Cell surface GRP78: An emerging imaging marker and therapeutic target for cancer

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Article

Reference

Cell surface GRP78: An emerging imaging marker and therapeutic target for cancer

FARSHBAF, Masoud, et al.

Abstract

As one of the deadliest diseases, cancer frequently resists existing therapeutics because they do not target all cells within a progressing tumor, for example both tumor stem and proliferating cells. This frequently results in enrichment of invasive and metastatic drug-resistant tumor cells subpopulations, cancer recurrence and eventually, patient mortality.

Thus, there is an urgent need to identify specific markers, by which the targeted imaging and/or therapeutic "guided missile"-like agents can specifically detect and/or eradicate all cancer cells within a heterogeneous tumor, while leaving the normal cells intact. As a member of heat shock protein 70 (HSP70) superfamily, glucose regulated protein 78 (GRP78) has been documented as a molecular chaperone in the endoplasmic reticulum (ER) which mainly responds to ER stresses in normal cells. There is over-expression of GRP78 on the surface of cancer cells and angiogenic endothelial cells, which makes it a promising target for different types of peptides and antibodies that can be employed for targeted cancer therapy or imaging. In this review, we discuss the biological [...]

FARSHBAF, Masoud, et al . Cell surface GRP78: An emerging imaging marker and therapeutic target for cancer. Journal of Controlled Release , 2020, vol. 328, p. 932-941

DOI : 10.1016/j.jconrel.2020.10.055 PMID : 33129921

Available at:

http://archive-ouverte.unige.ch/unige:148776

Disclaimer: layout of this document may differ from the published version.

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Journal of Controlled Release 328 (2020) 932–941

Available online 29 October 2020

Review article

Cell surface GRP78: An emerging imaging marker and therapeutic target for cancer

Masoud Farshbaf

^a^,^b

, Ahmad Yari Khosroushahi

^a^,^b

, Solmaz Mojarad-Jabali

^c

, Amir Zarebkohan

^a

, Hadi Valizadeh

^d^,^**

, Paul R. Walker

^e^,^*

aDepartment of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

bDrug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

cDepartment of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

dDepartment of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

eCenter for Translational Research in Onco-Hematology, Division of Oncology, Geneva University Hospitals and University of Geneva, Geneva, Switzerland

A R T I C L E I N F O Keywords:

GRP78 Cancer Nanomedicine Active targeting Therapy Imaging

A B S T R A C T

As one of the deadliest diseases, cancer frequently resists existing therapeutics because they do not target all cells within a progressing tumor, for example both tumor stem and proliferating cells. This frequently results in enrichment of invasive and metastatic drug-resistant tumor cells subpopulations, cancer recurrence and eventually, patient mortality. Thus, there is an urgent need to identify specific markers, by which the targeted imaging and/or therapeutic “guided missile”-like agents can specifically detect and/or eradicate all cancer cells within a heterogeneous tumor, while leaving the normal cells intact. As a member of heat shock protein 70 (HSP70) superfamily, glucose regulated protein 78 (GRP78) has been documented as a molecular chaperone in the endoplasmic reticulum (ER) which mainly responds to ER stresses in normal cells. There is over-expression of GRP78 on the surface of cancer cells and angiogenic endothelial cells, which makes it a promising target for different types of peptides and antibodies that can be employed for targeted cancer therapy or imaging. In this review, we discuss the biological processes, functional importance and translocation mechanisms of cell surface GRP78 (csGRP78) in tumor cells. As a cancer biomarker, we also review the potential applications of csGRP78 targeted therapy and imaging and finally we suggest a brief roadmap ahead of csGRP78 targeting for targeted theranostic implications.

1. Introduction

In recent years, targeted delivery has been developed to selectively deliver therapeutic or imaging agents to tumor cells resulting in increased local concentrations of the agents without affecting healthy cells [1]. Moreover, active targeting may overcome drug-resistance induced by tumor stem and/or proliferating cells, by directing the cargo directly to the vast majority of cancer cells at the site of interest.

Taking into account different behavioral, microenvironmental and proteomic profiles of cancer cells, these may lead to distinctive cell surface characteristics compared to normal cells, which could be employed as potential markers for diagnosis as well as for therapeutic targeting [2]. These include cell surface receptors such as integrins

[3,4], folate receptor [4,5], transferrin receptor [6,7], and insulin-like growth factor 1 (IGF-1) receptor [8,9], which are over-expressed by cancer cells, and play a vital role in targeted cancer therapy and diagnosis. Though their applications as potential targets have widely been explored, many of these receptors are also expressed by the normal cells, which reduces the specificity of targeted therapeutics towards tumor cells and enhances on-target off-tumor cytotoxicity [10]. This limitation has spurred the investigation of more specific targets that are mainly associated with cancer stem and proliferating cells. In this regard and as a relevant molecular target expressed in metastatic tumors, cell surface glucose-regulated protein 78 kDa (csGRP78) was firstly identified by fingerprinting the circulating repertoire of antibodies from cancer patients [11]. GRP78 is a member of the heat shock protein 70 (HSP70)

* Correspondence to: P. R. Walker, Centre for Translational Research in Onco-haematology, University of Geneva, University Medical Centre (CMU), 1 rue Michel- Servet, CH-1205 Geneva, Switzerland.

** Correspondence to: H. Valizadeh, Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz 51664, Iran.

E-mail addresses: [email protected] (H. Valizadeh), [email protected] (P.R. Walker).

Contents lists available at ScienceDirect

Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

https://doi.org/10.1016/j.jconrel.2020.10.055

Received 2 September 2020; Received in revised form 23 October 2020; Accepted 25 October 2020

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super-family that mainly resides in the endoplasmic reticulum (ER) lumen [12]. This protein is also known as BiP or HSPA5. GRP78 is one of the most important ER chaperones functioning as a vital component in many cellular processes, including protein assembly and folding and translocation across the ER membrane. It also targets misfolded proteins for proteosomal degradation and regulates calcium homeostasis in normal cells [13]. On the other hand, recent findings evidence the overexpression of GRP78 in cancer stem cells, tumor-associated endo- thelium and proliferating cancer cells as a result of stress conditions such as severe glucose starvation, lactic acidosis, and hypoxia, which are well-known hallmarks of tumor microenvironment. This elevated expression also results in the active translocation of GRP78 to the surface of tumor cells, which mainly correlates with cancer cells that are proliferative, malignant, metastatic, anti-apoptotic and drug resistant [14]. The profile of GPRP78 gene expression showed that among the diverse cancer types, the highly malignant and invasive glioblastoma has the highest overexpression of csGRP78, with lung, breast, colon, and liver cancers, as well as angiogenic endothelial cells also reported to have high expression [12,15]. These findings confirm the fact that csGRP78 could be present on both malignant cells and endothelial cells, but with rare expression on normal cells, suggesting csGRP78 as a promising cancer cell-specific biomarker and target for cancer imaging and therapy [16,17]. A wide range of targeting ligands comprising antibodies and peptides has been employed to target csGRP78 and to direct the cargo of interest to the tumor cells, or even to possess therapeutic function alone, as shown for certain antibodies [18]. Here we review the biological processes, functional importance and translocation mechanism of csGRP78 in cancer cells, discuss its recent applications in cancer- targeted therapy and imaging and investigate the related ongoing clinical trials. We finally give a brief insight towards the future of csGRP78 targeting by the means of targeted therapy and imaging.

2. Transduction of GRP78 to the surface of stressed cancer cells and structural considerations

As reported earlier, most transmembrane proteins like calnexin are able to embed into the cell membrane bilayer and consequently anchor to the cell surface [19,20]. Although being considered as an ER lumen protein, a subfraction of ER GRP78 was found to be insensitive to

alkaline carbonate extraction [21]. This characteristic is common among the transmembrane proteins, suggesting a subfraction of GRP78 acts as a transmembrane protein spanning the ER membrane. However, GRP78 does not contain classical transmembrane domains and config- uration, rather, it preferentially exists as a peripheral protein on the plasma membrane of stressed cancer cells through interaction with other cell surface proteins like glycosylphosphatidylinositol-anchored proteins [22]. This feature may facilitate the designing of new antibodies that can recognize the N-, and C-terminal and/or middle region of csGRP78 in targeted therapy. As the plasma membrane originates from ER membrane, it is speculated that the ER membrane-spanned sub- population of GRP78 transduces to the cell surface of stressed cancer cells and turns into csGRP78 which is never or rarely observed in normal cells. It is important to note that the overall active trafficking mechanism of GRP78 to the cell surface may vary for different cancer cell lines and is cell-context dependent and associated with its client proteins [22].

The 3D structure of human csGRP78 has been elucidated by X-ray crystallography and homology modeling, which showed an ATPase domain (Val31 to Gly407) and peptide-binding domain (Thr423 to Leu654) consisting of two major domains of csGRP78 (Fig. 1A) [23–25].

The crystal structures of csGRP78 indicated that most of the amino acid residues at binding sites were conserved, except one variation between GRP78 (Ile61) and HSP70 (Thr37) [25]. This forms a more hydrophobic binding site in csGRP78, facilitating design of targeting ligands that selectively bind to csGRP78 rather than other HSP70 isoforms.

Furthermore, the peptide-binding domain of csGRP78 consists of two subdomains: (I) helix-bundle subdomain with numerous tandem helices, and (II) β-sandwich subdomain comprised of two layers of β-sheets (Fig. 1B) [24]. Of note, csGRP78 has a negative charge at physiological pH (7.4), which is mostly distributed in the peptide-binding domain [23]. Interestingly, the binding of ATPase domain to ADP closes the peptide-binding domain which further traps the peptide ligand, while the peptide-binding is open when the ATPase domain binds ATP, which results in the detachment of peptide ligands [26].

Fig. 1. Simulated modeling of the 3D structure of human csGRP78. (A) The csGRP78 includes a peptide-binding domain (light green) which is predicted to bind a csGRP78-taargeting peptide, and an ATPase domain (light blue) linked by a loop (red). (B) The peptide-binding domain of csGRP78 is composed of a helix-bundle subdomain at the c-terminal and a β-sandwich subdomain. Adapted with permission from ref. [23]. Copyright 2016, Elsevier. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Journal of Controlled Release 328 (2020) 932–941

934 3. Biological functions and signaling pathways of csGRP78 in cancer

3.1. Cell survival and proliferation

As previously discussed, stress conditions like hypoxia or glucose starvation significantly enhance csGRP78 expression in cancer cells. The csGRP78 serves as a high-affinity receptor for α₂-macroglobulin and this interaction enhances cell survival, proliferation, and metastasis of prostate cancer cells through prostate-specific antigen (PSA) upregulation [27]. Secreted PSA forms a complex with α₂-macroglobulin and further binds to csGRP78, resulting in proliferative signaling pathway activation [28]. Furthermore, the csGRP78/α₂-macroglobulin complex further induces csGRP78 expression by two-fold, via upregulation of TFII-1 [29]. It is also documented that the α₂-macroglobulin/csGRP78 complex plays an important role in upstream regulating of PDK1/PLK1 signaling pathways that activates the transcription of proliferative genes [30]. Moreover, the interaction of α₂-macroglobulin with csGRP78 results in its phosphorylation, triggering RAS/MAPK, and PI3-kinase/Akt/

mTOR downstream signaling cascades, promoting cellular proliferation and survival [28,31,32]. In cancer patients, some circulating autoantibodies mimic α₂-macroglobulin and bind to the NH2 terminal of csGRP78, which modulates the regulation of AKT signaling that promotes tumor growth [33]. Furthermore, csGRP78/Cripto in cancer cells can regulate TGF-β signaling that induces cell proliferation [34]. The IGF-1R activation and resulting hepatoma cell proliferation can also be regulated by csGRP78 [35].

3.2. Apoptosis

csGRP78 can act as a double-edged sword, by promoting either apoptotic or anti-apoptotic signaling in cancer cells (Fig. 2). Interest- ingly, the autoantibodies including C38 and C107 that act against COOH terminal of csGRP78, can effectively activate P53 apoptotic signaling and suppress tumor growth [36]. On the other hand, Kringle 5 of human plasminogen, is known to have high affinity towards csGRP78 [37].

Once bound to csGRP78, Kringle 5 promotes the intrinsic apoptosis pathway of proliferating endothelial cells and tumor cells; this occurs through procaspase 7 binding with the ATP-binding domain of ER GRP78 and subsequent caspase 7 activation. In contrast, the ubiqui- tously expressed pro-apoptotic tumor suppressor, the secreted prostate apoptosis response 4 (Par-4) protein, can bind with NH2 terminal of csGRP78, activate caspases 3 and 8 and engage the extrinsic apoptotic pathway [38]. Isthmin is a secreted 60-kDa protein ligand with proapoptotic and anti-angiogenesis characteristic that selectively targets alphavbeta5 (αvβ5) integrin on the surface of endothelial cells [39] and the NH2 terminal of csGRP78 on cancer cells with high affinity [40], leading isthmin into the cytosol through clathrin-mediated endocytosis and subsequently to mitochondria [41]. This trafficking results in mitochondria dysfunction via blocking ATP/ADP exchange on the mitochondrial membrane and eventually activating the intrinsic apoptotic pathways. In a self-destructive manner, the secreted GRP78 itself, existing in the extracellular environment and serum, can remarkably act as a cell death ligand of csGRP78 to activate a proapoptotic pathway in stressed pancreatic beta cells [42]. The GRP78 secretion in beta cells was found to be as a result of ER stress induction

Fig. 2.Schematic illustration of apoptotic and anti-apoptotic ligands of csGRP78 and their mechanism of action. GALNT6, Par-4, and mAb stand for N-acetylga- lactosaminyltransferase, prostate apoptosis response 4, and monoclonal antibody, respectively.

M. Farshbaf et al.

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by pro-inflammatory cytokines [43]. Concerning cancer cell anti- apoptosis behavior, this can be explained by the activation of AKT and NF-κB signaling through csGRP78/α2-macroglobulin complex [31]. It has also been identified that upregulation of N-acetylgalactosaminyl- transferase 6 (GALNT6) in cancer cells and its following elevated activity results in O-glycosylation, the consequently enhanced stability of csGRP78 then promotes its anti-apoptotic function [44]. Furthermore, GRP78 may play a critical role in Golgi-to-ER relocation of GALNT6, which further enhances GALNT6 activity in carcinogenesis [44]. Despite the fact that the intracellular GRP78 plays a crucial role in the cross-talk between autophagy and apoptosis of malignant cancer cells [45,46], it is unknown how csGRP78 mediates between prosurvival or prodeath outcomes of autophagy.

3.3. Invasion and migration

Phosphates and kinases are the main signaling molecules that are involved in the process of tumor metastasis, in which they facilitate the degradation of the extracellular matrix (ECM) and promote the motility and penetration of highly malignant cells towards new tissues. Meta- static cancer cells express high levels of csGRP78 which is also positively implicated in lymph node metastasis [47]. Accompanied by cofilin and LIMK1 phosphorylation, csGRP78/α2-macroglobulin ligation activates p21-activated kinase-2 (PAK2) and focal adhesion kinase (FAK), resulting in enhanced motility of metastatic cells [48–50]. csGRP78 is reported to link urokinase receptor (uPAR) and urokinase-type plasminogen activator (uPA) and to act as a binding site for plasminogen.

Once bound, plasminogen transforms to plasmin, and promotes ECM degradation and subsequent cell invasion [51]. Furthermore, the aggressive and metastatic phenotype of prostate cancer cells is regulated through csGRP78 and its signaling oncoprotein partner, CRIPTO, where knockdown of either GRP78 or CRIPTO inhibited proliferation, bone metastasis and clonogenicity [52]. Additionally, It was found that high levels of csGRP78 results in high phosphorylation levels of STAT3 that promotes breast cancer cell growth and migration [53].

4. csGRP78 targeting moieties and their therapeutic implications

4.1. Peptides

Since 2003, many different synthetic peptides have been designed for csGRP87 targeting with different therapeutic implications including apoptosis induction, proliferation inhibition and anti-angiogenesis [54].

The peptides WIFPWIQL and WDLAWMFRLPVG are the most employed peptides that have shown superior targeting activity and specificity towards csGRP78 compared to other members of HSP70 and HSP90 in different cancer cells [55]. Upon binding, csGRP78 further mediates the internalization of WIFPWIQL and WDLAWMFRLPVG peptide ligands into the cancer cell via a receptor-mediated process [56]. A synthetic chimeric protein containing WIFPWIQL and mung bean trypsin inhibi- tor, mTI, has been shown to specifically inhibit cell growth via G1 phase arrest and induce apoptosis in xenografted human colorectal carcinoma while it had little or no significant effects on normal cells [57]. This could be attributed to the specific targeting ability of WIFPWIQL and underlines csGRP78 as a unique target in cancer treatment. Miao et al.

also fused WIFPWIQL to a proapoptotic moiety (_D(KLAKLAK)₂) [58]

known as bone metastasis targeting peptide 78 (BMTP78, WIFPWIQL- GG-D(KLAKLAK)2); this complex was employed to selectively destroy mouse breast cancer cells [59]. BMTP78 resulted in significant death of 4 T1.2 mouse mammary tumor cells in a dose-dependent manner, while control peptides with no proapoptotic motif, showed no therapeutic effects. Furthermore, BMTP78 has shown promising results by delaying the aggressive tumor outgrowth in mice bearing either 4 T1.2 mammary tumor or human MDAMB-231 cancer cells and promoting the overall survival in patients with severe metastatic cancer.

BC71 is a cyclic peptide which harbors the RKD motif in the isthmin adhesion-associated domain with efficient ability to bind to csGRP78 and suppress tumor growth in mice [60]. BC71 can also confer the extrinsic apoptosis through activating caspase-8 and p53 signaling pathways in human umbilical vein endothelial cells (HUVECs), highlighting its anti-angiogenic properties against tumor blood vessel endothelial cells along with its anticancer activity. Recently, a hex- apeptide, GIRLRG, has shown much potential in targeted imaging and delivery considering its high affinity for ATPase domain of csGRP78 (2.16 ×10⁻³M) in nude mice bearing esophageal (OE33), cervical (HT3), lung (A549), pancreatic (BXPC3), and glioma (D54) tumors [61,62]. The conjugation of GIRLRG with polyethylene glycol (PEG) was also shown to increase its blood circulation time in mice [61]. Given that the multi-drug resistance of various types of cancer cells reduces the effectiveness of cancer clinical treatments, Kang et al. have derived a selective targeting peptide, GMBP1 (ETAPLSTMLSPY) from a phage display library that was shown to specifically bind to csGRP78 on gastric cancer multi-drug resistant (MDR) cells, reversing the MDR phenotypes and re-sensitizing them to a variety of chemotherapeutics including adriamycin, cisplatin, vincristine and 5-fluorouracil (5-FU) [63]. These findings support the fact that the pre-incubation of GMBP1 in MDR cancer cells, significantly inhibits MDR1, GRP78 and BCL-2 expression, while up-regulating Bax expression. It has been shown that the transferrin-related pathway is the main route through which csGRP78 can mediate the internalization of GMBP1 into human MDR gastric adenocarcinoma SGC7901/ADR cells [64].

The hepta-peptide, PRKLYDY, derived from lysine-binding site of Kringle 5 of human plasminogen has been shown to direct Kringle 5 towards csGRP78 on proliferating endothelial cells and stressed tumor cells, such as HT1080 fibrosarcoma cells; this is responsible for the antiangiogenic and antitumor activities of Kringle 5, which were discussed earlier [37]. L-peptide is a 12-mer peptide (RLLDTNRPLLPY), identified with a phage displayed random peptide library and capable of binding to the csGRP78 on the tumor cells of most nasopharyngeal carcinoma cell lines, sparing normal cells, including mucosal cells [65,66]. L-peptide was observed to bind csGRP78 with a slow dissociation rate compared to the fast dissociation of WIFPWIQL at equal concentrations (50 μM) [66]. This is due to the strong connection between L-peptide and csGRP78 via seven hydrogen bonds, while, there is only one hydrogen bond between WIFPWIQL peptide and csGRP78. In this regard, fluorescent liposomes modified with L-peptide showed superior cellular uptake in tumor cells compared to non-modified liposomes, highlighting the potential of L-peptide for targeted drug delivery [65]. Using whole cell panning against human melanoma cell line Me6652/4, Kim et al. identified a cyclic 13-mer Pep42, CTVALPG- GYVRVC, with preferential internalization into the malignant cancer cells that was mediated by csGRP78, while there was no cellular uptake in a human dermal fibroblast cell line [67]. Furthermore, Pep42 and the involved receptor-mediated translocation mechanism were shown to evade lysosomes and their harsh enzymatic and acidic conditions, which allows the respective cargo to remain intact on its trafficking into the cytoplasm or even the nucleus. Notably, the cyclic structure of Pep42 is a critical element for internalization, as the linear form of the same peptide sequence, Lin42, showed no cellular uptake. Temperature is another important element that affects active internalization, as at 4 ^◦C, the lipid membrane rigidifies and inhibits Pep42 endocytosis. Joseph et al.

showed that structural modification of Pep42 with varying lengths (3− 12) of poly(arginine) sequences endows Pep42 with a secondary structure containing helical and turn conformations that improves its thermal stability and retains its csGRP78 binding ability on HepG2 cells [68].

Kitahara et al. investigated a cholangiocarcinoma (CCA)-binding oligopeptide, COP35, which was confirmed by nano LC-ESI-QTOF MS- MS analysis to selectively bind to csGRP78 and clathrin heavy chain on RBE and MMNK-1 cancer cells, but not to normal cells [69]. Further- more, COP35 could significantly enhance the tumor inhibitory effect of

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936 the frequently used anticancer agent 5-FU, when used together against RBE cells, while COP35 alone did not show such an effect. As a proof of concept, siRNA-mediated silencing of the GRP78 encoding gene elimi- nated the COP35 capacity to enhance the tumor inhibitory effect of 5- FU. L-VAP (SNTRVAP), is the most recently discovered csGRP78 targeting peptide, characterised in a study investigating androgen- independent PC-3 prostate cancer cells [70]. To further enhance the physiological stability of peptides against enzymatic proteolysis, it is common to use the retro-inverso method, in which the original sequence is reversed and all the L-amino acids are replaced with their D-forms [71]. In this regard, Ran et al. synthesized the retro-inverso structure of L-VAP, named RI-VAP (^DP^DA^DV^DR^DT^DN^DS) and explored its targeting ability towards csGRP78 [72]. The resulting RI-VAP possessed a similar affinity (K_i=2.91 (nM)) to csGRP78 as with the L-VAP (K_i=2.72 nM).

Interestingly, RI-VAP showed no obvious perceptible degradation in 50% mouse plasma, while L-VAP was almost totally degraded after 4 h incubation. Furthermore, RI-VAP-modified micelles showed unique tumor homing ability and high penetration depth both in U87MG glioblastoma tumor spheroids and in U87MG bearing mice.

The latter study emphasized the importance of peptide stability in the context of a real biological environment. This is important when the peptide ligand is designed to bind to its receptor before enzymatic degradation. Therefore, structurally stable csGRP78-targeted peptides may experience prolonged blood circulation time, with higher capacity to reach the target cells. On the other hand the adsorption of serum proteins on such peptides can result in a “protein corona” formation, and induce immune responses which could significantly increase their clearance and subsequently lower their therapeutic efficiency [73].

Hence, the optimum circulation time should be taken into consideration during the designing of csGRP78-targeted peptides to avoid the mentioned drawbacks and facilitate their clinical utility. It is also worth noting that a new class of pro-apoptotic small molecules has recently been described; one such molecule, FL5, showed strong binding affinity to csGRP78 and robust anti-cancer activity. This class of therapeutic may have advantages over peptides, as they are less prone to affect the ATPase activity of GRP78 and consequently show less cytotoxicity of normal cells [74].

4.2. Antibodies

Monoclonal antibodies (mAbs) against cell-surface markers have long been used in cancer treatment, even before peptides have been designed and tested. Despite the expense in production and purification, mAbs can offer high antigen specificity and in some cases, yield therapeutic efficacy through engagement of immune effector mechanisms [75]. Therapeutic targeting of csGRP78 has been frequently achieved using various types of mAbs, including the natural human IgM antibody PAT-SM6, which has been extensively investigated due to its high avidity to csGRP78 and oxidized low-density lipoprotein [76,77].

Through this specificity for csRP78, PAT-SM6 dose-dependently induces potent apoptosis and to a lesser extent, promotes complement dependent cytotoxicity in relapsed or refractory multiple myeloma, while sparing normal plasma cells [78,79]. A phase 1 trial with PAT-SM6 for immunotherapy of multiple myeloma started at the end of 2012 with promising results according to pharmacokinetic analysis [80]. The trial demonstrated that PAT-SM6 reduced the risk of host-protective immune responses, it had an acceptable terminal half-life, a favorable apparent volume of distribution and clearance, and was well tolerated with modest clinical activity in multiple myeloma. The combination of PAT- SM6 with anticancer agents such as lenalidomide and bortezomib, has resulted in further expression of csGRP78 and significant synergistic anti–multiple myeloma effects when used with these respective drugs [81]. Ab39 is another well-known candidate with high affinity to csGRP78 expressed by cancers of different origins including breast, lung and melanoma, with good tumor cell specificity [82]. Despite its

superior selectivity to cancer cells, Ab39 was proven to be ineffective for reducing growth of breast tumor cells. The single chain variable frag- ments (scFvs) of Ab39 bind to a region of csGRP78 that is not associated with its apoptosis domain, consistent with the observed therapeutic outcomes. Thus, Ab39 should be considered just as a targeting ligand rather than a therapeutic agent. In contrast, the IgG antibody, C107, targeted the COOH-terminal domain of csGRP78, induced apoptosis in tumor cells and increased the median survival time of tumor-bearing mice up to 6 days [36]. MAb159 mAb has been utilized to attenuate the PI3-kinase signaling pathway without compensatory MAPK pathway activation, and to suppress tumor growth and metastasis via binding to csGRP78 with high affinity (Kd =1.7 nmol/L) in vitro and in vivo [83].

Moreover, csGRP78 facilitated Mab159 endocytosis through the clathrin-mediated pathway, localizing to early endosomes. While being entirely non-toxic for normal cells, Mab159 triggered caspases 8 and 9 in breast and colon cancer cells and tumor xenograft models, suggesting its ability to activate both intrinsic and extrinsic apoptotic pathways in tumor cells. V80 is a single-domain antibody, also known as a nanobody that has been recently developed with high affinity (Kd =2.1 ×10⁻⁷M) to the C-terminus of csGRP78 on lung and liver cancer cells, with no cross-reactivity with other proteins [84]. This nanobody showed mini- mal interaction with csGRP78-negative cells. This pioneering study should encourage further exploration of csGRP78 targeting nanobodies in future studies. Indeed, nanobodies present a variety of advantages over conventional therapeutic mAbs; these include low immunoge- nicity, small size (nanoscale), high penetration in various tissues, stability under harsh situations (e.g. low pH or high temperature), and the potential for high affinity interactions with cell-surface as well as intracellular antigens [85].

Intensive investigations have shown complexity regarding the efficacy of csGRP78 antibodies, where mAb against C-terminus domain of csGRP78 act as a receptor antagonists, resulting in enhanced apoptosis, and tumor growth inhibition in ovarian, prostate and melanoma cancer patients [86,87]. In contrast, csGRP78 autoantibodies purified from prostate cancer patients promoted tumor cell proliferation [88]. Hoesen et al. proposed that these opposing effects of anti-csGRP78 antibodies depended upon the csGRP78 epitope targeted [89]. Among the six different main epitopes recognized by csGRP78 autoantibodies purified from serum of ovarian cancer patients, csGRP78559-579 (IDTRNELE- SYAYSLNKNQIGD) was responsible for cell growth inhibition.

5. Applications of csGRP78 targeting moieties in cancer therapy and diagnosis

5.1. Targeted drug delivery via csGRP78 targeting peptides

Apart from being a targeting moiety with or without therapeutic effects, csGRP78 targeting peptides and antibodies can also act as a targeting vehicle for a wide range of chemotherapeutics. This enables higher therapeutic doses to be used without adverse off-target effects [90–92]. WIFPWIQL peptide has gained much attention for targeted delivery of different anticancer agents, for example, after conjugation to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer for the selec- tive delivery of geldanamycin to prostate cancer tumors [93]. The targeted conjugates presented similar targeting ability and superior cytotoxicity compared to free peptide and non-targeted conjugates, respectively. In order to inhibit cell proliferation, doxorubicin (DOX) needs to selectively enter the tumor cell’s nucleus and bind to DNA [94].

Recently, a complex containing DOX prodrug and WIFPWIQL peptide was utilized against colorectal cancer cells [95]; this targeted DOX entered the nucleus more efficiently and exhibited enhanced anticancer behavior in colorectal cancer xenografts compared to free DOX, without the off-target degenerative effects seen with free DOX.

In order to inhibit the angiogenesis which is vital for aggressive tumor growth and metastasis, Katanasaka et al. examined the therapeutic effect of DOX-loaded WIFPWIQL-modified liposomes on the M. Farshbaf et al.

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angiogenic endothelial cells, HUVECs, especially those treated with vascular endothelial growth factor (VEGF), which induces csGRP78 expression [96]. Unlike unmodified liposomes that leaked out of angiogenic vessels, the targeted nanoparticles displayed enhanced accumulation within angiogenic cells, significantly inhibited tumor growth and increased the survival of colon cancer bearing mice [96]. In a similar study, WIFPWIQL-functionalized DOX-loaded polymeric micelles could target cargos into gastric cancer MKN45 cells and thereby augment therapeutic efficiency in vivo [97]. The 100 nm csGRP78- targeted nanoparticles showed higher accumulation in tumor cells compared to plain nanoparticles judged by intracellular fluorescence intensity of up-taken fluorescent-tagged nanoparticles. When incubated with serum, WIFPWIQL either alone or on the surface of nanoparticles, preserved its stability and functionality and the drug was released from the nanoparticles in a controlled manner. Interestingly, the targeted nanoparticles improved the therapeutic efficiency of DOX and decreased its therapeutic concentration, both in vitro and in vivo.

The WIFPWIQL has also been used to modify the surface of DOX- loaded mesoporous silica nanoparticles (MSNPs) to confer them with selective tumor cell targeting property towards colorectal cancer cells [98]. Furthermore, polyethylenimine facilitated the endosomal escape of nanoparticles through proton sponge phenomena and detachment of the nanoparticles in the reducing environment within the tumor cells to release DOX from the targeted MSNPs. The targeted MSNPs showed five- fold higher accumulation at the interior region of murine tumors compared to those treated with non-functionalized MSNPs, which was clearly attributed to the elevated expression of csGRP78. This preferential targeting ability resulted in dramatic improvement of DOX therapeutic efficiency, with prolonged survival of tumor-bearing mice treated with csGRP78-targeted MSNPs. In a similar study the surface modification of pH/ROS-sensitive MSNPs with WIFPWIQL promoted their cellular uptake and DOX concentration in cytoplasm and nucleus of breast cancer cells [99]. The in vivo results were consistent with those observed in vitro, with the DOX-loaded csGRP78-targeted MSNPs significantly reducing the tumor volume compared to those treated with non-targeted NPs.

Taking advantage of high intensity focused ultrasound (HIFU) and following hyperthermia generated in tumor tissue that further upregu- lated csGRP78, WDLAWMFRLPVG has been chosen to target the doce- taxel (DOC)-polymer conjugates into the target cells in a murine prostate tumor xenograft model [100]. csGRP78 upregulation was apparent in mice treated with heat for 500 s at 43 ^◦C compared to those that did not undergo HIFU. This strategy offers the possibility to guide the csGRP78- targeted nanoparticles to even non-cancerous diseases, if hyperthermia can be induced. Besides DOC, aminohexylgeldanamycin (AHGDM) and cisplatin have also been targeted into DU145 human prostate cancer cells [101]. The only difference with previous study is that the hyperthermia procedure and subsequent csGRP78 upregulation were mediated by gold nanorods, which was shown to have significant impact on decreasing the IC50 of DOC down to 1.8 nM, compared to 13.1 nM for its non-targeted form in vitro. However, the csGRP78-targeted form of AHGDM (IC50 1.81–2.76 μM), or cisplatin (IC50 4.4–44 μM) did not exhibit much higher cytotoxicity compared to their non-targeted forms, for reasons that have not been clearly elucidated.

The csGRP78 targeting peptide, SP94 displayed on Pyrococcus fur- iosus ferritin (HccFn), has also led to ultrahigh delivery of DOX into metastatic hepatocellular carcinoma cells [102]. This selective delivery resulted in effective eradication of cancer cells, enhancement of DOX maximum tolerated dose by six-fold compared to its free form and significant reduction of DOX accumulation in healthy organs in vivo. These promising results are mainly related to superior binding affinity of targeted HccFn (Kd <1 nM) to csGRP78, even in comparison to free SP94 (Kd >700 nM). The pharmacokinetic studies showed that the designed nanostructures mainly accumulated in kidney, liver, and tumor site, 1 and 4 h post-injection, however, except for the tumor site, there was no sign of nanoparticles in kidney and liver 24 h post-injection, indicating

the persistence of csGRP78-targeted carriers at the tumor site. Pep42 has been frequently utilized for conjugation with anticancer agents like Taxol and DOX [67,103]. In order to achieve a cleavable delivery system, paclitaxel or DOX was linked to Pep42 through a cathepsin B- sensitive linker containing a Val-Cit motif, which could be cleaved in the presence of cathepsin B inside the lysosome, resulting in release of drug into the cell medium [103]. In comparison to their free form, both paclitaxel and DOX showed robust cytotoxicity against SJSA-1 cells when conjugated to Pep42, where their IC50 values dropped to 1.1 and 1.7 nM, respectively. The internalization mechanism of Pep42-modified drug delivery vehicles involved clathrin-mediated endocytosis and lysosomal trafficking [104], justifying use of enzyme and pH-sensitive spacers in the conjugate design.

The optimized form of L-peptide which was represented by advanced homologous modeling and molecular docking has also generated encouraging results with a significant reduction of cancer stem cells after treatment with csGRP78-taegeted liposomal DOX in xenografts of breast cancer [23]. The targeting ability of L-peptide has been further exploited in dual-responsive nanoparticles to enhance the anticancer efficiency of paclitaxel against human breast cancer cells [105]. Judging from confocal microscopy images and anti-tumor evaluations and compared to non-targeted nanoparticles, the csGRP78-targeted nanoparticles were mostly taken up by cancer cells, which were killed through both apoptosis and necrosis, with a consequent increased in overall survival of mice bearing tumor xenografts, with few cytotoxic effects on normal cells. It was demonstrated that subcutaneously implanted GL261 murine glioblastoma and MDA-MB-231 human breast cancer cells that received 3 Gy ionizing radiation (XRT), overexpress csGRP78 at a higher level compared to untreated cancer cells [16]. This treatment combination led to enhanced accumulation of paclitaxel- loaded nanoparticles surface modified with GIRLRG peptide at the tumor site and increased the drug concentration and resulting apoptosis of the irradiated cancer cells. The tumor tripling time of XRT-irradiated glioblastoma and breast cancer bearing nude mice that received a single dose of csGRP78-targeted paclitaxel nanocarriers was delayed by 12 and 55 days, respectively. Taken together, these results indicate that the novel csGRP78 targeting recombinant peptides, often expressed at the surface of various drug-encapsulated nano-vehicles, are excellent can- didates to improve the efficacy and reduce adverse effects of current chemotherapy approaches.

5.2. Targeted drug delivery via csGRP78 targeting antibodies

Regarding csGRP78 targeting antibodies as a drug delivery modality, their applications have to date been limited, although production costs and immunogenic consequences are nowadays probably less limiting.

The anti-KDEL (carboxy-terminal ER retention domain) functionalized polymeric nanoparticles more efficiently targeted csGRP78 expressing prostate cancer cells to paclitaxel compared to non-targeted nanoparticles and free drug [106]. In another study, the csGRP78 mAb was used to decorate the surface of bovine serum albumin (BSA) nanoparticles loaded with 5-FU; this promoted its selective delivery to csGRP78-expressing human hepatocellular carcinoma cells and inhibited their adhesion and invasion in vitro [107]. Cancer cell viability was reduced through caspase-3 mediated apoptosis. The targeted BSA nanoparticles entered the cancer cell through micropinocytosis (mostly) and clathrin-mediated endocytosis (occasionally) and finally were broken down via secreted lysosomal proteinases, releasing the 5-FU into the cytoplasm; nanoparticle uptake was reduced if cells were pre-treated with csGRP78 inhibitors, highlighting the substantial role of csGRP78.

Overall, csGRP78 is fulfilling its promise as a specific target for cancer targeted drug delivery. However, more studies are required to clarify whether csGRP78 targeting is limited to late-stage cancer, and to explore new strategies to translate csGRP78-tageted drug delivery to a successful clinical cancer treatment. Furthermore, the degree of protein corona formation and its impact on the interaction of csGRP78-tageted

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938 nanocarriers with their receptor is yet to be investigated,

5.3. Imaging

Accurate cancer imaging is essential for patient assessment, before, during and after treatment. It is most challenging at the very early stages of cancer. Thus, many advanced molecular imaging approaches including magnetic resonance imaging (MRI), computed tomography (CT), positron-emission tomography (PET), single-photon emission computerized tomography (SPECT), and fluorescence imaging are under constant development to improve diagnosis and to assess therapy efficacy [108,109]. As for selective delivery systems, cancer imaging agents should specifically locate the target to enhance the image contrast at the tumor site [110]. This again requires targeting ligands to bind to receptors expressed specifically on cancer cells. As csGRP78 meets this requirement, it can be considered as a candidate receptor for wide range of clinically applied imaging tracers coupled with csGRP78 targeting peptides or antibodies. Micro-SPECT/CT imaging has been recently employed to monitor the cellular and whole-body distribution of L- peptide-modified ¹⁸⁸Re-labled liposomes and its non-targeted counter- part in xenografts of breast cancer; the csGRP78-targeted liposomes showed higher and faster cellular uptake that was mostly distributed at the tumor site rather than normal organs, at 6 h and 24 h post-injection [23]. Similarly, ¹¹¹In-tagged polymeric micelles surface modified with WIFPWIQL peptide enhanced the SPECT γ-signals at the tumor site of gastric cancer xenografts and highlighted the tumor shape from the surrounding healthy tissues, which was evident in SPECT images [111].

While non-targeted radioactive nanoparticles mostly accumulated in liver and spleen rather than tumor tissue. Nano-SPECT/CT imaging of different tumor-bearing mice revealed that ¹¹¹In-labeled PEG(40 kDa)- GIRLRG probe specifically bound to cervical, esophageal, lung and glioma cells with an affinity constant of 2.16 × 10⁻³M and showed significantly higher (P <0.001) distribution at every tumor site 96 h post-injection compared to non-targeted ¹¹¹In-labeled PEG_{(40 kDa)}[61].

Besides csGRP78 targeting ability of imaging probes, blood persistence is also important, which was achieved using 40 kDa PEG in preference to 5 and 10 kDa PEG. Nevertheless, there was still some ¹¹¹In-labeled PEG(40-kDa)-GIRLRG entrapped in liver that might be associated with the molecular weight of PEG chains. Recently, Zhao et al. developed a csGRP78-targeted PET probe for accurate molecular imaging of triple- negative breast cancer (TNBC) cells which was designed based on DOTA-L-VAP conjugates radiolabeled with ⁶⁸Ga [112]. The selective targeting by the L-VAP peptide conferred good biodistribution and la- beling of tumor cells; moreover, since the expression level of csGRP78 in TNBC cells (MDA-MB-231) was higher than non-TNBC cells (MCF7), these two subtypes of breast cancer cells could be easily distinguished from each other in PET images. However, as observed for the PEG₍₄₀

kDa)-GIRLRG probe, a relatively high percentage of DOTA-L-VAP ⁶⁸Ga probe was detected in the liver which might be due to the fact that ⁶⁸Ga is mostly excreted via the liver [113].

The csGRP78 targeting antibodies have also demonstrated potential application in cancer imaging, where anti-csGRP78 mAb, MAb159 was linked to the ⁶⁴Cu-DOTA PET probe and utilized for whole-body imaging of BXPC3 pancreatic cancer xenografts [114]. The ⁶⁴Cu-DOTA-MAb159 radiotracer showed dominant tumor accumulation (18.3 ±1.0% 48 h post-injection), while ⁶⁴Cu-DOTA-human IgG showed significantly lower tumor accumulation (4.6 ±0.8%) after the same time interval.

Furthermore, the biodistribution patterns for both probes showed that regardless of the antibody, they both showed similar accumulation in normal tissues, including liver, kidney, and muscle. Anti-csGRP78 scFvs were used to efficiency deliver fluorescent semiconductor quantum dots (Qdots) to breast cancer xenografts in mice for advanced multi-color optical imaging [115]. Tumor growth inhibition could be clearly visu- alized through fluorescence imaging. Fluorescence was below the level of detection in kidney, heart, liver, spleen and lung, indicating good tumor targeting. These imaging studies confirm the interest of csGRP78

as target in clinical cancer imaging, though additional studies should aim to further improve specificity and to limit the accumulation of csGRP78-targeted probes in normal organs, to open the way for clinical acceptance.

5.4. Other applications

In addition to drug delivery and imaging, csGRP78 has started to be explored as a target for other medical applications, including nucleic acid delivery and photodynamic therapy (PDT). The microRNA (miRNA)-145 was incorporated in WIFPWIQL-capped MSNs to be specifically delivered to the cytosol of colorectal cancer cells, and to inhibit p70S6K1 post-transcriptional expression by binding to its 3^′-UTR [98].

This binding inhibited the expression of two downstream factor of p70S6K1, hypoxia-inducible factor 1α (HIF-1α) and VEGF, which significantly suppressed tumor angiogenesis and growth in the ortho- topic SW480 colorectal tumor model, compared to little effect of free miRNA-145. Superior synergic effects were achieved when co-delivered with DOX. As many photosensitizers (PS) applied for PDT suffer from poor solubility and selectivity for tumors, Battogtokh Et al. employed WDLAWMFRLPVG to modify PS-HPMA conjugates to target the csGRP78 of MCF-7 breast cancer cells; tumor cell hyperthermia was employed to improve cancer cell sensitivity to the targeted PDT [116].

The csGRP78-targeted conjugates showed greater cancer cell selectivity than untargeted conjugates and free PS, consistent with the superior phototoxicity of targeted conjugates after 670 nm laser and hyperthermia treatment. Despite being promising, since this study was in vitro, in vivo investigations will be needed before future clinical potential can be evaluated.

6. Concluding remarks and future prospect

Though many efforts have been made to translate a wide range of cancer therapeutics from research level to clinic, few make it to market [117,118]. In this regard, there is no exception for csGRP78 targeting peptides or antibodies aimed for cancer therapy or diagnosis, as there is only one such agent in early phase of clinical trials employed for cancer therapy. This impediment is mainly due to the fact that all the csGRP78 targeting agents must be clinically efficient, biocompatible, easy to manufacture and available to different sections of consumers [119,120].

Based on a search in clinicaltrials.gov for: (i) ‘78-kDa glucose-regulated protein’, (ii) ‘GRP78’, (iii) ‘Cell surface GRP78’, and (iv) each of aforementioned csGRP78 targeting peptides or antibodies, it is only PAT-SM6 antibody that has been evaluated by means of safety and tolerability in subjects with relapsed or refractory multiple myeloma [80] in a phase 1 clinical trial in 2014 [105]. As discussed earlier, the overall outcomes were encouraging in phase 1, where the PAT-SM6 was well-tolerated with modest clinical activity in relapsed or refractory multiple myeloma, however, according to the International Myeloma Working Group (IMWG) standards, objective responses were not seen [81]. This could explain why the phases 2 and 3 were not pursued.

Nevertheless, the same group further explored PAT-SM6 therapeutic efficiency in combination with current myeloma therapies, which could provide some good insights for future trials. Hence it is essential to prioritize fundamental studies for clinical development and to bring more of the previously introduced csGRP78 targeting peptides or antibodies integrated on nanocarriers to clinical trials, where they can be investigated for cancer-targeted theranostic applications.

In spite of significant progress related to employment of csGRP78 in targeted cancer therapy and diagnosis, there is still a long journey ahead to fully comprehend GRP78 trafficking and anchoring to the cell surface.

Furthermore, it remains to be clarified how csGRP7-related therapeutic resistance might develop. We also need to better understand tumor cross-talk and metastatic processes and to identify all csGRP78 pathways and involved partners, otherwise, successful clinical trials are unlikely.

It is also important to identify the signaling pathways, by which M. Farshbaf et al.

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csGRP78 leads to prosurvival autophagy and/or dormancy of cancer cells, and eventually cancer recurrence. More efforts should be made to understand the underlying mechanisms of drug-resistance in aggressive cancer cells developed by csGRP78 and to develop effective anti- csGRP78 combination therapies to tackle this challenge in clinic.

Despite numerous types of peptides and antibodies developed for csGRP78 targeting, there is no reported study to date involving a csGRP78 targeting aptamer sequence with therapeutic impact. Thus, profound investigations on csGRP78 targeting aptamers would be beneficial, as they are less immunogenic and show more specificity towards their targets compared to antibodies and peptides, respectively.

Unlike most other tumor markers which can also be identified on the surface of normal cells, csGRP78 is principally expressed on aggressive, proliferative cancer cells and cancer stem cells. Nevertheless, few or very limited studies have investigated csGRP78 as a valuable target for gene delivery or cancer vaccination. Therefore, there are valuable opportunities to focus studies on csGRP78 for cancer immunotherapy and gene delivery, rather than just drug delivery and tumor imaging.

Regarding tumor imaging, as MRI possesses unlimited tissue depth of penetration and high resolution, it is surprising to see little evidence for the design and production of csGRP78-targeted magnetic nanoparticles or any other MRI contrast agents that would further improve MRI contrast and facilitate tumor visualization at early stages. Moreover, the combination of csGRP78 targeting ligands with two well-known features of gold nanorods including attenuating of X-ray signal and surface plasmon resonance, could offer opportunities for advanced CT imaging, and highly sensitive cancer biosensors with improved limits of detection and quantification. Finally, the wealth of past, present, and future investigations on csGRP78 should provide further insight into establishing promising csGRP78-targeted therapeutic and imaging agents in advanced cancer care centers.

Declaration of Competing Interest

The authors declare no conflicts of interest in this work.

Acknowledgment

This paper is written based on a dataset of PhD dissertation sub- mitted by Masoud Farshbaf in the Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences (No. 61513). The financial support from the “Faculty of Advanced Medical Sciences” and “Drug Applied Research Center” of Tabriz University of Medical Sciences is greatly acknowledged. The authors are also grateful for the financial support from the Iran National Science Foundation (INSF) [Grant No.

97024873].

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