Nuclear hormone receptors that are not eliminated by small molecule Sigma1 inhibitor (IPAG).

RNAi mediated knockdown of ATG5 does not prevent IPAG induced decrease in AR levels.

CT-189 Chemical, ADME, and Pharmacokinetics Properties.

Quantification of immunoblot band densities in Figure 3B.

Multiple Sigma1 shRNA and absence of general protein level changes - evidence of selective actions.

Specificity of ARV7 and ARv567es antibodies.

Abstract Emerging evidence suggests that Sigma1 (also known as sigma1 receptor) is a unique ligand-operated integral membrane chaperone or scaffolding protein that contributes to cellular protein homeostasis. Previously, we found that treatment of various cancer cell lines with some prototypic small molecule modulators of Sigma1 can engage endoplasmic reticulum (ER) associated protein homeostasis pathways including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type 1 integral membrane glycoprotein that is processed and transported through the ER and secretory pathway of tumor cells. PD-L1 expressed at the surface of tumor cells can act as a T-cell inhibitory checkpoint molecule that inactivates tumor infiltrating immune cells that express PD-1, its cognate receptor. Here, we show that Sigma1 physically associates with PD-L1. In triple negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels are suppressed by both RNAi mediated knockdown of Sigma1 and pharmacological modulation of Sigma1. We observe decreased cell surface and intracellular levels of PD-L1 by flow cytometry and biochemical subcellular fractionation respectively, which corresponds with a dose-responsive decrease in functional PD-L1/PD-1 interaction in a co-culture of cancer cells and T-cells. Inhibitors of autophagy block this suppression of PD-L1 protein levels, suggesting PD-L1 is degraded away by autophagy after Sigma1 modulation. Through confocal microscopy, we show that Sigma1 modulation results in colocalization of PD-L1 and GFP-LC3, a marker of autophagosomes. From these conclusions, we hypothesize that autophagic degradation of nascent PD-L1 after Sigma1 modulation plays a key role in preventing the transport of functional PD-L1 to the plasma membrane. Together, these data demonstrate that Sigma1 modulators have the potential to act as novel therapeutic agents in PD-1/PD-L1 blockade strategies. Citation Format: Christina M. Maher, Jeffrey D. Thomas, Charles G. Longen, Derick A. Haas, Halley M. Oyer, Jane Y. Tong, Felix J. Kim. Pharmacologic modulation of Sigma1 induces autophagic degradation of programmed death-ligand 1 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3202. doi:10.1158/1538-7445.AM2017-3202

Abstract Suppression of androgen receptor (AR) activity in prostate cancer by androgen depletion or direct AR antagonist treatment, although initially effective, leads to incurable castration-resistant prostate cancer (CRPC) via compensatory mechanisms including resurgence of AR and AR splice variant (ARV) signaling. Emerging evidence suggests that Sigma1 (also known as sigma-1 receptor) is a unique chaperone or scaffolding protein that contributes to cellular protein homeostasis. We reported previously that some Sigma1-selective small molecules can be used to pharmacologically modulate protein homeostasis pathways. We hypothesized that these Sigma1-mediated responses could be exploited to suppress AR protein levels and activity. Here we demonstrate that treatment with a small-molecule Sigma1 inhibitor prevented 5α- dihydrotestosterone-mediated nuclear translocation of AR and induced proteasomal degradation of AR and ARV, suppressing the transcriptional activity and protein levels of both full-length and splice-variant AR. Consistent with these data, RNAi knockdown of Sigma1 resulted in decreased AR levels and transcriptional activity. Furthermore, Sigma1 physically associated with ARV7 and ARv567es as well as full-length AR. Treatment of mice xenografted with ARV-driven CRPC tumors with a drug-like small-molecule Sigma1 inhibitor significantly inhibited tumor growth associated with elimination of AR and ARV7 in responsive tumors. Together, our data show that Sigma1 modulators can be used to suppress AR/ARV–driven prostate cancer cells via regulation of pharmacologically responsive Sigma1-AR/ARV interactions, both in vitro and in vivo. Cancer Res; 77(9); 2439–52. ©2017 AACR.

Abstract Emerging evidence suggests that Sigma1 (SIGMAR1, also known as sigma-1 receptor) is a unique ligand-regulated integral membrane scaffolding protein that contributes to cellular protein and lipid homeostasis. Previously, we demonstrated that some small-molecule modulators of Sigma1 alter endoplasmic reticulum (ER)–associated protein homeostasis pathways in cancer cells, including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type I integral membrane glycoprotein that is cotranslationally inserted into the ER and is processed and transported through the secretory pathway. Once at the surface of cancer cells, PD-L1 acts as a T-cell inhibitory checkpoint molecule and suppresses antitumor immunity. Here, we demonstrate that in Sigma1-expressing triple-negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels were suppressed by RNAi knockdown of Sigma1 and by small-molecule inhibition of Sigma1. Sigma1-mediated action was confirmed by pharmacologic competition between Sigma1-selective inhibitor and activator ligands. When administered alone, the Sigma1 inhibitor decreased cell surface PD-L1 expression and suppressed functional interaction of PD-1 and PD-L1 in a coculture of T cells and cancer cells. Conversely, the Sigma1 activator increased PD-L1 cell surface expression, demonstrating the ability to positively and negatively modulate Sigma1 associated PD-L1 processing. We discovered that the Sigma1 inhibitor induced degradation of PD-L1 via autophagy, by a mechanism distinct from bulk macroautophagy or general ER stress–associated autophagy. Finally, the Sigma1 inhibitor suppressed IFNγ-induced PD-L1. Our data demonstrate that small-molecule Sigma1 modulators can be used to regulate PD-L1 in cancer cells and trigger its degradation by selective autophagy. Implications: Sigma1 modulators sequester and eliminate PD-L1 by autophagy, thus preventing functional PD-L1 expression at the cell surface. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies that regulate the tumor immune microenvironment. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/2/243/F1.large.jpg. Mol Cancer Res; 16(2); 243–55. ©2017 AACR.

Genome-wide association studies have identified thousands of loci for common diseases, but, for the majority of these, the mechanisms underlying disease susceptibility remain unknown. Most associated variants are not correlated with protein-coding changes, suggesting that polymorphisms in regulatory regions probably contribute to many disease phenotypes. Here we describe the Genotype-Tissue Expression (GTEx) project, which will establish a resource database and associated tissue bank for the scientific community to study the relationship between genetic variation and gene expression in human tissues.

In vitro and in vivo experimental studies have demonstrated the role of lysophosphatidic acid (LPA) signaling in tumor proliferation, invasiveness, and metastasis. Among LPA receptors, the overexpression of LPA receptor 3 (LPAR3) in transgenic mice has resulted in the highest rate of breast cancer metastasis. Our goal is to evaluate the LPA-producing enzyme autotaxin and LPAR3 as potential therapeutic targets in breast cancer patients. The expression of autotaxin and LPAR3 was examined by immunohistochemical analysis of 87 invasive human breast carcinomas. Carcinomas were more frequently positive for autotaxin and LPAR3 (24.4 and 43 %, respectively) compared to adjacent normal breast tissue (6.1 and 2.9 %, respectively). Increased stromal autotaxin expression was found in 16.3 % of the tumors. LPAR3 overexpression was associated with less differentiated tumors, human epidermal growth factor receptor 2 expression, and absence of progesterone receptors. The luminal type A carcinomas showed the lowest frequency of autotaxin and LPAR3 expression. Strong desmoplastic stromal reaction was more frequent among the carcinomas with autotaxin-positive tumor cells or autotaxin-positive stroma. Patients with carcinomas overexpressing LPAR3 in epithelial cells or autotaxin in stromal cells were more likely to have larger tumors, nodal involvement, and higher stage disease. Autotaxin overexpression in tumor cells also correlated with tumor size and clinical stage. Our data indicate that the increased expression of LPAR3 and autotaxin in human breast cancer is associated with tumor aggressiveness. They also suggest that LPA mediates tumor metastatic ability and peritumoral desmoplastic reaction through autocrine–paracrine mechanisms. A substantial portion of breast cancer patients might benefit from autotoxin/LPA receptor-targeted therapies.

Translation is often repressed in cell lines that are exposed to hypoxic conditions (0.5% - 1.5% O2) but this repression requires prolonged exposure (> 16 h). We report here that prolonged exposure to hypoxia results in the depletion of glucose from the media and that the loss of glucose correlates with the shut down in translation. Furthermore, we show that the addition of glucose or reoxygenation restores translation in hypoxic PC3 cells. This indicates that both glucose depletion and hypoxia are required for translational repression. We also show that eIF2alpha phosphorylation is reversed by glucose addition. Moreover, we present data that strongly indicate that eIF2alpha phosphorylation as well as the translational inhibition that occurs when cells are grown under conditions of glucose depletion and hypoxia is pancreatic eIF2alpha kinase (PERK) independent. We believe this is the first report to show that glucose depletion is required for translational repression under hypoxic conditions and that this explains why prolonged exposure to hypoxia is required for this inhibition. Since the physiological conditions that lead to tumor hypoxia would also likely lead to reduced glucose levels, understanding the interplay of glucose and hypoxia in regulating tumor metabolism will provide important information on the growth and development of solid tumors.

Tumors must adapt to the hypoxic environment in order to grow beyond a benign microscopic mass. In addition to transcriptional activation mediated by HIF-1alpha, hypoxia has also been reported to inhibit translation. The degree of translational inhibition is dependent on the duration as well as the severity of the hypoxic insult. Anoxia (<0.02% O(2)) seems to have a more rapid and dramatic effect on translation as compared to hypoxia. We show here that prolonged hypoxia dramatically and reversibly inhibits translation in PC-3 cells. We also found that mTOR is inactivated and eIF-2alpha is phosphorylated during hypoxic treatment but only the eIF-2alpha phosphorylation correlates with the translational repression. We further used polysome analysis and microarray technology to analyze the impact of this translational repression on gene expression. We found that 33 mRNAs were refractory to this translational repression and that there was no correlation between mRNA induction and the ability to recruit ribosomes during hypoxia. We also found that ribosomal protein encoding mRNAs are more sensitive to this translational repression as compared to the majority of mRNAs. Although other reports have analyzed the effect of translation inhibition on gene expression under anoxic conditions, we believe that this is the first report in hypoxic cells. Our results show that the translational repression that occurs during hypoxia does impact gene expression in the highly transformed prostate cancer cell line, PC-3.

Prostate adenocarcinoma metastasizes to the skeleton more frequently than any other organ. An underlying cause of this phenomenon may be the ability of bone-produced factors to specifically select disseminated prostate cancer cells that are susceptible to their trophic effects. Platelet-derived growth factor (PDGF), a potent mitogen for both normal and tumor cells, is produced in several tissues including bone, where it is synthesized by both osteoblasts and osteoclasts. Here, we show that PDGF causes a significantly stronger activation of the Akt/PKB survival pathway in bone-metastatic prostate cancer cells compared to nonmetastatic cells. Normal prostate epithelial cells and DU-145 prostate cells, originally derived from a brain metastasis, are not responsive to PDGF. In contrast, epidermal growth factor stimulates Akt to the same extent in all prostate cells tested. This difference in PDGF responsiveness depends on the higher expression of α -PDGFR in bone-metastatic compared to nonmetastatic prostate cells and the lack of α -PDGFR expression in normal and metastatic prostate cells derived from tissues other than bone. Thus, α -PDGFR expression might identify prostate cancer cells with the highest propensity to metastasize to the skeleton.