Analysis of coding microsatellite frameshift mutations in MSI-H colorectal carcinomas and characterization of their effects on the cellular glycosylation machinery
Final Report Abstract
Microsatellite instability (MSI-H), i.e. length variations of repetitive DNA sequences (microsatellites) occurs in colorectal cancer (CRC) due to defects in the DNA mismatch repair (MMR) system and is a hallmark of tumors associated with hereditary non-polyposis colorectal cancer (HNPCC, 1 – 5% of total CRC) as well as sporadic CRC (15% of total CRC). Instability in coding region microsatellites (cMNR) of expressed genes leads to frameshift mutations and a subsequent loss of protein function or the translation of a truncated protein. Despite the obvious significance of altered glycoprotein synthesis, transport and glycosylation patterns in colorectal cancer, aberrant glycosylation and glycosylation pathways have not been investigated in MSI-H colorectal tumors. The goal of the present project was the identification of frameshift mutations in candidate genes with an involvement in the glycosylation machinery and the understanding of their role in MSI-H tumorigenesis. Using a bioinformatics-based approach 28 cMNR harboring candidate genes were identified that encode proteins of the cellular glycosylation machinery. Coding MNR mutation analysis in MSI-H CRC cell lines revealed a high mutation frequency in two genes: the glycoprotein transporter gene LMAN1/ERGIC53 (52%; 12/23) and the xylosyltransferase gene XYLT2 (35%; 8/23) that catalyzes the first step in proteoglycan synthesis. Apart from their occurrence in cultured cell lines these genetic alterations were also found at similar frequency in MSI-H colorectal adenomas (LMAN1: 40%, 8/20; XYLT2: 21%, 4/19) as well as carcinomas (LMAN1: 46%, 78/170; XYLT2: 26%, 27/105). Biallelic mutations, abrogating normal protein function, were detected in MSI-H CRC cell lines (LMAN1, XYLT2) and in primary colorectal tumors (LMAN1). In biallelically mutated cells LMAN1 was transcribed but not translated into a stable protein in MSI-H CRC cell lines or LMAN1-mutated areas in tumors. MSI-H CRC cell lines with biallelic LMAN1 mutations behaved differently from LMAN1-proficient CRC cell lines. LMAN1-deficient cell lines exhibited severely reduced transport and secretion of the LMAN1 cargo protein alpha-1-antitrypsin (A1AT), an inhibitor of angiogenesis and tumor growth, but impaired A1AT secretion was restored upon LMAN1 re-expression. A strong correlation between lower local levels of A1AT and enhanced tumor growth has been described and this phenomenon might be caused by a lost transport function of LMAN1. Furthermore, re-expression of LMAN1 in LMAN1-deficient LoVo cells led to a cell surface glycoprotein pattern different from the pattern observed on LoVo cells lacking LMAN1. Increased PHA-L lectin binding to the cell surface suggested a glycosylation pattern known to be involved in the progression of cancer from a tumorigenic to a metastatic phenotype. In addition to cell surface changes in LMAN1-deficient MSI-H CRC cell lines, we could also report changes in proteoglycan synthesis in XYLT2-deficient MSI-H CRC cell lines. These changes were observed by a radioactive incorporation assay in a XYLT2-deficient MSI-H CRC cell line transiently transfected with a XYLT2-construct. Overall, two members of the cellular glycosylation machinery, a glycoprotein transporter and a glycosyltransferase, appear to be involved in MSI-H tumorigenesis. Genetic alterations in LMAN1 and XYLT2 mark early events already detectable in preneoplastic lesions. Furthermore, loss of LMAN1 and XYLT2 protein function changed the secretion of proteins and the cell surface glycosylation pattern. Both genes might influence MSI-H tumor progression by changing cell-cell communication and interactions. In summary, the present project provided an initial basis for the understanding of the function of glycoproteins in MSI tumorigenesis as well as for the identification of MSI-H specific glycopeptides for diagnostic or therapeutic applications.
Publications
- (2009) High frequency of LMAN1 abnormalities in colorectal tumors with microsatellite instability. Cancer Res 69:292-299
Roeckel N, Woerner SM, Kloor M, Yuan YP, Patsos G, Gromes R, Kopitz J, Gebert J
