMO
New target molecules for diagnosis of and drug development for colorectal cancer (CRC) are always in great demand. Previously, we identified a new colorectal cancer-specific protein, TMEM180, and successfully developed an anti-TMEM180 monoclonal antibody (mAb) for the diagnosis and treatment of CRC. Although TMEM180 is classified as a member of the cation symporter family and multi-pass membrane protein, little is known about its function. In this study, we examined topology of this membrane protein and analyzed its function. Using a homology model of human TMEM180, we experimentally determined that the protein has 12 transmembrane domains, and that its N-terminal and C-termini are exposed extracellularly. Moreover, we found that the putative cation-binding site of TMEM180 is conserved among orthologs, and that its position is similar to that of melibiose transporter MelB. These results suggest that TMEM180 acts as a cation symporter. Our topological analysis based on the homology model provides insight into functional and structural roles of TMEM180 that may help to elucidate the pathology of CRC.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas de Membrana/química , Sequência de Aminoácidos , Anticorpos Monoclonais , Sítios de Ligação , Neoplasias Colorretais/genética , Simulação por Computador , Sequência Conservada , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Regulação para CimaRESUMO
As the proper binding of CCCTC-binding factor (CTCF) in the boundaries of topological association domains (TADs) was important for chromatin structures and gene regulation, we hypothesized that single nucleotide polymorphisms (SNPs) affecting CTCF binding in TAD boundaries might contribute to pancreatic cancer (PC) susceptibility. We first genome widely screened out potential SNPs via bioinformatics analysis on Hi-C data, ChIP-seq data, and CTCF binding motif, then tested their associations with PC risk in a previous genome-wide association studies (GWASs) data set (981 cases and 1,991 controls), followed by another independent replication set (1,208 cases and 1,465 controls). Electrophoretic mobility shift assays (EMSAs), expression Quantitative Trait Loci (eQTL) analyses and cell proliferation experiments were performed to uncover the biological mechanisms. The positive SNP rs2001389 was found significantly associated with PC risk with odds ratio (OR) being 1.166 (95% confidence interval (CI) = 1.075-1.264, P = 2.143E-04) in the combined study. The allele G of rs2001389 weakened the binding activity with CTCF, and it was related to the lower expression of a putative antioncogene MFSD13A whose knockdown promoted proliferation of PC cells. By integrating analysis on multiomics data, association studies and functional assays, we proposed that the common variant rs2001389 and the gene MFSD13A might be genetic modifiers of PC tumorigenesis.
Assuntos
Fator de Ligação a CCCTC/genética , Carcinogênese/genética , Proteínas de Membrana/genética , Neoplasias Pancreáticas/genética , Adulto , Idoso , Sítios de Ligação/genética , Proliferação de Células/genética , Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/patologia , Polimorfismo de Nucleotídeo Único/genética , Domínios Proteicos/genética , Locos de Características Quantitativas/genética , Fatores de RiscoRESUMO
TMEM180, a novel colon cancer-specific protein with a 12-transmembrane topology, is upregulated at low oxygen. Previously, we established a humanized monoclonal antibody against TMEM180 aimed at clinical trials. Prior to such trials, it is necessary to clarify the function of TMEM180 in cancer. To compare SW480 human colon cancer cells and their TMEM180-knockdown derivatives, we analyzed proliferation and oxygen consumption, and also performed phosphorylation proteomics, metabolomics, and next-generation sequencing (NGS). The preliminary results revealed that TMEM180 appeared to promote the growth of colon cancer but had almost no effect on oxygen consumption or expression of phosphorylated proteins. By contrast, glycolysis differed dramatically between SW480 and TMEM180-knockdown cells. The NGS analysis revealed that TMEM180 promotes enzyme expression in nitric oxide (NO) synthesis system, suggesting that it promotes glucose and glutamine metabolism, thereby contributing to cancer growth. Overall, the results of this study warrant further basic studies of TMEM180 molecule.