The Aberrant GSL Of Breast Cancer And Its Biological Function

Part I The expression of Fuc-ɑ1,2LacCer in tissues of breast cancer patients.Objective: The expression of major neutral GSLs in tissue samples of breastcancer patients was analyzed. The expression profiles between the tumoral tissues andperitumoral tissues were compared to find the abnormal GSLs of breast cancer. Andthen its relevance to breast cancer and biological function was discussed. In furtherstudy, we explored the correlation of the aberrant GSL with breast cancer and itsbiological function.Methods:(1) The GSLs were extracted four times by sonication with organic solvents frombreast cancer patients’ samples both in tumoral tissues and paired peritumoraltissues. The neutral and acid GSLs were separated by Sephadex A-25columnafter the extraction step.(2) Per-N,O-methylation of neutral GSLs was performed before detected by theESI-LIT-MS. MS1and MSnanalysis were applied to identify the certain structure ofmajor neutral GSLs. The abnormal GSLs were found by compared the GSLs’expression profiles of thetumoral tissues and paired peritumoral tissues.(3) Firstly, the MS spectrum of the potential aberrant GSLs was in comparison ofthe standard samples. Then the neutral GSLs were analyzed by liquidchromatography-MS (LC-MS) after per-N,O-methylation. The potential aberrantGSLs were analyzed by MS after treated with ɑ1,2fucosidase. All these analysis allowed us to confirm the aberrant GSLs.(4) We made the contrast of the relative content of the major neutral GSLs and aberrantGSL between tumoral tissues and paired peritumoral tissues. Parent-ion scanningwas applied to analyze the expression of the aberrant GSL. The values ofsignal-to-noise (SNR) of the aberrant GSL were compared among breast cancertumoral tissues, paired peritumoral tissues, leukemia bone marrow samples andliver cancer tissue samples. The receiver operating characteristic curve (ROC)analysis was taken to figure outthe significance of clinical diagnostic of theaberrant GSLs. Comparative analysis of the abnormal GSL were taken indifferent molecular classification of breast cancer. All the data above confirmedthe correlation between the aberrant GSL and breast cancer.Results:(1) Major neutral GSLs in tumoral tissues and paired peritumoral tissues wereidentified as GlcCer, LacCer, globotrihexosylceramide (Gb3), globo-tetraglycosylceramide (Gb4) by MS1and MSnanalysis. The ion peak m/z1184in theMS1spectrum of breast cancer tumor tissue was confirmed as Fucosyl-Lactoceramide(Fuc-ɑ1,2LacCer). Besides, the potential breast cancer biomarker Globo-H(Fucosyl-Gb4) previous studied was also detected in breast cancer tumor tissue.(2) The results of the relative content of the major neutral GSLs was concludedthat GlcCer and Gb3overexpressed in peritumoral tissues while LacCer, Globo-Hand Fuc-ɑ1,2LacCer overexpressed in tumoral tissues.(3) The conclusion of SNR analysis was Fuc-ɑ1,2LacCer overexpressed specific inbreast cancer tumoral tissues compared with leukemia bone marrow samples andliver cancer tissue samples. ROC analysis suggested that Fuc-ɑ1,2LacCer hadcertain sensitivity and specificity considered as a clinical diagnosis index.Conclusion: Fuc-ɑ1,2LacCer has a highly expression which is specific in breastcancer tumoral tissues. Considered as a clinical diagnosis index, Fuc-ɑ1,2LacCer has certain sensitivity and specificity. Part II The expression and functionof Fuc-ɑ1,2LacCer on breast cancer cellsObjective: The expression of Fuc-ɑ1,2LacCer will be detected in various breastcancer cell lines. We will study the functional role of alternation of the relevantsynthetase of breast cancer cells. We’ll further explore the biological significance ofFuc-ɑ1,2LacCer and related synthetase on the genesis and development of breastcancer.Methods:(1) The expression of Fuc-ɑ1,2LacCer was contrasted among various human breastcancer cell lines, human leukemic cell, human hepatomacarcinomacell lineshuman peripheral blood mononuclear cells (PBMC).(2) The level of the synthetase responsible for Fuc-ɑ1,2LacCer, ɑ1,2fucosyl-transferase1(FUT1), was detected in different breast cancer cell lines byRT-PCR and Realtime PCR. The FUT1high-expressed and low-expressed celllines were selected as research tools.(3) The expression of FUT1was detected by western blot in breast cancer tumoraltissues.(4) The FUT1was knocked-down by RNAi in FUT1high-expressed cell linewhile overexpressed by transfected with pEGFP-N1in low-expressed cell line.The GSLs of the transfected cells were analyzed by MS, and the motility of thecells were assessed after transforming.Results:(1) The relative content of Fuc-ɑ1,2LacCer was contrasted among different tumor cell lines and normal cells. The result showed the level of Fuc-ɑ1,2LacCer wasmuch higher in breast cancer cell lines.(2) The expression of FUT1was higher in MDA-MB-157cells and MDA-MB-453cells while lower in MDA-MB-231cells and MCF-7cells. MDA-MB-453cellsand MCF-7cells were selected as tools for gene knockout and geneoverexpressing.(3) FUT1was highly expressed in breast cancer tumor tissues compared with pairedperitumoral tissues.(4) The level of Fuc-ɑ1,2LacCer was downregulated in MDA-MB-453cells afterthe FUT1silenced by RNAi. The level of Fuc-ɑ1,2LacCer was upregulated inMCF7cells after the FUT1overexpressed and with a higher motility. Theresults indicated the upregulation of FUT1might contribute to motility ofcancer cells.Conclusion: The expression of Fuc-ɑ1,2LacCer is high and specific in breastcancer cell lines. Accordingly, the level of FUT1is much higher in breast cancer celllines and breast cancer tissue samples. FUT1contributes to the motility in breast cancercell lines.