Master Regulator Of Unfolded Protein Response Research Articles

Programmed cell death regulator BAP2 is required for IRE1-mediated unfolded protein response in Arabidopsis

Environmental and physiological situations can challenge the balance between protein synthesis and folding capacity of the endoplasmic reticulum (ER) and cause ER stress, a potentially lethal condition. The unfolded protein response (UPR) restores ER homeostasis or actuates programmed cell death (PCD) when ER stress is unresolved. The cell fate determination mechanisms of the UPR are not well understood, especially in plants. Here, we integrate genetics and ER stress profiling with natural variation and quantitative trait locus analysis of 350 natural accessions of the model species Arabidopsis thaliana. Our analyses implicate a single nucleotide polymorphism to the loss of function of the general PCD regulator BON-ASSOCIATED PROTEIN2 (BAP2) in UPR outcomes. We establish that ER stress-induced BAP2 expression is antagonistically regulated by the UPR master regulator, inositol-requiring enzyme 1 (IRE1), and that BAP2 controls adaptive UPR amplitude in ER stress and ignites pro-death mechanisms in conditions of UPR insufficiency.

Abstract P5-14-19: Targeting the unfolded protein response components differentially sensitizes breast tumors to doxorubicin while modulating chemotherapy-induced cardiotoxicities

Abstract Background: The unfolded protein response (UPR) is an endoplasmic reticulum pathway stimulated when the protein folding demand of a cell surpasses the bio-energetic capacity. UPR master regulator and protein chaperone, GRP78, binds to the three signaling arms (PERK, ATF6, and IRE1) rendering them inactive. When unfolded protein accumulate, GRP78 unbinds from these components enabling UPR activation. We, among other, previously showed elevated UPR in breast tumors promoted therapeutic resistance. However, studies exploring whether combinatoral therapies targeting UPR coupled with standard of care chemotherapeutics modulates the development of co-morbidities remain under-explored. Methods: 4T1-luciferase triple negative breast cancer cells were injected into the mammary fat pad of BALBc mice. Tumor-bearing mice were treated with 1 x weekly 30 µM GRP78, PERK, or IRE1 morpholino +/- four 1 x weekly 2.5 mg/kg doxorubicin (cumulative dose 10 mg/kg). Tumor volume was measured by calipers and IVIS. Cardiac function was determined by VEVO ultrasound in morpholino + DOX treated BALBc and WT/GRP78 heterozygous mice. In vitro and ex-vivo viability experiments were performed using ACEA xCELLigence RTCA system. Results: Targeting GRP78 or IRE1 potentiated doxorubicin-mediated tumor killing while targeting PERK enhanced 4T1 tumor growth and prevented chemotherapy efficacy. Targeting GRP78 or PERK reduced cardiac function in response to doxorubicin resulting in poor overall survival. However, targeting XBP1 either through upstream IRE1 protein reduction or inhibition of IRE1 activity protected in vivo heart function. In vitro pre-treatment of cardiomyoblast H9C2 cells with KIRA8 (a XBP1 inhibitor) reduced doxorubicin-mediated cell death and reduced chemotherapy-induced apoptosis as measured by cleaved caspase-3 and cleaved PARP. Ex-vivo treatment of primary cardiac cells isolated from wild-type and GRP78 heterozygous mice indicate inhibition of XBP1 ameliorated GRP78 heterozygous sensitization to doxorubicin, suggesting the elevated cardiotoxicities observed by systemic GRP78 targeting is resulting from elevated IRE1 activation. Conclusions: We now show that while targeting GRP78 and IRE1 in vivo enhances tumor doxorubicin responsiveness, targeting these proteins results in differential effects on chemotherapy-mediated cardiotoxicities. Targeting PERK enhanced tumor growth and reduced chemotherapy responsiveness. Our study highlights the importance of testing these pre-clinical drugs on cardiac function to reduce future clinical trial side effects. Citation Format: David Soto-Pantoja, Kenysha Clear, Katherine Loree Cook. Targeting the unfolded protein response components differentially sensitizes breast tumors to doxorubicin while modulating chemotherapy-induced cardiotoxicities [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-14-19.

Cell surface GRP78 expression on T and NK cell sub-populations of breast cancer patients.

11561 Background: The targeting of unfolded protein response (UPR) in tumor cells has received much attention. However, data are sparse on the impact of UPR on T and NK cells. The master regulator of UPR is the glucose-regulated protein 78 (GRP78) that is expressed in some tumor cells or normal stressed cells. There are few studies concerning GRP78 expression on T and NK cells in cancer and its relationship to stress induction by chemotherapy. We aimed to reveal the effect of UPR activation on the peripheral T and NK cells of breast cancer patients by the evaluation of cell surface GRP78 expression on T and NK cells before and after neoadjuvant chemotherapy. Methods: Forty-seven patients with triple negative, ER positive/Her2 negative and Her2 positive breast cancer were included. FACS analysis of their blood specimens before and after neoadjuvant treatment was performed. For multicolor FACS analysis, anti-CD3, CD4, CD8, CD56, CD16, NKG2D, CD45RA, CD45RO, CCR7 CD62L and anti-GRP78 antibody (AF488) were added to one of the tubes. A second tube was incubated with IgG-AF488 as isotype control. Analysis of the different T and NK subpopulations that expressed cell surface GRP78 were analyzed with the Gallios Flow cytometer and Kaluza Flow Analysis Software (Beckman Coulter, Inc.). Results: The percentage of cell surface GRP78 baseline expression in CD3 (1.8±0.9), CD8 (2.9±1.5), NKG2D (4.8±2) and CD45RO/CD62L/CCR7 active T memory cells (2.1±0.5) in Her2 positive patients were significantly higher than in triple negative and ER positive/Her2 negative patients. GRP78 expression on CD56, CD16 and NKG2D cells measured after neoadjuvant treatment was significantly higher in patients with complete response (CR) compared to patients without CR. 89% of the CR patients presented with Her2+ subtype. The non-CR patients include triple negative and Her2 negative subtypes. Conclusions: GRP78 was found to be expressed in the different T and NK sub-populations. The level of expression changed with each breast cancer subtype and response to chemotherapy. These novel findings suggest that GRP78 may be used as a new predictive biomarker. It sets the stage for understanding the mechanism of UPR activation on the immune system in breast cancer.

AKT inhibition mitigates GRP78 (glucose‐regulated protein) expression and contribution to chemoresistance in endometrial cancers

Overexpression of the unfolded protein response master regulator GRP78 is associated with poor prognosis and therapeutic resistance in numerous human cancers, yet its role in endometrial cancers (EC) is undefined. To better understand the contribution of GRP78 to EC, we examined its expression levels in EC patient samples and EC cell lines. We demonstrate that GRP78 overexpression occurs more frequently in EC tissues compared with that found in normal endometrium, and that GRP78 expression occurs in most EC cell lines examined. Functional analysis demonstrated that GRP78 is inducible by cisplatin in EC cells, and siRNA knockdown of GRP78 augments chemotherapy-mediated cell death. Examination of AKT and GRP78 expression demonstrated that inhibition of AKT activity by MK2206 blocks GRP78 expression in EC cells. SiRNA studies also revealed that knockdown of GRP78 reduces but does not abrogate AKT activity, demonstrating that GRP78 is required for optimal AKT activity. In the presence of MK2206, siRNA knockdown of GRP78 does not augment AKT mediated survival in response to cisplatin treatment, suggesting that GRP78's antiapoptosis functions are part of the AKT survival pathway. Targeted therapies that reduce GRP78 expression or activity in cancers may serve to increase the effectiveness of current therapies for EC patients.

Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis

Neurodegenerative diseases are often associated with dysfunction in protein quality control. The endoplasmic reticulum (ER), a key site for protein synthesis, senses stressful conditions by activating the unfolded protein response (UPR). Here we report the creation of a novel mouse model where GRP78/BiP, a major ER chaperone and master regulator of UPR, is specifically eliminated in the Purkinje cells (PCs). GRP78 depleted PCs activate UPR including induction of GRP94, PDI, CHOP and GADD34, feedback suppression of eIF2α phosphorylation and apoptotic cell death. In contrast to current models of protein misfolding where abnormal accumulation of ubiquitinated protein is prominent, cytosolic ubiquitin staining is dramatically reduced in GRP78 null PCs. Ultrastructural evaluation reveals that the ER shows prominent dilatation with focal accumulation of electron-dense material within the ER. The mice show retarded growth and severe motor coordination defect by week 5 and cerebellar atrophy by week 13. Our studies uncover a novel link between GRP78 depletion and reduction in cytosolic ubiquitination and establish a novel mouse model of accelerated cerebellar degeneration with basic and clinical applications.