Glucose regulated proteins in cancer: molecular mechanisms and therapeutic potential

Proliferation

GRP78 expression levels correlated with proliferative rates of human glioma cell lines and knockdown of GRP78 by small interfering RNA (siRNA) suppresses their growth³⁸. In a mouse mammary tumour virus (MMTV)-Polyoma middle T (PyT) mammary tumour model, Grp78 heterozygosity was sufficient to prolong the latency period and impede cancer growth in part through suppression of tumour cell proliferation³⁹ (Table 1). How might GRP78 facilitate proliferation? As an ER chaperone, GRP78 controls processing and maturation of a wide variety of cell surface receptors and secretory proteins crucial for the ability of cancer cells to respond to extrinsic proliferative signals²⁰. GRP78 is also a regulator of Wnt-β-catenin proliferative signaling through its stabilization of Wnt in the ER. When GRP78 was dissociated from Wnt under hypoxic conditions, Wnt was not properly processed, leading to its proteasomal degradation and reduced Wnt secretion⁴⁰.

GRP78 might also promote cell proliferation from the cell surface. ER stress or ectopic expression of GRP78 leads to localization of a subfraction of GRP78 on the cell surface¹⁵. Specific proteins have been reported to transport GRP78 to the cell surface in different cell types, such as the carrier protein MTJ-1 in macrophages and the tumour suppressor prostate apoptosis response 4 (PAR-4; also known as PAWR) in the prostate cancer cell line PC-3^41,42.

Cell surface GRP78 acts as a multifunctional receptor impacting both cell proliferation and viability^11-14. For example, in prostate cancer cells, cell surface GRP78 serves as receptor for the activated form of the plasma proteinase inhibitor α2-macroglobulin (α2-M*)⁴³, triggering ERK and AKT activation and increased DNA and protein synthesis⁴⁴. AKT signaling, which promotes proliferation and inhibits apoptosis, is also triggered by autoantibodies against the N-terminus of GRP78 that are found in cancer patients⁴⁵. How might GRP78 regulate AKT activation? Cell surface GRP78 co-localizes with PI3K, an activator of AKT, and co-immunoprecipitates with PI3K subunits^16,17. Furthermore, in cell culture model systems, overexpression of GRP78 leads to increased PIP3 production (a signaling molecule downstream of PI3K) and mutation of the N-terminal region of GRP78 reduced both the binding of cell surface GRP78 to PI3K and PIP3 production¹⁶. A requirement for GRP78 in a serum-stimulated increase in PIP3 production has also been reported in human leukemic cells³⁴.

PTEN (phosphatase and tension homolog deleted in chromosome 10), which encodes a plasma membrane lipid phosphatase that antagonizes the PI3K signaling pathway, is a major tumour suppressor gene in human cancer⁴⁶. A biallelic conditional knockout mouse model of Grp78 and Pten in the prostate epithelium or bone marrow showed that GRP78 deficiency reduces PI3K-AKT activation, which normally occurs as a result of PTEN loss in these cells, and potently inhibits prostate tumorigenesis⁴⁷ and leukemogenesis⁴⁸, respectively (Table 1). Although cell surface GRP78 has been shown to regulate PI3K signaling, further studies are required to determine whether GRP78 in the ER or other cellular locations might also regulate PI3K-AKT signaling. Recent studies revealed GRP78 is a downstream target of the IGF-1R-PI3K signaling pathway in mouse embryo fibroblasts, as well as in cancer cell lines^18,19, and this could represent a feedback regulatory mechanism that balances GRP78 expression and cancer cell proliferation.

Another pro-proliferative mechanism of GRP78 is the interaction of cell surface GRP78 with Cripto-1 (also known as teratocarcinoma-derived growth factor 1), a glycosylphosphatidylinositol (GPI)-anchored, developmentally regulated, oncoprotein.⁴⁹ Disruption of cell surface GRP78 and Cripto complex blocked Cripto activation of MAPK and PI3K pathways and modulation of activin-A, activin-B, nodal and transforming growth factor-β1 signaling⁵⁰. Thus, cell surface GRP78 is a necessary mediator of Cripto proliferative signaling in human cancer.

GRP94 controls the maturation and secretion of IGFs, which are important mitogenic factors⁵¹, and binding of IGF-1 or IGF-2 to the IGF-1R leads to PI3K-AKT activation. GRP94 regulates the processing of the low density lipoprotein receptor-related protein 6 (LRP6), a Wnt co-receptor⁵². Without GRP94, LRP6 is not exported from the ER to the cell surface, leading to attenuation of the pro-proliferative and pro-survival Wnt-β-catenin signaling pathway. This is the proposed mechanism for the attenuation of multiple myeloma and inflammatory colorectal cancer in mouse models where Grp94 is deleted in B-cells⁵³ and macrophages⁵⁴, respectively (Table 1). In breast cancer cells that are able to proliferate under chronic exposure to reactive oxygen species (ROS) in vitro, the expression of GRP94, but not HSP90 or GRP78, is increased⁵⁵. ROS are counteracted by the production of antioxidants and the formation of disulphide bonds in proteins in the ER, which is promoted by GRP94.

Overexpression of GRP75 in mouse fibroblasts leads to anchorage-independent growth, and formation of tumours when transplanted into nude mice⁵⁶. Contributing factors might include the role of GRP75 as a mitochondrial protein importer and its ability to retain p53 in the cytoplasm, leading to down-regulation of p53 target genes such as Cdkn1a and Mdm2. This effect on p53 has been shown in a subset of neuroblastomas⁵⁷. Another client protein of GRP75 is fibroblast growth factor-1 (FGF-1) which possesses broad mitogenic activities and functions as a modifier of endothelial cell migration and proliferation and is therefore pro-angiogenic⁵⁸.

Apoptosis

In general, the GRPs are suppressors of apoptosis²⁰. Caspase-7, an executioner caspase that is associated with the ER, can be activated by the chemotherapeutic agent etoposide, and GRP78, in a manner dependent on its ATP binding activity, forms complex with caspase-7 (Figure 1) and protects cells from apoptosis induced by etoposide^59,60. Recently, a functional relationship was uncovered at the outer surface of the ER between GRP78, the pro-apoptotic protein BIK and the anti-apoptotic protein BCL-2^61,62. GRP78 and BCL-2 form separate complexes with different domains of BIK. BIK sequestration of BCL-2 reduces BCL-2 interaction with the ER, leading to ER Ca²⁺ release, translocation of the pro-apoptotic protein BAX to the mitochondria and the release of cytochrome c to the cytosol, which initiates apoptosis. However, high levels of GRP78 sequester BIK, which releases the inhibition of BCL‐2, thereby suppressing apoptosis⁶².

These observations, however, raise the important question of how GRP78 as an ER lumen protein, can interact with cytosolic proteins that associate with the outer ER membrane. Intriguingly, two independent studies showed that a subpopulation of GRP78 from isolated microsomes was resistant to sodium carbonate extraction and existed as a partially protease resistant (presumably transmembrane) protein^59,60. However, despite the presence of some weak hydrophobic motifs supporting this possibility, the primary amino sequence of GRP78 does not predict a traditional transmembrane configuration under normal physiological conditions. Thus, the interaction between GRP78 and cytosolic proteins will have to be mediated either by an unconventional form of GRP78 that spans the ER membrane or lumenal GRP78 in complex with other ER transmembrane proteins; this issue remains to be resolved.

ER stress induces alternative splicing of GRP78, generating a cytosolic isoform (GRP78va) that regulates PERK signaling and enhances leukemia cell survival⁶³. ER stress also promotes GRP78 localization to the mitochondria, which are physically and functionally interconnected with the ER (Box 3). Mitochondria-associated GRP78 can bind to RAF1 and this interaction is involved in the maintenance of mitochondrial permeability and thus protective against ER-stress-induced apoptosis⁶⁴. In support of the anti-apoptotic functions of GRP78, knockout of Grp78 in various tissues led to caspase activation and tissue atrophy^33,34,65,66 (Supplemental Table 2). In breast, prostate and leukemic cancer models, heterozygous and/or homozygous knockout of Grp78 increased tumour apoptosis and impeded tumour progression^39,47,48 (Table 1).

Through interaction with α2-M*, cell surface GRP78 promotes 1-LN prostate cancer cell survival by activating the AKT and NF-κB signaling cascades⁶⁷. In hypoxic HT1080 fibrosarcoma cells, cell surface GRP78 serves as a receptor for Kringle 5, a human plasminogen factor, which upon internalization, competes with procaspase-7 binding to the ATP binding domain of ER GRP78, leading to caspase-7 activation and tumor cell apoptosis⁶⁸. However, one study indicates that cell surface GRP78, along with PAR4 has a pro-apoptotic function through mediating TNF-related apoptosis-inducing ligand (TRAIL; also known as TNFSF10) activation, triggering the extrinsic apoptotic pathway in PC3, HeLa and H460 cells⁴². Nonetheless, in MCF-10A and MDA-MB-231 cells, GRP78 prevents TRAIL-induced apoptosis and is therefore a pro-survival factor⁶⁹. Thus, the effect of GRP78 on TRAIL-induced apoptosis may be context dependent. Besides modulating apoptosis, GRP78 has been implicated in protective autophagy through maintenance of ER structural integrity³², and modulation of mTOR signaling in estrogen resistant breast cancer cells⁷⁰.

GRP94 maintains ER Ca²⁺ homeostasis and protects cancer cells from apoptosis⁷¹. GRP94 deficiency in human multiple myeloma cells resulted in apoptosis through inhibition of the Wnt-survivin pathway⁵³. In the SkBr3 breast cancer cell line that abundantly expressed cell surface HER2 (also known as ERBB2), pharmacological inactivation of GRP94 destabilized HER2 and inhibited RAF1-MAPK survival signaling at the cell membrane⁷². In cancer cells, GRP170 is upregulated by hypoxia and by drugs such as celecoxib and proteasome inhibitors, and knockdown of GRP170 activated the expression of the UPR pro-apoptotic factor CHOP and stimulated apoptosis^73,74. GRP170 may also protect cancer cells against cell death through blocking ER Ca²⁺ release or delaying the onset of UPR by binding to the ER stress sensors^75,76.

Angiogenesis

Eliminating the tumour vasculature, which supplies nutrients and oxygen within the tumour, is a key strategy for anti-cancer therapy. Tumour-associated endothelial cells are physiologically and functionally different from endothelial cells derived from normal tissues, and express high levels of GRP78 compared with normal organs^38,77,78. Grp78^+/- mice, as well as mice with conditional heterozygous knockdown of Grp78 in the host endothelial cells, showed a dramatic reduction in tumour microvessel density (MVD) while having no effect on the MVD of normal organs³⁹ (Table 1). Grp78 knockdown impairs immortalized endothelial cell proliferation, survival and migration in vitro, supporting its requirement for neoangiogenesis in primary tumour and metastatic growth³⁹.

GRP78 is expressed on the surface of proliferating endothelial cells^68,79. Cell surface GRP78 assoicates with the GPI (glycosylphosphatidylinositol) anchored T-cadherin and mediates T-cadherin-dependent endothelial cell survival⁸⁰. In exploiting cell surface GRP78 for anti-angiogeneis therapy, plasminogen Kringle 5 is reported to bind GRP78 in glioma endothelial cells for induction of apoptosis⁶⁸, which can be sensitized by radiation dependent on internalization of cell surface GRP78 by the low-density receptor-related protein and activation of p38 MAK⁷⁸. Other studies however suggest that the apoptotic effect of Kringle 5 in proliferating endothelial cells and 1-LN prostate cancer cells was mediated by the cell surface voltage-dependent anion channel which co-localizes with GRP78 and is regulated by GRP78⁸¹. VEGF can induce cell surface expression of GRP78 in endothelial cells and knockdown of GRP78 suppressed VEGF-mediated MAPK signaling and endothelial cell proliferation⁸². On the other hand, knockdown of GRP170, while having no effect on GRP78 and GRP94 expression, resulted in retention of VEGF in the ER and blocked its secretion⁸³. Collectively, these studies suggest that targeting GRP78 and GRP170 could achieve a dual effect in suppressing tumour growth as well as tumour angiogenesis.

Invasion and metastasis

Tumour metastasis is a multistep process and requires enhancement of specific tumour cell properties including degradation of the extracellular matrix (ECM), migration, invasion, angiogenesis and survival. The level of intracellular GRP78, as well as cell surface GRP78, is increased in metastatic cancer cell lines, lymph node metastases and human metastatic lesions^6,84-86. Knockdown of GRP78 suppresses tumour cell invasion in vitro and suppresses metastatic growth in xenograft and syngeneic tumour models^87-89. In addition to protecting metastatic tumour cells from the adverse host environment and promoting angiogenesis, GRP78 has been shown to promote cell motility. One mechanism is through cell surface GRP78 acting as co-receptor for ligands signaling the activation of kinases known to enhance migration, such as AKT, focal adhesion kinase (FAK) and p21-activated kinase 2 (PAK2)^88,90. It has also been proposed that cell surface GRP78 acts as a bridge protein for the urokinase-type plasminogen activator (uPA-uPAR) protease system, which can mediate degradation of the ECM and promote invasion⁸⁸. Like GRP78, GRP94 overexpression is associated with lymph node metastasis and carcinoma recurrence, and silencing of GRP94 inhibits migration and proliferation of MDA-MB-231 breast cancer cells in vitro^55,91. GRP94 client proteins include cell interaction and cell matrix component, such as integrins, which might explain its influence on cell invasion. Recently, it was demonstrated that a cell-permeable peptide that competitively inhibited the interaction between GRP94 and integrins blocked cell invasion⁹². GRP75 overexpression is associated with liver cancer metastasis⁹³, and GRP170 upregulation is observed in invasive breast cancer⁹⁴ (Supplemental Table 1). Thus, the GRPs are novel protein targets for the inhibition of cancer metastasis.

Inflammation and immunity

ER stress can drive a pro-inflammatory program in tumour cells and macrophages that facilitates tumour progression. Additionally, stressed tumour cells secrete mediators that stimulate macrophages to produce pro-inflammatory cytokines, further amplifying the pro-inflammatory response of tumour cells^20,95. On the other hand, cancer cell survival requires resistance against host immune defenses. GRP78 regulates inflammation and immunity through multiple mechanisms^20,96. As a major ER chaperone, GRP78 facilitates the processing and secretion of cytokines and chemokines^96,97. Acute ablation of Grp78 in adult mice results in alteration of their chemokine and cytokine profile³⁴ (Supplemental Table 2). In terms of immune evasion, GRP78 protects fibrosarcoma cells from lysis by cytotoxic T lymphocytes (CTL) and tumour necrosis factor in vitro, and when fibrosarcoma cells incapable of inducing GRP78 were injected into mice, tumours were either not formed or rapidly regressed with evidence of cytotoxic T cell response⁹⁸.

GRP78 is an obligatory binding partner for cell surface major histocompatibility complex (MHC) class I molecules⁹⁹. Acting as the α2M* cell surface signaling receptor, GRP78 regulates the Gs-mediated cAMP production and the pro-inflammatory COX-2-PGE-cAMP signaling cascade^100,101. Regulatory T cells (Tregs) are a subpopulation of T cells that drive immune suppression. In some cancers, increased numbers of Tregs promote cancer progression by active suppression of the immune responses against the tumour. Cell surface GRP78 in T cells forms a complex with and confers stabilization to cell surface TGF-β which is an immune regulator and inducer of Tregs¹⁰². Some cancer cells secrete GRP78, which modulates human monocyte differentiation into mature dendritic cells and subsequent recruitment of T cells leads to generation of Tregs¹⁰³.

GRP94 has an important role in immunity by facilitating MHC class I molecule-mediated antigen presentation; by inducing the maturation and activation of various cells involved in innate and adaptive immune responses; and by secretion of proinflammatory cytokines²⁰. GRP94 is the unique and obligatory chaperone of Toll-like receptors (TLRs), facilitating their maturation and translocation to the cell surface. Macrophage-specific knockout of Grp94 resulted in lack of response to TLR ligands and loss of innate immune function¹⁰⁴ (Supplemental Table 2), and exhibited reduced colitis and inflammation-associated colon tumorigenesis⁵⁴. Thus, GRPs regulate inflammation and immunity in both tumour cells and through interactions with the tumour microenvironment (Figure 2).

Inhibitors of the GRPs

In principle, agents that inhibit the synthesis, stability or activity of the GRPs can simultaneously suppress their function at various cellular locations. The challenge is to minimize toxicity to normal organs. Various heterozygous knockout mouse models revealed that a 50% decrease in GRP78 expression has no effect on normal organs but significantly impedes tumour growth and angiogenesis⁸⁹ (Table 1). This implies that agents that selectively block the stress induction of GRP78 will affect tumours that require a high level of GRP78 and spare normal organs. Natural compounds with anti-cancer properties that suppress GRP78 induction have been reported, however, they exert pleiotrophic effects^5,120. Specific cancers also express microRNAs that can act cooperatively to suppress GRP78 translation (Figure 3) and reverse chemoresistance¹²¹. However, specific inhibitors of GRP78 stress induction remain to be identified.

Alternatively, as the GRP78 promoter is highly active in aggressive solid tumours, this offers the opportunity to use the GRP78 promoter to direct expression of suicide genes, immunosuppressors and tumour suppressors in anti-cancer therapy. As proof-of-principle, the Grp78 promoter driving the expression of the herpes simplex kinase suicide gene in a retroviral system results in eradication of sizable tumours^122,123. Recently, systemic administration of a dual tumour-targeted phage containing the RGD tumour homing ligand and the Grp78 promoter, showed persistent transgene expression in vitro and significant killing of therapy resistant tumours in vivo¹²⁴. Likewise, cancer-inducible transgene expression can be directed by the Grp94 promoter in tumours of various origins and cancer-associated macrophages¹²⁵.

Selective destruction of GRP78 at the protein level might be possible owing to the discovery of a bacterial toxin SubAB which cleaves GRP78 at a single site (L416-L417) in the hinge region connecting the ATPase and the substrate binding domain of the molecule, thereby inactivating it¹²⁶. Systemic delivery of an engineered fusion protein combining epidermal growth factor (EGF) and SubAB was toxic to EGFR expressing cancer cells in vitro and caused a delay in the growth of human breast, prostate and glioblastoma xenografts in mice^127,128. One of these studies¹²⁸ also showed that modest cleavage of GRP78 in normal mouse liver cells due to EGFR expression did not lead to weight loss, consistent with the findings in genetic models that normal organs including the liver can tolerate partial decrease in GRP78 levels³⁹.

Since the ATPase catalytic activity of GRP78 is necessary for its anti-apoptotic function⁶⁰, targeting its ATP binding domain can effectively inactivate GRP78 in cancer. Several plant compounds including (-)epigallocatechin gallate (EGCG), honokiol and aspirin (also known as salicylate) directly bind to this domain and inhibit the ATPase activity (Figure 3), and this is associated with the sensitization of cancer cells to chemotoxic agents^129-131. Furthermore, an unconjugated peptide derived from the co-chaperone BAG1 binds to the substrate binding domain of GRP78 and inhibits its protein refolding activity, and prostate cancer cells stably expressing this peptide showed reduced growth and apoptosis in xenograft models in a manner dependent on binding to GRP78¹³². In glioblastoma cells, GRP78 can also be inactivated via acetylation by vorinostat, a deacetylase inhibitor with anti-tumour activity¹³³.

Recently, specific inhibitors against GRP94 function have been identified based on its unique secondary nucleotide binding pocket^72,134. One of the compounds, PU-WS13, has been shown to reduce the viability of breast cancer cells expressing high level of cell surface ERBB2⁷² and human multiple myeloma cells⁵³ in vitro. Interestingly, honokiol induces calpain-mediated cleavage GRP94 in human gastric cancer cells, associating with apoptosis and reduction in tumour growth¹³⁵. GPM1, a chemical that can bind GRP94, suppresses its surface presentation through increased ER retention. This chemical was shown to compromise the immune functions of GRP94 in vivo; however, its efficacy in cancer is not known¹³⁶. MKT-077, a cationic rhodacyanine dye, binds the nucleotide-binding domain of GRP75, abrogates its interaction with p53 and reactivates the transcriptional and pro-apoptotic activities of p53 in cancer cells, but not in normal cells in vitro^137-139. Additionally, virtual screening of a drug database has revealed several small molecule inhibitors that are able to interrupt the p53-GRP75 complex¹⁴⁰.

Collectively, these proof-of-concept studies demonstrate that GRP inhibitors can selectively confer toxicity to cancer cells in vitro and in vivo, warranting further development and validation.

Cytotoxic agents targeting cell surface GRP

Preferential expression of GRP78 on the surface of tumour cells in vivo enables specific tumour targeting with minimal harmful effects on normal cells^11,13,141. As cell surface GRP78 expression is further detected in some TICs and increased in metastatic and drug resistant tumours^6,16,117 and in hypoxic endothelial cells supporting tumour growth^68,78, cytotoxic agents against cell surface GRP78 have the potential to target these cells in addition to the primary tumour.

Several synthetic peptides composed of GRP78 binding motifs fused to cell-death inducing peptides or cytotoxic drugs are able to promote apoptosis in cancer cells in vitro, including human prostate, and breast cancer cells, human melanoma, chemotherapy-resistant B lineage acute lymphoblastic leukemia cells and multidrug resistant gastric cells^6,142-148 (Figure 3). Furthermore, xenograft and isogenic mouse models were used to validate the efficacy of the peptides in suppressing tumour growth of human prostate and breast cancer, melanoma, as well as bone metastasis with no apparent toxicity^6,142,145. The GRP78 binding peptides have been conjugated to nanoparticles or liposomes for more efficient drug delivery^145,149, and such agents are able to home to endothelial cells in tumours, suppressing growth and prolonged survival of colon carcinoma bearing mice⁸². Furthermore, a reconstructed protein containing GRP78 binding peptide and mung bean trypsin inhibitor displays targeted anti-cancer effects both in vitro and in vivo in colorectal cancer¹⁵⁰.

Recently, MAb159, a high affinity GRP78 specific mouse monoclonal IgG antibody was identified that triggers endocytosis and degradation of cell surface GRP78, and activates both intrinsic and extrinsic apoptosis¹⁷. MAb159 causes cancer cell death and suppresses the growth of colon and lung xenografts, the metastatic growth of human breast and melanoma xenografts and the growth of prostate cancer and leukemia in genetically engineered mouse models, at least in part through inhibition of the PI3K signaling pathway¹⁷. MAb159 also synergizes with Irinotecan, a topoisomerase I inhibitor, in suppressing human colon cancer xenograft growth. A humanized MAb159 retains anti-tumour activity with no toxicity in mice and exhibits favorable pharmacokinetics¹⁷. In principle, this antibody can also be used as an in vivo imaging agent for selection of patients expressing cell surface GRP78 and to determine whether that predicts disease progression and response to therapy. Another screen yielded a mouse monoclonal IgG antibody targeting the carboxyl-terminal domain of GRP78, C107, which is capable of inducing apoptosis in melanoma cells in vitro and slowing their growth as xenografts in mice¹⁵¹. A human monoclonal IgM antibody (PAT-SM6) isolated from a patient with gastric cancer that can simultaneously bind low density lipoproteins and multiple GRP78 molecules on the surface of tumour cells, induces lipid accumulation and apoptosis in human multiple myeloma cells^152,153, and suppresses human melanoma growth both in vitro and in xenografts¹⁵⁴. Based on favorable safety profiles in phase I studies, the efficacy of PAT-SM6 is being tested in clinical trials¹⁵⁴. It has also been reported that autoantibodies against GRP78 from ovarian cancer patients promote apoptosis and decrease the invasiveness of ovarian cancer cells¹⁵⁵. In another study, autoantibodies against GRP78 from prostate cancer patients trigger ER Ca²⁺ release in human bladder carcinoma cells and increase tissue factor procoagulant activity, implying that blocking cell surface GRP78 signaling could potentially reduce the risk of cancer-related thrombotic events¹⁵⁶. As proof-of-principle that cell surface GRP75 may also be amendable to therapy, intra-tumoural and intra-peritoneal injections of an anti-GRP75 antibody results in tumour growth suppression²².

This work is supported in part by NIH grants CA027607 and AG034906. I thank Drs. Larry Hightower, Zihai Li, John Subjeck, Xiang-Yang Wang, and John Luk for helpful discussions, Lee lab members in particular, Jieli Shen and Yi Zhang, and Esther and Sarah Olivo for assistance.