CXCR4 mediated chemotaxis is regulated by 5T4 oncofetal glycoprotein in mouse embryonic cells.

5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site.

The removal of multiplicative, systematic bias allows integration of breast cancer gene expression datasets - improving meta-analysis and prediction of prognosis.

BACKGROUND: The number of gene expression studies in the public domain is rapidly increasing, representing a highly valuable resource. However, dataset-specific bias precludes meta-analysis at the raw transcript level, even when the RNA is from comparable sources and has been processed on the same microarray platform using similar protocols. Here, we demonstrate, using Affymetrix data, that much of this bias can be removed, allowing multiple datasets to be legitimately combined for meaningful meta-analyses. RESULTS: A series of validation datasets comparing breast cancer and normal breast cell lines (MCF7 and MCF10A) were generated to examine the variability between datasets generated using different amounts of starting RNA, alternative protocols, different generations of Affymetrix GeneChip or scanning hardware. We demonstrate that systematic, multiplicative biases are introduced at the RNA, hybridization and image-capture stages of a microarray experiment. Simple batch mean-centering was found to significantly reduce the level of inter-experimental variation, allowing raw transcript levels to be compared across datasets with confidence. By accounting for dataset-specific bias, we were able to assemble the largest gene expression dataset of primary breast tumours to-date (1107), from six previously published studies. Using this meta-dataset, we demonstrate that combining greater numbers of datasets or tumours leads to a greater overlap in differentially expressed genes and more accurate prognostic predictions. However, this is highly dependent upon the composition of the datasets and patient characteristics. CONCLUSION: Multiplicative, systematic biases are introduced at many stages of microarray experiments. When these are reconciled, raw data can be directly integrated from different gene expression datasets leading to new biological findings with increased statistical power.