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LARGE2 mediates O-glycosylation in HT-29 colorectal cancer cells

ABSTRACT: The O-glycosyltransferase LARGE2 attaches a laminin-binding matriglycan structure on alpha-Dystroglycan (α-DG) to mediate the interaction of different cell types with their laminin-containing basement membrane. We used HT-29 CRC cells, characterized by low endogenous LARGE2 gene expression, to ectopically express LARGE2 and to drive O glycosylation of its protein substrates. Immunoblot analyses had revealed that the addition of matriglycan-structures on LARGE2 substrate proteins substantially increased their molecular weight (MW). In order to take into account this shift in MW of candidate proteins, glycoprotein-enriched fractions from HT-29 cells with or without ectopic LARGE2 expression were separated by gel electrophoresis and subsequently fractionated in 6 different molecular weight windows (kDa: 0-25, 25-55, 55-75, 75-110, 110-160, 160<). Subsequently, samples were prepared for quantitative mass spectrometry analysis (qLC-MS/MS) analysis according to standard procedures. qLC-MS-MS analysis identified α-DG–related peptides within higher MW protein fractions only when LARGE2 was overexpressed. HT-29 cell secreted Laminin-2 was exclusively detected together with these high MW forms of α-DG, which demonstrated the affinity of Laminin towards the LARGE2-synthesized matriglycan structure on α-DG. Additional candidate proteins identified in this study need further experimental confirmation. Overall, we show that size fractionation after gel electrophoresis followed by quantitative mass spectrometry, performed separately on different MW windows, represents an elegant approach to reveal potential substrates of the O-glycosyltransferase LARGE2.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Colorectal Adenocarcinoma

SUBMITTER: Svenja Wiechmann

LAB HEAD: Bernhard Kuster

PROVIDER: PXD013800 | Pride | 2020-06-28

REPOSITORIES: Pride

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			Action	DRS
	03122_BA1_P035310_A00_B01_R1.raw	Raw
	03122_BA2_P035311_A00_B01_R1.raw	Raw
	03122_BA3_P035312_A00_B01_R1.raw	Raw
	03122_BA4_P035313_A00_B01_R1.raw	Raw
	03122_BA5_P035314_A00_B01_R1.raw	Raw

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Project description:Metastasis and drug-resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil (5-FU), methotrexate (MTX), doxorubicin (DOX) or oxaliplatin (OXA). Drug-resistant invasive cells were compared to non invasive cells using cDNA microarray, qRT-PCR, flow cytometry, immunoblots and ELISA. Functional and cellular signaling analyses were undertaken using pharmacological inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-FU- and MTX-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behaviour in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, mAb 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 is associated with up-regulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2a and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from colon cancer patients treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis can potentially counteract the emergence of the invasive metastatic behaviour in clonal derivatives of drug-resistant colon cancer cells. Samples: HT-29 cell clones were obtained by limiting dilution from HT-29 subpopulations resistant to methothrexate (HT-29 5M21 cell clone) and or to 5-Fluoro-uracil (HT-29 5F7 and HT-29 5F31). Samples were provided by Dr. T Lesuffleur. Xenografts: HT-29 5M21, HT-29 5F7, and HT-29 5F31 xenografts were obtained by a first subcutaneous inoculation of cells in Nude mice and then fragments of subcutaneous tumors were reimplantated in SCID mice. First experiment of microarrays: HT-29 5M21 and HT-29 5F7 cells (that are invasive in in vitro assays on type I collagen) were compared to HT-29 5F31 cells (that are not invasive in vitro on type I collagen). Samples were co-hybridized on Agilent Human 44K GEP arrays (respectively 5F31 vs 5M21, 5F31 vs 5F7, 5M21 vs 5F31 and 5F7 vs 5F31). Second experiment of microarrays: HT-29 5M21 and HT-29 5F7 xenografts (that develop metastases in immuno-deficient mice lungs) were compared to HT-29 5F31 xenografts (that do not develop metastases in immuno-deficient mice lungs). Samples were consequently co-hybridized on human and mouse 4x44K GEP arrays (respectively AG41_5F31-5M21-138869, AG39_5F31-5F7-138867, AG42_5M21-5F31-138870 and AG40_5F7-5F31-138868 for human microarrays; 5M21_5F31_27033_4, 5F31_5M21_27033_3, 5F7_5F31_27033_2 and 5F31_5F7_27033_1_1 for mouse microarrays).

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LARGE2 mediates O-glycosylation in HT-29 colorectal cancer cells

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