Figure S1. (A) Representative images of triple IHC staining of Integrin αvβ3 (green), Gal-3 (red), and E-cadherin (brown) in normal prostate and tumor tissues from PCa patients with different grades. Deconvoluted images of Integrin αvβ3 are shown on the right; bars, 50 µm. (B) Quantification of Integrin αvβ3 H-score at PM from samples in A. Dunn Test (1964) Kruskal-Wallis multiple comparisons, p-adjusted using Benjamini-Hochberg; ****p<0.0001 and *p<0.05, median ± SD. (C) Quantification of Integrin αvβ3 and Gal-3 colocalization at PM in normal prostate and PCa with grades 2-5. Kruskal-Wallis test; *p<0.05, mean ± SD.
Funding
Foundation for the National Institutes of Health (FNIH)
National Institute of General Medical Sciences (NIGMS)
United States Department of Health and Human Services
Find out more...Government Council on Grants, Russian Federation
ARTICLE ABSTRACT
Prostate cancer 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 IHC, we found a strong association of integrin αv and Gal-3 at the plasma membrane (PM) in primary prostate cancer 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 patient with prostate cancer samples aggravates Golgi scattering, activates MGAT5, and enhances integrin expression at PM. This explains known link between alcohol consumption and prostate cancer 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.
Combined ablation of ATF6 and autophagy can serve as new mCRPC therapeutic.
