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The Tabula Muris ConsortiumWe have created a compendium of single cell transcriptome data from the model organism Mus musculus comprising more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, revealing gene expression in poorly characterized cell populations and allowing for direct and controlled comparison of gene expression in cell types shared between tissues, such as T-lymphocytes and endothelial cells from distinct anatomical locations. Two distinct technical approaches were used for most tissues: one approach, microfluidic droplet-based 3’-end counting, enabled the survey of thousands of cells at relatively low coverage, while the other, FACS-based full length transcript analysis, enabled characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

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.

The link between prostate cancer (PCa) risk and alcohol consumption has long been debated. In our recent analysis of the epidemiologic evidence for this link, we found that, since the onset of the PSA testing era, a preponderance of studies indicates ethanol (EtOH) consumption is strongly associated with PCa risk. Despite epidemiologic support for this relationship, little is known about the underlying mechanisms. Our group introduced the concept of “onco-Golgi,” where the Golgi becomes fragmented alongside activating transcription factor 6 (ATF6)-mediated Endoplasmic Reticulum (ER) stress. This results in increased plasma membrane (PM) expression of abnormally glycosylated αv Integrins by Golgi glycosyltransferase, N-acetylglucosaminyltransferase-V (MGAT5). Importantly, these MGAT5-modified integrins bind to pentameric Galectin-3, resulting in clustering and increased retention on the PM. This, in turn, modulates tumor cell behavior, including adhesion to extracellular matrix and migration, leading to acceleration of prostate tumor dissemination to lymph nodes and distant organs, including bones. We have also found the EtOH treatment causes further Golgi disorganization. PCa tissues from heavy alcoholic patients demonstrate higher MGAT5 expression and elevated Integrin αv levels on the PM. We propose that EtOH promotes PCa lethality by increasing Integrin αv-mediated PCa progression. Altered glycosylation of Integrin αv in the onco-Golgi is expected to be exacerbated by alcohol’s disorganizing effect on Golgi. We have found a positive correlation between the number of Golgi fragments and the intensity of Integrin αv on the PM in both docetaxel-resistant PC-3 and DU145 cells after EtOH treatment. Importantly, we revealed that Golgi disorganization in advanced PCa cells is an autophagy-driven process. We found that autophagy inhibitor, Hydroxychloroquine (HCQ), restores compact Golgi in both PC-3 and DU145 cells, as well as in mice orthotopic PC-3-derived tumors. The effect of HCQ was strengthened by depletion of ATF6, suggesting the synergetic effect of autophagy inhibition and alleviation of ER stress. Significantly, HCQ treatment attenuates the EtOH-induced Golgi fragmentation and restores the level of PM Integrin αv to that of control cells. We expect that our established protocol to restore Golgi morphology and inhibit ER stress by HCQ treatment and ATF6 depletion, respectively, will attenuate the impact of EtOH on PCa cells. Overall, these data shed light on alcohol-promoting effects on prostate tumor growth and metastasis and provide a potentially effective therapeutic strategy. Citation Format: Amanda Macke, Artem Pachikov, Taylor Divita, Armen Petrosyan. Golgi disorganization and ER stress: the mechanism underlying alcohol-mediated prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1330.