Supplementary Figure 4 from Targeting the ATF6-mediated ER stress response and autophagy blocks integrin-driven prostate cancer progression

Macke, Amanda J.; Pachikov, Artem N.; Divita, Taylor E.; Morris, Mary E.; LaGrange, Chad A.; Holzapfel, Melissa S.; Kubyshkin, Anatoly V.; Zyablitskaya, Evgeniya Y.; Makalish, Tatiana P.; Eremenko, Sergey N.; Qiu, Haowen; Riethoven, Jean-Jack M.; Hemstreet, George P.; Petrosyan, Armen

doi:10.1158/1541-7786.27035080.v1

Supplementary Figure 4 from Targeting the ATF6-mediated ER stress response and autophagy blocks integrin-driven prostate cancer progression

journal contribution

posted on 2024-09-16, 11:46 authored by Amanda J. Macke, Artem N. Pachikov, Taylor E. Divita, Mary E. Morris, Chad A. LaGrange, Melissa S. Holzapfel, Anatoly V. Kubyshkin, Evgeniya Y. Zyablitskaya, Tatiana P. Makalish, Sergey N. Eremenko, Haowen Qiu, Jean-Jack M. Riethoven, George P. Hemstreet, Armen Petrosyan

Figure S4. (A) Representative images of Golgi in DU145 cells treated with 60 µM HCQ for 72 h and stained by GM130 (green); bars, 20 µm. (B) Quantification of Golgi spots per cell in samples from A. Unpaired t test; **p<0.01, mean ± SD; n represents a number of cells counted. (C) Representative images of Golgi in DU145 cells treated with control or ATF6α siRNAs and stained by GM130 (green); bars, 20 µm. (D) Quantification of Golgi spots per cell in samples from C. Unpaired t test; ***p<0.001, mean ± SD; n represents number of cells counted. (E) ATF6 W-B of the lysates of DU145 cells transfected with control or ATF6α siRNAs; β-actin is a loading control. (F) Representative images of Golgi in PC-3 cells treated with control or ATG5 siRNAs and stained by GM130 (green); bars, 20 µm. (G) Quantification of Golgi spots per cell in samples from F. Unpaired t test; ****p<0.0001, mean ± SD; n represents number of cells counted. (H) ATG5 W-B of the lysates of PC-3 cells transfected with control or ATG5 siRNAs; γ-tubulin is a loading control. (I) Representative images of Golgi in DU145 cells treated with control or ATG5 siRNAs and stained by GM130 (green); bars, 20 µm. (J) Quantification of Golgi spots per cell in samples from I. Unpaired t test; ****p<0.0001, mean ± SD; n represents number of cells counted. (K) ATG5 W-B of the lysates of DU145 cells transfected with control or ATG5 siRNAs; γ-tubulin is a loading control. (L) Representative images of Golgi in PC-3 cells treated with Bafilomycin A1 (10 µM for 72 h) or appropriate amount of DMSO and stained by GM130 (green); bars, 20 µm. (M) Quantification of Golgi spots per cell in samples from L. Unpaired t test; ****p<0.0001, mean ± SD; n represents number of cells counted. (N-R) GRASP65 (N), GM130 (O), Giantin (P), Golgin-245 (Q), and TGN46 (R) W-Bs of the lysates of PC-3 cells treated with HCQ; β-actin is a loading control. All data presented are representative of at least three independent experiments.

History

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.