GRP78: Drug Discovery

ICC/IF of GRP78 Antibody in HeLa Cells
Immunofluorescent detection of GRP78 in HeLa cells using Anti-GRP78 (6H4-2G7 monoclonal antibody at 1:100

Grp78/BiP is the main regulatory molecule of the unfolded protein response (UPR). Numerous physiological and patholgical conditions are known to induce acute ER stress leading to the activation of the UPR. By using Grp78/BiP knockout mice, it became obvious that Grp78/BiP is essential for embryonic development as homozygous Grp78/BiP knockout was found to lead to embryonic lethality 292. Interestingly, heterozygous Grp78/BiP +/- mice were viable, remained phenotypically inconspicous but expressed Grp78/BiP at a markedly reduced level 292. In a transgenic mouse model for aggressive metastatic breast cancer, GRP78/BIP heterozygosity led to extended latency and decelerating tumor progression 293. GRP78/BIP heterozygosity thus represents a reliable tool to study the impact of Grp78/BiP in human disease progression.

A growing body of evidence demonstrates the pivotal role of Grp78/BiP in the pathogenesis of neurodegeneration and age-related diseases, autoimmune and auto-inflammatory diseases, microbial infections and cancer. With respect to cancer, Grp78/BiP has been found as being critically implicated in tumor progression, angiogenesis, malignancy, metastasis as well as chemoresistance. As outlined before, the UPR can be divided into two phases: pro-survival (early) and pro-apoptotic (late). Chronic activation of this pathway as found in several types of cancer is associated with the inactivation of the late (pro-apoptotic) branch thereby blocking the elimination of tumor cells. The concomitant stimulation of the early (pro-survival) branch then promotes the growth of tumors.

While the expression of Grp78/BiP in adult tissues is usually low under non-stress conditions, the expression of Grp78/BiP is strikingly elevated in cancer cells, solid tumors and human tumor biopsies 123, 294. The expression of Grp78/BiP is considerably up-regulated in primary tumors in comparison to benign tissues 294. Apart from its location to the ER, Grp78/BiP can also be found in the cytosol and the plasma membrane. Grp78/BiP translocation has been reported as being associated with cell transformation and the development of chemoresistance in cancer cells 295. The identification of Grp78/BiP on the cell membrane of tumor cells, but not on normal cells, implies a pro-survival function of the chaperone when localized outside of the ER. Since Grp78/BiP plays a crucial role in the induction of the UPR which is highly activated in refractory tumors, one can speculate that tumor cells expressing Grp78/BiP at a higher level would respond less markedly to chemotherapy. New evidence accumulates implying that Grp78/BiP expression can be considered as a prognostic and diagnostic marker for chemoresistance. Several investigations revealed that up-regulated Grp78/BiP levels may predict greater response to adjuvant therapy in breast cancer patients. A retrospective cohort study with 127 stage II and stage III breast cancer patients subjected to doxorubicin monotherapy identified a positive correlation between Grp78/BiP levels and resistance to chemotherapy 201. In contrast, Grp78/BiP expression in breast cancer specimens was found to predict a longer recurrence-free survival in patients who sequentially received doxorubicin and taxanes, rendering Grp78/BiP expression as being a promising candidate to predict doxorubicin resistance in breast cancer 296.

Recently, Grp78/BiP was introduced as a prognostic indicator and a potential therapeutic target in prostate cancer 202, 203. Fu et al. reported that targeted knockout of Grp78/BiP in mouse prostate epithelium abolished prostate carcinogenesis and blocked AKT activation without interfering with prostate development and growth 297. Based on these findings, inactivation of Grp78/BiP might be considered as being a novel approach to halt prostate cancer and potentially other malignancies 297. In this context, knockdown of Grp78/BiP in colorectal cancer cells significantly inhibited cell proliferation, colony formation and tumorigenesis in vitro and in vivo by down-regulating the phosphorylation of AKT and ERK-1/-2 298, thereby elucidating the Grp78/BiP-mediated down-regulation of oncogenic AKT signaling in tumors.

A significantly up-regulated expression of Grp78/BiP was also noted in malignant glioma and glioma cell lines 199, correlating with tumor grade, chemoresistance and poor survival 208. Remarkably, Grp78/BiP knockout sensitized glioma cells to chemotherapy 199. Grp78/BiP is generally up-regulated in the vasculature derived from human glioma specimens in comparison to normal brain tissues and blood vessels 210. Knockdown of Grp78/BiP using small interfering RNA or chemical inhibitors enhanced chemosensitivity of blood vessels from malignant glioma tissues 210. Combinatorial treatment of human stomach cancer xenografts with compounds targeting Grp78/BiP significantly increased drug sensitivity without affecting the phenotype of the mice 299. From these data one can conclude that targeting Grp78/BiP does not only delay tumor growth but also arterial genesis.

Since Grp78/BiP is expressed on the plasma membrane of malignant but not benign cells, surface Grp78/BiP might function as a cancer-specific target in tumor therapy. Kim and colleagues convincingly demonstrated that targeting Grp78/BiP using a cell-permeable peptidic Grp78/BiP ligand was able to induce apoptosis in the human melanoma cell line Me6652/4 300. Recombinant Kringle 5 (rK5) of plasminogen is also able to bind to surface Grp78/BiP and thus to inhibit angiogenesis and to induce caspase-dependent apoptosis in proliferating endothelial cells as well as in stressed tumor cells 8. Of note, ligation of cell surface Grp78/BiP by a commercially available polyclonal antibody directed against the C-terminus of Grp78/BiP diminished growth rate and blocked PI3K/AKT signaling in prostate cancer cells 139, 301. An antibody directed against the N-terminus of Grp78/BiP was found to suppress oncogenic Cripto signaling via MAPK/PI3K and Smad-2/-3 pathways by blocking of the cell surface Cripto/Grp78 complex in human tumor, mammary epithelial and embryonic stem cells 209.

As outlined before, most neurodegenerative disorders including Alzheimer’s disease (AD), and Parkinson’s disease (PD) are characterized by the activation of the unfolded protein response (UPR) and altered Grp78/BiP expression leading to the accumulation of aggregated or misfolded polypeptides 130, 213-215. By generating a knockin mouse model expressing a mutant isoform of Grp78/BiP devoid of the ER-retention signal KDEL, the group of Tomohiko Aoe investigated the physiological role of Grp78/BiP in neuron development. The data raised in this study revealed that neonates expressing mutant Grp78/BiP suffered from respiratory failure caused by impaired secretion of pulmonary surfactant by alveolar type II epithelial cells 302. Moreover, the mutant mice harbored disordered layer formation in the cerebral cortex and cerebellum, together with down-regulated levels and secretion of reelin 303. Reelin is a secretory glycoprotein which functions in cortical layer formation through binding to lipoprotein receptors on cortical neurons 304. Despite its function in brain development, reelin is crucially involved in the pathogenesis of human mental disorders such as AD 305. Since reelin has been reported to signal through ApoER2 in adult brains and to modulate synaptic plasticity and memory 306, the defective reelin signaling pathway thus appears to contribute to the pathogenesis of adult neurological disorders. The molecular BiP protein inducer X (BIX) was recently identified to induce the expression of Grp78/BiP via the ATF-6 pathway in retinal ganglia and neuroblastoma cells and to protect them from apoptosis 307, 308, suggesting that BIX might be a potential therapeutic agent for ER stress-induced cerebral and retinal diseases. A further study by the same group yielded reduced infarct volume and brain swelling as well as protection against apoptosis and acute ischemic neuronal damage under certain conditions 309. The expression of Grp78/BiP can also be induced in the substantia nigra pars compacta (SNc) of mice by administration of the methoxyflavone tangeretin. This treatment protected dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin which induces oxidative as well as ER stress 310. These data imply a crucial role of methoxyflavones in the regulation of ER stress, rendering methoxyflavones as being a promising therapeutic tool for ER stress-related diseases.  Notably, over-expression of Grp78/BiP mediated by adeno-associated viral (AAV) gene transfer into retinal degenerative photoreceptors in rats has been found to restore the visual function of the rhodopsin photoreceptors 311. In this transgenic rat model, Grp78/BiP over-expression impaired ER stress and diminished ATF-6 and PERK signaling pathways of the UPR as well as the level of pro-apoptotic CHOP protein, thereby attenuating the degree of apoptosis. Delivery of the Grp78/BiP cDNA by AAV vectors also led to a significant reduction in the α-Syn-induced loss of tyrosine hydoxylase-positive neurons in the SNc 311. It should be highlighted in this context that down-regulation of miR-181b in mouse brain induced neuroprotection against ischemic injury through negatively regulating the protein levels of Grp78/BiP and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) 159. Cervical cancer models clearly revealed the suppressive capacity of miR-181a in cervical cancer development via down-regulating Grp78/BiP 312. Nonetheless, up-regulation of Grp78/BiP not only enhanced the tumor cell proliferation rate but also oxaliplatin resistance in these cancer models. In line with this finding, induction of Grp78/BiP by the proteasome inhibitor bortezomib promoted macroautophagy and improved chemoresistance and survival of multiple myeloma via inactivation of NF-κB 212.  In a suicide gene therapy approach, the tyrosine kinase gene from herpes simplex virus (HSV-tk) under control of the stress-inducible HSPA5 promoter was transduced into GC cells and inoculated into nude mice 313. The HSV-tk gene acts a suicide gene when administered together with the prodrug ganciclovir (GCV). Intriguingly, GRP78-tk/GCV treatment resulted in complete tumor elimination in GC cells without inducing p53 mutations in vitro and in vivo 313. Collectively, these data validate Grp78/BiP as a prospective novel and key therapeutic target for future treatments of a potentially wide spectrum of disorders. For additional information see also section Disease Relevance.