Salubrinal Exposes Anticancer Properties in Inflammatory Breast Cancer Cells by Manipulating the Endoplasmic Reticulum Stress Pathway

Abstract

The endoplasmic reticulum (ER) regulates protein folding, post-translational modifications, lipid synthesis, and calcium signaling to attenuate the accumulation of misfolded proteins causing ER stress and maintains cellular homeostasis. The tumor microenvironment is rich in soluble cytokines, chemokines, growth, and angiogenic factors and can drive the ER’s abnormal functioning in healthy cells. Cancer cells adapt well to the tumor microenvironment induced ER stress. We identified that the inflammatory breast cancer (IBC) cells abundantly express osteoprotegerin (OPG) and their tumor microenvironment is rich in OPG protein. OPG also called osteoclast differentiation factor/osteoclastogenesis inhibitory factor (OCIF) is a soluble decoy receptor for receptor activator of nuclear factor-kappa B ligand (RANKL). Employing mass spectrometry analysis, we identified a set of ER chaperones associated with OPG in IBC cell lysates (SUM149PT, SUM1315MO2) compared to healthy human mammary epithelial cells (HMEC). Proximity ligation assay (PLA) and immunoprecipitation assay validated the interaction between OPG and ER chaperone and master regulator of unfolded protein response (UPR) GRP78/BiP (glucose-regulated protein/Binding immunoglobulin protein). We detected remarkably high gene expression of CCAAT enhancer-binding protein homologous protein (CHOP), inositol-requiring enzyme 1 (IRE1α), protein disulfide-isomerase (PDI), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP-1) and growth arrest and DNA damage-inducible protein (GADD34) in SUM149PT and SUM190PT cells when compared to HMEC. Similarly, tissue sections of human IBC expressed high levels of ER stress proteins. We evaluated cell death and apoptosis upon Salubrinal and phenylbutyrate treatment in healthy and IBC cells by caspase-3 activity and cleaved poly (ADP-ribose) polymerase (PARP) protein assay. IBC (SUM149PT and SUM190PT) cells were chemosensitive to Salubrinal treatment, possibly via inhibition in OPG secretion, upregulating ATF4, and CHOP, thus ultimately driving caspase-3 mediated IBC cell death. Salubrinal treatment upregulated PDI, which connects ER stress to oxidative stress. We observed increased ROS production and reduced cell proliferation of Salubrinal treated IBC cells. Treatment with antioxidants could rescue IBC cells from ROS and aborted cell proliferation. Our findings implicate that manipulating ER stress with Salubrinal may provide a safer and tailored strategy to target the growth of inflammatory and aggressive forms of breast cancer.

The roles of osteoprotegerin in cancer, far beyond a bone player

“…Lane et al reported that OPG inhibited the TRAIL-mediated apoptosis of ovarian cancer cells in an α v β 3 and α v β 5 integrin-dependent manner [ 46 ]. In inflammatory breast cancer, OPG interacted with glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP), the endoplasmic reticulum (ER) chaperone and master regulator of the unfolded protein response, to promote cell apoptosis and prolong the survival of inflammatory breast cancer cells [ 47 ]. By forming complexes with the lipid raft core protein Cav-1 and EGFR, RANK promoted gastric cancer cell migration, and this effect was mitigated by OPG [ 48 ].…”

Endoplasmic reticulum stress in innate immune cells - a significant contribution to non-alcoholic fatty liver disease

“…Pre-clinical studies have shown that carbon monoxide upregulates sestrin-2 through the PERK-eIF2α-ATF4 signaling pathway and alleviates dietary methionine/choline deficiency induced hepatic steatosis ( 23 ). Salubrinal is a selective inhibitor of eIF2α dephosphorylation, which maintains the phosphorylation state of eIF2α and protects cells from ER stress-induced apoptosis ( 24 ). By inhibiting the dephosphorylation of eIF2α in ER stress, Salubrinal reduces hepatic steatosis and fat deposition ( 25 ).…”

ZNF354C Mediated by DNMT1 Ameliorates Lung Ischemia-Reperfusion Oxidative Stress Injury by Reducing TFPI Promoter Methylation to Upregulate TFPI

“…ROS Detection. Following previous research [35], the level of reactive oxygen species (ROS) was analyzed using a DCFDA/H2DCFDA-Cellular ROS Assay (ab113851; Abcam, Cambridge, UK) according to the manufacturer's instruction.…”