Data Supplement from Restoration of Compact Golgi Morphology in Advanced Prostate Cancer Enhances Susceptibility to Galectin-1–Induced Apoptosis by Modifying Mucin O-Glycan Synthesis

Petrosyan, Armen; Holzapfel, Melissa S.; Muirhead, David E.; Cheng, Pi-Wan

doi:10.1158/1541-7786.22510747.v1

Data Supplement from Restoration of Compact Golgi Morphology in Advanced Prostate Cancer Enhances Susceptibility to Galectin-1–Induced Apoptosis by Modifying Mucin O-Glycan Synthesis

journal contribution

posted on 2023-04-03, 16:20 authored by Armen Petrosyan, Melissa S. Holzapfel, David E. Muirhead, Pi-Wan Cheng

Figure S1. (A, B) Confocal immunofluorescence microscopic images showing colocalization of C1GalT1, ST3Gal1 and C2GnT-L with ER marker HSP70 in LNCaP, PC-3 and DU145 cells, in normal prostate tissues and different grades of prostatic adenocarcinoma. White boxes in each panel are enlarged and shown at the right side as green, red and merged images. All confocal images were acquired with same imaging parameters; bar, 10 μm. Figure S2. (A, B) Confocal immunofluorescence microscopic images showing colocalization of C1GalT1, ST3Gal1 and C2GnT-L with Golgi matrix protein GM130 and Golgi-specific GTPase Rab6a in PC-3 and DU145 cells. All confocal images were acquired with same imaging parameters; bar, 10 μm. Figure S3. (A, B) Confocal immunofluorescence microscopic images showing colocalization of C1GalT1 with Golgi matrix protein GM130 in PC-3 and DU145 cells treated with scramble, giantin, GM130 and GRASP65 siRNAs. (C, D) Confocal immunofluorescence microscopic images showing colocalization of C1GalT1 with giantin in PC-3 and DU145 cells treated with GM130 and GRASP65 siRNAs. The images inside the white boxes are enlarged and shown as green and red channels at the bottom of each panel. All confocal images were acquired with same imaging parameters; bar, 10 μm. Figure S4. (A) Confocal immunofluorescence microscopy shows colocalization of C2GnT-L and ST3Gal1 with giantin in DU145 cells treated with DMSO, Blebbistatin or NMIIA siRNAs. Images in the white boxes are amplified and placed at the right side of each panel. (B) Confocal immunofluorescence images of giantin in DU145 cells treated with scramble and Plk3 siRNAs followed by treatment with Blebbistatin, or Plk3, NMIIA, or NMIIA plus Plk3 siRNAs. Bar, 10 μm. Figure S5. (A, C) Confocal fluorescence images of immunostained giantin, and MAA lectin stained α2-3Sia or LEA lectin stained polylactosamine in DU145 cells treated with Blebbistatin or NMIIA siRNAs. Control cells were treated with DMSO. (B, D) Quantification of α-2,3-Sia- and polylactosamine-specific average integrated fluorescence (in a.u.) in DU145 cells shown in A and C. (E) The 8% SDS PAGE of DU145 cells plasma membrane fractions pulled-down by MAA and LEA lectins followed by capturing with Streptavidin magnet beads. (F) DAPI staining of DU145 cells treated with DMSO, Blebbistatin, or scramble or NMIIA siRNAs and then with or without treatment with 10 μM galectin-1. (G, H) The percentage apoptotic cells and caspase-3 activity in cells are presented in F. Apoptotic nuclei were counted in 30 fields each of three independent experiments and expressed as percentage apoptotic cells out of total number of cells. Caspase-3 activity is expressed as relative mean fluorescence units (RFU) from three independent experiments. Bar, 10 μm; *, p<0.001.

History

ARTICLE ABSTRACT

Prostate cancer progression is associated with upregulation of sialyl-T antigen produced by β-galactoside α-2,3-sialyltransferase-1 (ST3Gal1) but not with core 2-associated polylactosamine despite expression of core 2 N-acetylglucosaminyltransferase-L (C2GnT-L/GCNT1). This property allows androgen-refractory prostate cancer cells to evade galectin-1 (LGALS1)–induced apoptosis, but the mechanism is not known. We have recently reported that Golgi targeting of glycosyltransferases is mediated by golgins: giantin (GOLGB1) for C2GnT-M (GCNT3) and GM130 (GOLGA2)-GRASP65 (GORASP1) or GM130-giantin for core 1 synthase. Here, we show that for Golgi targeting, C2GnT-L also uses giantin exclusively whereas ST3Gal1 uses either giantin or GM130-GRASP65. In addition, the compact Golgi morphology is detected in both androgen-sensitive prostate cancer and normal prostate cells, but fragmented Golgi and mislocalization of C2GnT-L are found in androgen-refractory cells as well as primary prostate tumors (Gleason grade 2–4). Furthermore, failure of giantin monomers to be phosphorylated and dimerized prevents Golgi from forming compact morphology and C2GnT-L from targeting the Golgi. On the other hand, ST3Gal1 reaches the Golgi by an alternate site, GM130-GRASP65. Interestingly, inhibition or knockdown of non-muscle myosin IIA (MYH9) motor protein frees up Rab6a GTPase to promote phosphorylation of giantin by polo-like kinase 3 (PLK3), which is followed by dimerization of giantin assisted by protein disulfide isomerase A3 (PDIA3), and restoration of compact Golgi morphology and targeting of C2GnT-L. Finally, the Golgi relocation of C2GnT-L in androgen-refractory cells results in their increased susceptibility to galectin-1–induced apoptosis by replacing sialyl-T antigen with polylactosamine.Implications: This study demonstrates the importance of Golgi morphology and regulation of glycosylation and provides insight into how the Golgi influences cancer progression and metastasis. Mol Cancer Res; 12(12); 1704–16. ©2014 AACR.