Figure S9. (A) Left Panels: representative images of triple IHC staining of Na+/K+-ATPase (green), Integrin αv (red), and Gal-3 (brown) in the orthotopic tumor samples from the indicated group of mice; bars, 100 µm. Middle panel: IF of Golgi stained by GM130 in the prostate tumor foci; bars, 10 µm. White boxes indicate areas enlarged at the right. (B) Quantification of Golgi fragments per cell in tumor foci from samples in A; median ± SD. (C) ATF6 IF in the orthotopic tumor samples from the indicated group of mice; bars, 20 µm. Areas of mice prostate epithelium is highlighted by dotted lines. (D) Quantification of ATF6 IF intensity from samples in C; median ± SD. For all graphs: pairwise comparisons using Wilcoxon rank sum exact test, p-adjusted using Benjamini-Hochberg; ****p<0.0001, **p<0.01, and *p<0.05; n indicates the number of foci counted. (E) IF staining of the prostate tumor foci from the indicated group of mice to visualize Na+/K+-ATPase (magenta), Integrin αv (red), and Gal-3 (green); bars, 10 µm. (F, G) Quantification of IF intensity of Integrin αv (F) and Gal-3 (G) at the PM in samples from C; median ± SD.
ARTICLE ABSTRACT
Prostate cancer (PCa) progression to the lethal metastatic castration-resistant phenotype (mCRPC) is driven by αv integrins and is associated with Golgi disorganization and activation of the ATF6 branch of unfolded protein response (UPR). Overexpression of integrins requires N-acetylglucosaminyltransferase-V (MGAT5)-mediated glycosylation and subsequent cluster formation with Galectin-3 (Gal-3). However, the mechanism underlying this altered glycosylation is missing. For the first time, using HALO analysis of immunohistochemistry, we found a strong association of Integrin αv and Gal-3 at the plasma membrane (PM) in primary PCa and mCRPC samples. We discovered that MGAT5 activation is caused by Golgi fragmentation and mislocalization of its competitor, N-acetylglucosaminyltransferase-III, MGAT3, from Golgi to the endoplasmic reticulum (ER). This was validated in an ethanol-induced model of ER stress, where alcohol treatment in androgen-refractory PC-3 and DU145 cells or alcohol consumption in PCa patient samples aggravates Golgi scattering, activates MGAT5, and enhances integrin expression at PM. This explains known link between alcohol consumption and PCa mortality. ATF6 depletion significantly blocks UPR and reduces the number of Golgi fragments in both PC-3 and DU145 cells. Inhibition of autophagy by hydroxychloroquine (HCQ) restores compact Golgi, rescues MGAT3 intra-Golgi localization, blocks glycan modification via MGAT5, and abrogates delivery of Gal-3 to the cell surface. Importantly, the loss of Gal-3 leads to reduced integrins at PM and their accelerated internalization. ATF6 depletion and HCQ treatment synergistically decrease Integrin αv and Gal-3 expression and temper orthotopic tumor growth and metastasis.
Implications: Combined ablation of ATF6 and autophagy can serve as new mCRPC therapeutic.
