Elevated Golgi pH in breast and colorectal cancer cells correlates with the expression of oncofetal carbohydrate T‐antigen

Abstract

Altered glycosylation has turned out to be a universal feature of cancer cells, and in many cases, to correlate with altered expression or localization of relevant glycosyltransferases. However, no such correlation exists between observed enzymatic changes and the expression of the oncofetal Thomsen-Friedenreich (T)-antigen, a core 1 (Gal-beta1 --> 3-GalNAc-ser/thr) carbohydrate structure. Here we report that T-antigen expression, instead, correlates with elevated Golgi pH in cancer cells. Firstly, using a Golgi-targeted green fluorescent protein (GT-EGFP) as a probe, we show that the medial/trans-Golgi pH (pHG) in a high proportion of breast (MCF-7) and colorectal (HT-29, SW-48) cancer cells is significantly more alkaline (pHG > or = 6.75) than that of control cells (pHG 5.9-6.5). The pH gradient between the cytoplasm and the Golgi lumen is also markedly reduced in MCF-7 cells, suggesting a Golgi acidification defect. Secondly, we show that T-antigen expression is highly sensitive to changes in Golgi pH, as only a 0.2 pH unit increase was sufficient to increase T-antigen expression in control cells. Thirdly, we found that T-antigen expressing MCF-7 cells have 0.3 pH units more alkaline Golgi pH than non-expressing MCF-7 cells. Fourthly, in all cell types examined, we observed significant correlation between the number of T-antigen expressing cells and cells with a markedly elevated Golgi pH (pHG > or = 6.75). Consistent with these observations in cultured cells, cells in solid tumors also heterogenously expressed the T-antigen. Thus, elevated Golgi pH appears to be directly linked to T-antigen expression in cancer cells, but it may also act as a more general factor for altered glycosylation in cancer by affecting the distribution of Golgi-localized glycosyltransferases.

Organizational Interplay of Golgi N-Glycosyltransferases Involves Organelle Microenvironment-Dependent Transitions between Enzyme Homo- and Heteromers

“…4D) in the Golgi both at normal and at slightly elevated Golgi pH. For this purpose we utilized 40 M CQ, which raises Golgi pH by only 0.4 pH units without causing any major structural changes in the Golgi (43). We transfected cells with the two highly pH-sensitive heteromeric FRET enzyme constructs (GalT-I/ST3Gal-III and ppGalNAcT-6/C3GnT-1) together with an HA-tagged homomeric construct.…”

Functional Organization of Golgi N- and O-Glycosylation Pathways Involves pH-dependent Complex Formation That Is Impaired in Cancer Cells

“…Complex Formation in Cancer Cells-The Golgi pH is also elevated in many cancer cells (36). Therefore, we anticipated that complex formation might be affected.…”

“…Therefore, we anticipated that complex formation might be affected. To clarify this, we performed FRET measurements using acidification-defective cancer cells MCF7 and SW48 (36) as targets. COS7 and HeLa cells, which both have a normal Golgi pH, were used as control cells.…”

Ratiometric Imaging of pH Probes

“…Predominantly generated by ion exchangers, pumps, and channels, proton fluxes across the plasma membrane regulate dynamic changes in cytosolic or intracellular pH (pHi) that contribute to cell proliferation, cytoskeleton remodeling, and glycolytic metabolism (Casey, Grinstein, & Orlowski, 2010;Webb, Chimenti, Jacobson, & Barber, 2011). Proton fluxes across membranes of intracellular organelles generate pH gradients that drive vesicle trafficking and posttranslational modification and sorting of cargo proteins (Marshansky & Futai, 2008;Rivinoja, Kokkonen, Kellokumpu, & Kellokumpu, 2006;Vavassori et al, 2013). Although in normal conditions proton fluxes are homeostatically controlled to maintain pHi and organelle pH within narrow physiological ranges, dysregulated proton fluxes enable many diseases and pathologies, including cancer (Cardone, Casavola, & Reshkin, 2005;Stock & Schwab, 2009;Webb et al, 2011), neurodegenerative disorders (Harguindey, Reshkin, Orive, Arranz, & Anitua, 2007), and a number of myopathies and cardiovascular dysfunctions (VaughanJones, Spitzer, & Swietach, 2009).…”