Linking cell-surface GRP78 to cancer: From basic research to clinical value of GRP78 antibodies

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Article

Reference

Linking cell-surface GRP78 to cancer: From basic research to clinical value of GRP78 antibodies

HERNANDEZ, Isabelle, COHEN, Marie-Benoîte

Abstract

Glucose-related protein 78 (GRP78) is a chaperone protein localized primarily in the endoplasmic reticulum (ER) lumen, where it helps in proper protein folding by targeting misfolded proteins and facilitating protein assembly. In stressed cells, GRP78 is translocated to the cell surface (csGRP78) where it binds to various ligands and triggers different intracellular pathways. Thus, csGRP78 expression is associated with cancer, involved in the maintenance and progression of the disease. Extracellular exposition of csGRP78 leads to the production of autoantibodies as observed in patients with prostate or ovarian cancer, in which the ability to target csGRP78 affects the tumor development. Present on the surface of cancer cells and not normal cells in vivo, csGRP78 represents an interesting target for therapeutic antibody strategies. Here we give an overview of the csGRP78 function in the cell and its role in oncogenesis, thereby providing insight into the clinical value of GRP78 monoclonal antibodies for cancer prognosis and treatment.

HERNANDEZ, Isabelle, COHEN, Marie-Benoîte. Linking cell-surface GRP78 to cancer: From basic research to clinical value of GRP78 antibodies. Cancer letters , 2021, vol. 524, p. 1-14

DOI : 10.1016/j.canlet.2021.10.004 PMID : 34637844

Available at:

http://archive-ouverte.unige.ch/unige:155895

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

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Cancer Letters 524 (2022) 1–14

Available online 9 October 2021

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Linking cell-surface GRP78 to cancer: From basic research to clinical value of GRP78 antibodies

Isabelle Hernandez

^a^,^b

, Marie Cohen

^a^,^b^,^*

aDepartment of Pediatrics, Gynecology and Obstetrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland

bTranslational Research Centre in Oncohaematology, Faculty of Medicine, University of Geneva, Geneva, Switzerland

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

Cell-surface GRP78 GRP78 antibodies Tumor development Prognosis Therapy

A B S T R A C T

Glucose-related protein 78 (GRP78) is a chaperone protein localized primarily in the endoplasmic reticulum (ER) lumen, where it helps in proper protein folding by targeting misfolded proteins and facilitating protein assembly.

In stressed cells, GRP78 is translocated to the cell surface (csGRP78) where it binds to various ligands and triggers different intracellular pathways. Thus, csGRP78 expression is associated with cancer, involved in the maintenance and progression of the disease. Extracellular exposition of csGRP78 leads to the production of autoantibodies as observed in patients with prostate or ovarian cancer, in which the ability to target csGRP78 affects the tumor development. Present on the surface of cancer cells and not normal cells in vivo, csGRP78 represents an interesting target for therapeutic antibody strategies. Here we give an overview of the csGRP78 function in the cell and its role in oncogenesis, thereby providing insight into the clinical value of GRP78 monoclonal antibodies for cancer prognosis and treatment.

1. Introduction

Glucose-related protein 78 (GRP78), also known as immunoglobulin heavy-chain binding protein (BiP) or heat shock protein (HSP) A5, is a chaperone protein belonging to the HSP70 family. It is a 78 kDa protein containing a nucleotide-binding domain (NBD) in the N-terminus and a substrate-binding domain (SBD) in the C-terminus [1] (Fig. 1). The GRP78 peptide sequence also contains a KDEL retention motif in the C-terminus, allowing for GRP78 storage in the endoplasmic reticulum (ER) compartment [2]. The protein is mainly localized in the ER [3], but in stressed cells, it is also found in cytosol, nucleus, and mitochondria and at the cell surface.

1.1. GRP78 function in the ER

The main functions of GRP78 are to assist proper protein folding by facilitating protein assembly, target misfolded proteins to prevent in- termediate aggregation in the ER lumen [4] and regulate the activation of the Unfolded Protein Response (UPR). The UPR is an adaptive process resulting in decreased protein synthesis, increased misfolded protein degradation and ultimately the onset of apoptosis during severe or prolonged ER stress [5]. In non-stressed mammalian cells, GRP78 binds

the following ER transmembrane proteins involved in the UPR, thereby blocking their activity: protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) [5]. With impaired ER homeostasis, GRP78 acts as a primary sensor and dissociates from these proteins, thus allowing their activation.

The signaling cascade triggered by PERK, IRE1 and ATF6 activation reduces the accumulation of misfolded proteins within the ER lumen by stimulating chaperone protein expression [6], decreasing mRNA trans- lation [7] and taking misfolded proteins into the cytosol for ubiquiti- nation and degradation via the ER-associated protein degradation mechanism [8]. IRE1 and ATF6 work in concert to promote the expression of chaperone proteins and thus prevent the accumulation of misfolded proteins.

1.2. Cytosolic, mitochondrial, and nuclear expression of GRP78

Although GRP78 plays a major role in the ER, it can also be found in other cell compartments (for review, see Ref. [9]). Under ER stress, a GRP78 isoform resulting from alternative splicing, named GRP78va, is translocated to the cytosol where it plays a pro-survival role by interacting with the PERK inhibitor P58^IPK[9]. With imbalanced ER homeostasis, newly synthetized GRP78 may be directed to mitochondria.

* Corresponding author. Rue Michel Servet 1, 1206, Geneva, Switzerland.

E-mail address: [email protected] (M. Cohen).

Contents lists available at ScienceDirect

Cancer Letters

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

https://doi.org/10.1016/j.canlet.2021.10.004

Received 13 July 2021; Received in revised form 10 September 2021; Accepted 5 October 2021

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From this location, it has a pro-apoptotic signaling function by coordi- nating the UPR between the two organelles [9]. GRP78 can also be directed to the nucleus [10], where it may play a role in reducing DNA damage-induced apoptosis [9].

1.3. Cell-surface GRP78

Cell-surface GRP78 (csGRP78) exists on the cell surface of many cell types, including endothelial [11], cancer (lymphoma, neuroblastoma, lymphoblastic leukemia, ovarian tumor, lung and colon adenocarcinoma) [12–16], and trophoblastic cells [17,18], which are characterized as stressed and/or pathological cells [19].

CsGRP78 is found in three different configurations: 1) as a membrane-embedded protein, 2) associated with a transmembrane protein, or 3) associated with a glycosylphosphatidylinositol (GPI)- anchored protein (e.g., teratocarcinoma-derived growth factor 1, also called Cripto, or T-cadherin) [20]. Several mechanisms have been proposed to explain the presence of GRP78 at the cell surface. Under normal conditions, GRP78 is retained in the ER because of its KDEL motif; de- leting the KDEL sequence from GRP78 promotes its relocation to the cell surface [19]. Consequently, the leading hypothesis is a “leak” of ER-lumen GRP78 retention owing to an overwhelming amount of

GRP78 synthetized, which escapes the KDEL retrieval mechanism [21].

Another suggested mechanism relies on the conformational unavail- ability of the KDEL retention sequence to bind the ER retention receptors because of steric hindrance [12].

GRP78 localization on the cell surface has been observed only in specific cell types. CsGRP78 relocalization requires the presence of its SBD motif, which highlights the contribution of GRP78 ligands such as Murine Tumor cell DnaJ-like protein 1 [22] or Prostate Apoptosis Response-4 (PAR-4) [18,23,24] to its cell-surface transportation. PAR-4 and GRP78 co-localize in the ER before translocating to the membrane in response to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) exposure, which suggests that endogenous PAR-4 is involved in GRP78 localization [24]. This hypothesis was reinforced by the obser- vation that PAR-4 overexpression or silencing induced an increase or decrease, respectively, in csGRP78 expression in trophoblastic cells [18]

and SKOV-3 ovarian cancer cells [23].

CsGRP78 binding to extracellular ligands activates signaling pathways involved in cell survival, proliferation, or apoptosis (Fig. 2), which emphasizes its potential as a therapeutic target. CsGRP78 also triggers an immune response leading to the production of anti-GRP78 autoantibodies. Here, we review the signaling pathways activated by csGRP78 and the functional importance of its cell-surface localization in cancer.

Abbreviations

α2M alpha 2 macroglobulin

Akt RAC-alpha serine/threonine-protein kinase ATP/ADP adenosine triphosphate/adenosine diphosphate ATF6 activating transcription factor 6

BAD Bcl-2 associated agonist of cell death BAK Bcl-2 homologous antagonist/killer BAX Bcl-2 associated X

Bcl-2 B-cell lymphoma 2 protein Bip: Binding immunoglobulin protein

CLPTM1L: cleft lip and palate transmembrane 1-like protein Cripto teratocarcinoma-derived growth factor 1

csGRP78 cell-surface GRP78

c-Src proto-oncogene tyrosine-protein kinase Src DLC1 deleted in liver cancer-1

ER endoplasmic reticulum

Erdj5 Endoplasmic reticulum DNA J domain-containing protein ERK1/2 extracellular signal-regulated kinase 1/2 5

FOXO Forkhead box protein O

GADD153 Growth arrest and DNA damage-inducible gene 153 GSK3β Glycogen synthase kinase 3 beta

GPI glycosylphosphatidylinositol GRP78 glucose regulated protein 78 HSP Heat-shock 70 kDa protein

IKKβ inhibitor of nuclear factor kappa B subunit beta IRE1 inositol-requiring enzyme 1

JNK c-Jun N-terminal kinase LDL: low density lipoprotein LR linker region

MAPK mitogen-activated protein kinase

MKK3/6 mitogen-activated protein kinase kinase 3/6 MKK4 mitogen-activated protein kinase kinase 4 mTOR mammalian target of rapamycin

NBD nucleotide-binding domain NF-κB nuclear factor-kappa B NSCLC non-small-cell lung carcinoma p38MAPK p38 mitogen-activated protein kinase P21 WAF/CIP cyclin-dependent kinase inhibitor PAR-4 prostate apoptosis response-4

PARP-1 poly [ADP ribose] polymerase 1

PERK protein kinase RNA-like endoplasmic reticulum kinase PIK3-α/p110α/p85α phosphoinositide 3-kinase-alpha/subunit p110

alpha/p85alpha Rho Ras homologous protein

SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 SBD substrate binding domain

Smad2/3 mothers against decapentaplegic homolog 2/3 STAT3 signal transducer and activator of transcription 3 TGF-β: transforming growth factor-beta

TRAIL: tumor-necrosis factor-related apoptosis inducing ligand UPR unfolded protein response

VDAC voltage-dependent anion channel

YAP/TAZ Yes-associated protein/Transcriptional coactivator with PDZ-binding motif

Fig. 1.Schematic representation of GRP78 structure. GRP78 is a 78 kDa protein containing two major domains: NBD allowing for capture and hy- drolysis of ATP and SBD allowing interaction with proteins. GRP78 possesses a KDEL sequence causing its retention in the endoplasmic reticulum lumen.

ATP: adenosine triphosphate; Ca²⁺: calcium ion; C- term: C-terminus; GRP78: glucose-related protein 78; LR: linker region; NBD: nucleotide-binding domain; N-term: N-terminus; SBD: Substrate Bind- ing Domain.

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Cancer Letters 524 (2022) 1–14

We also provide insights into the clinical value of anti-GRP78 autoantibodies as prognostic markers and GRP78 antibodies for cancer targeting and/or targeted therapy.

2. CsGRP78 functions as a receptor

CsGRP78 can bind extracellular ligands such as alpha-2- macroglobulin (α2M), kringle-5, isthmin and PAR-4 [9]. The association of csGRP78 with these proteins activates several signaling pathways, thus promoting diverse responses such as cell survival, proliferation, or apoptosis (Fig. 2). Despite having opposite functions, some ligands such as Cripto, T-cadherin and PAR-4 share the same binding region in the N-terminus of GRP78 [25].

2.1. Pro-survival csGRP78 ligands: α2M, cripto, T-cadherin and secreted GRP78

GRP78 was identified as a signaling receptor for activated α2M in macrophages and choriocarcinoma and prostate cancer cells [26–28]. In prostate cancer cells, binding of activated α2M to csGRP78 is necessary and sufficient to induce a transduction signal [29] and promote proliferation [30] via activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK), p21RAS-dependent MAPK and PI3K/Akt, signaling pathways [28,29, 31]. It also increases cell survival by activating the UPR and nuclear factor-kappa B (NF-κB) signaling pathway [28] (Fig. 2).

CsGRP78 can also bind to Cripto [32] via its cysteine-rich

CRIPTO/FRL-1/CRYPTIC domain. The csGRP78–Cripto association inhibits transforming growth factor-beta (TGF-β) signaling, with a decrease in mothers against decapentaplegic homolog 2 (or Smad 2) phosphorylation [32,33]. Down-regulation of TGF-β signaling by Cripto binding to csGRP78 leads to cell proliferation by inhibiting the TGF-β pathway growth-inhibition effect [32]. The csGRP78 association with Cripto also leads to cell proliferation via ras/raf/ERK1/2 and PI3K/Akt pathways [33] (Fig. 2). Of note, inhibition of the TGF-β signaling cascade did not require csGRP78 binding to the TGF-β receptor type I and II but instead cooperation between csGRP78 and Cripto [32].

CsGRP78 can also be associated with GPI-anchored T-cadherin at the cell surface of endothelial cells, thus leading to cell survival via an Akt- dependent signaling pathway [34](Fig. 2). Furthermore, GRP78 itself can become a ligand of csGRP78. Secreted GRP78 can bind to csGRP78 and activate downstream cellular pathways such as PI3K/Akt and Wnt/β-catenin pathways in colon cancer cells, thus leading to cell proliferation [35] (Fig. 2).

2.2. Pro-apoptotic csGRP78 ligands: kringle 5, PAR-4 and isthmin Besides cell survival and pro-proliferative mechanisms associated with csGRP78 binding to its ligands, other proteins interact with csGRP78 and induce apoptosis. The binding of kringle 5 to csGRP78 in endothelial cells inhibits angiogenesis by inducing cell apoptosis [13]. Three mechanisms have been proposed to explain kringle 5-induced apoptosis. The first in- volves endocytosis of kringle 5 and its binding to the adenosine triphosphate (ATP)ase domain of GRP78, thus competing with the binding of Fig. 2. CsGRP78 ligands. CsGRP78 serves as receptor for several proteins, thereby mediating cell fate. Its binding to α2M, Cripto, and T-cadherin as well as GRP78 leads to the activation of pro-survival and pro-proliferative pathways, whereas csGRP78 binding to kringle 5, isthmin and PAR-4 binding triggers an apoptotic cascade. CsGRP78 also plays a role in virus internalization via direct interaction with pathogens and promotion of their internalization. GRP78 autoantibody binding to csGRP78 modulates either proliferation or apoptosis in tumor cells, and also promotes atherosclerosis lesion formation by increasing the expression of adhesion proteins. CsGRP78 binds to integrin-β1 or α-synuclein and may promote the onset of pathologies by facilitating an integrin-β1–mediated fibrotic response in renal cells or promoting cell degeneration in Parkinson’s models. α2M: alpha-2-macroglobulin; cas 7/8: caspase-7/8; ERK1/2: extracellular signal-regulated kinase 1/2;

FADD: Fas-associated protein with death domain; GRP78: glucose-related protein 78; ICAM-1: intracellular adhesion molecule-1; LRP1: Low density lipoprotein receptor-related protein 1; NF-κB: nuclear factor-kappa B; p38: p38 mitogen-activated protein kinase; Par-4: prostate apoptosis response-4; PI3K: phosphatidylinositol 3-kinase; T-cad: T-cadherin; UPR: unfolded protein response; VCAM-1: vascular cell adhesion molecule 1; VDAC: voltage-dependent anion channel.

I. Hernandez and M. Cohen

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procaspase-7 to GRP78 and activating caspase-7 signaling [13]. In human gastric carcinoma cells, kringle 5 decreases the expression of GRP78 by downregulating voltage-dependent anion channel (VDAC)-mediated ERK1/2 phosphorylation (Fig. 2). With less GRP78 available, procaspase-7 remains free within the cytosol and then is cleaved to caspase-7, which activates apoptosis [36]. The authors stated that the GRP78 decreased expression involved kringle 5 binding to VDAC and might also involve kringle 5 binding to csGRP78, although they did not demonstrate this. Nonetheless, Gonzalez-Gronow et al. demonstrated that csGPR78 and VDAC colocalize at the surface of 1-LN human prostate cancer cells and SK-N-SH human neuroblastoma cells [37,38]. Kringle 5 can also mediates the internalization of GRP78 via low-density lipoprotein receptor-related protein 1, thus activating p38 MAPK and apoptosis in dermal micro-vessel endothelial cells [39] (Fig. 2).

PAR-4 was initially described as a cytoplasmic and nuclear protein until Burikhanov et al. observed its secretion by cancer cells exposed to ER stress [24]. PAR-4 induces apoptosis by binding to csGRP78, which activates the Fas-associated protein with death domain/caspase- 8/caspase-3 pathway. TRAIL-induced apoptosis also depends on the PAR-4–GRP78 complex formation in BPH-1 epithelial cells and several cancer cell lines (PC-3/prostate, H460/lung and Hela/cervical cancer) [24] (Fig. 2).

Isthmin is a 60 kDa secreted angiogenesis inhibitor protein that binds to csGRP78 with high affinity and induces apoptosis of endothelial cells [40]. The binding of isthmin to csGRP78 triggers clathrin-dependent endocytosis. Once inside endothelial cells, the GRP78–isthmin complex moves to mitochondria, where it blocks mitochondrial ATP/adenosine disphosphate (ADP) carriers, thus restraining ATP transport from mitochondria to the cytosol and triggering apoptosis [40]. This pro-apoptotic effect was also observed in mouse models carrying sub- cutaneous 4T1 breast carcinoma or B16 melanoma and treated with recombinant isthmin [40] (Fig. 2).

2.3. Other ligands: GRP78 autoantibodies, α-synuclein, and integrin-β1 When exposed on the cell surface, csGRP78 can induce the production of GRP78 autoantibodies. CsGRP78 was observed at the surface of endothelial cells isolated from atherosclerotic lesions in ApoE⁻^/⁻ mice with endothelial dysfunction [11,41]. The binding of GRP78 autoantibodies to csGRP78 promotes atherosclerotic lesion growth by increasing NF-κB-induced intracellular adhesion molecule-1 and vascular cell adhesion molecule 1 expression [41] (Fig. 2). The presence of anti-GRP78 autoantibodies was also demonstrated in serum from patients with ovarian and prostate cancer [42–45], where they could contribute to modulating tumor growth [45,46] (Fig. 2). In prostate cancer cells, anti-GRP78 autoantibodies act as agonists of activated α₂M and stimulate prostate cancer cell proliferation; they also had a dose-dependent protective effect against apoptosis induced by tumor necrosis factor-alpha in the prostate cancer cell lines 1-LN, PC-3, LnCap and DU145 [46]. The various effects of these anti-GRP78 autoantibodies may depend on an epitope they recognize [42,45].

Other csGRP78 ligands such as α-synuclein and integrin-β1 were suggested to be involved in specific neurodegenerative and metabolic diseases. In a transgenic mouse model of Parkinson’s disease and in dopaminergic differentiated cell lines, csGRP78 bound to α-synuclein, a protein that aggregates in the ER of dopaminergic neurons. When the aggregate bound to GRP78, the UPR is activated, ultimately leading to cell degeneration in Parkinson’s disease [47]. Extracellular α-synuclein can also bind to csGRP78, thus forming clusters at the surface of dysfunctional neurons, which triggers a transduction signal resulting in defective actin turnover [48] (Fig. 2). CsGRP78 interaction with integrin-β1 was described in non-cancerous renal mesangial primary cells exposed to high-glucose treatment, therefore resulting in activated PI3K/Akt signaling, which in turn triggered a fibrotic response (Fig. 2).

Hence, csGRP78 may have a role in diabetic nephropathy [49].

2.4. Viruses

CsGRP78 assists the cellular entry of different viruses such as dengue [50,51], coxsackievirus A9 [52], Borna disease [53], Japanese enceph- alitis [54] and Ebola viruses (Fig. 2). It also facilitates the virus–host cell interaction for different types of coronaviruses such as middle east respiratory syndrome coronavirus and bat coronavirus [55]. Recently, an in silico study showed that the viral spike protein of SARS-CoV-2 poten- tially interacts with csGRP78 as a host-cell receptor [56] (Fig. 2).

3. Involvement of csGRP78 in oncogenic processes

A stressed ER results in UPR activation and overexpression of GRP78.

Excess GRP78 in the ER is then expressed on the cell surface [21].

Stressed cells exhibit the presence of GRP78 on the cell surface; therefore, csGRP78 is a hallmark of some cancer cells (ovarian, prostate, brain and breast cancer, myeloma, melanoma, and lymphoma), and more generally stressed cells.

In some specific cancer types, csGRP78 expression was found correlated with chemotherapy resistance, tumor recurrence and prognosis [42,57–61]. However, other studies associated csGRP78 expression with a favorable response to chemotherapy and better prognosis [62–65]. Hence, deciphering the underlining mechanisms of the csGRP78 involvement in neoplastic properties is a priority interest in research.

3.1. Proliferation, migration, and invasion

The pro-oncogenic properties of csGRP78 rely in part on its ability to promote tumor cell proliferation and reduce apoptosis (Fig. 3). There is evidence that in prostate cancer cells, the N-terminus of csGRP78 is involved in inducing proliferation and inhibiting apoptosis by activating Akt, forkhead box protein O, glycogen synthase kinase 3 beta and MAPK pathways [28]. Cell survival is also promoted by the association of csGRP78 with α2M, which increases histone acetylation via Akt pathway activation [66]. The GRP78-α2M–induced proliferation may also result from increased activation of mammalian target of rapamycin complex 1/2 [67], MAPK and NF-kB pathways and inhibited apoptosis [28].

Furthermore, prostate tumor growth can be stimulated by tissue factor procoagulant activation via the binding of anti-GRP78 autoantibodies to csGRP78 [68]. In the MCF-7 human breast cancer cell line, the overexpression of GRP78 led to increased csGRP78 expression as well as increased level of phosphorylated STAT3 [58]. CsGRP78 expression in these cells increases their proliferation and migration. GRP78 antibody suppressed STAT3 phosphorylation and reversed the effect of csGRP78 expression on proliferation and migration [58].

Indeed, in some situations, csGRP78 assists in migration of malignant cells (Fig. 3). In hepatoma cells, csGRP78 expression was enhanced by insulin-like growth factor I, thus resulting in a malignant cell phenotype by promoting cell proliferation and migration [69]. Furthermore, csGRP78-mediated activation of the Hippo–Yes-associated protein/transcriptional coactivator with PDZ-biding motif (YAP/TAZ) axis increased cell motility and invasion in pancreatic ductal adenocarcinoma [70].

CsGRP78 has a role in modulating cell invasiveness in several other types of cancer (Fig. 3). In nasopharyngeal and pharyngeal squamous carcinoma cell lines, csGRP78 associating with p85α and Rac family small GTPase 1 proteins led to an increased cell invasion via the NF-κB pathway [71]. These pro-invasive properties were also observed in colorectal cancer, where csGRP78 interacting with integrin-β1 promoted the modulation of focal adhesion kinase and therefore cell migration and invasion via csGRP78 interacting with the urokinase-type plasminogen activator-urokinase type plasminogen activator receptor (uPA-uPAR) protease system [57]. In hepatocellular carcinoma cells, treatment with GRP78 antibody inhibited cell invasion and decreased matrix metalloproteinase 2 secretion and activity [72]. Finally, α2M interacting with csGRP78 promoted metastasis of hepatocellular

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carcinoma by enabling direct interaction of csGRP78 with c-Src, which activates the epidermal growth factor receptor and promotes the invasion of cancer cells [73].

3.2. Response to treatment

The role of csGRP78 as a prognostic marker has been explored in part because of its role in conferring tumoral cell resistance to treatment.

This property depends on the cancer cell type, as summarized in Fig. 3.

In prostate and breast cancer, csGRP78 promoted resistance to hormonal therapy by activating PI3K/Akt signaling [74]. In pancreatic ductal adenocarcinoma, the association of csGRP78 with cleft lip and palate transmembrane 1-like protein (CLPTM1L) and PIK3-α/p110α inhibited sensitivity to chemotherapy [59].

Nevertheless, the association of csGRP78 and chemotherapy resistance should be explored in detail and specifically evaluated for each cancer type, because csGRP78 expression in breast cancer cells has also been found associated with a positive response to chemotherapy [63]

and could be used as a biomarker for positive prognosis. Indeed, in triple-negative breast cell lines, doxorubicin induced csGRP78 expression and thus promoted tumor cell apoptosis [75].

The csGRP78-mediated response to chemotherapy might also involve immune cells: 15 different peripheral blood mononuclear cell subpopulations from breast cancer patients who underwent neoadjuvant therapy before surgery were analyzed by flow cytometry. The mononuclear cell subpopulation showed increased expression of GRP78 from patients receiving taxane and with a “pathological complete response”

(i.e., disappearance of any invasive disease) [76]. Hence, csGRP78 may be used as a predictor of taxane benefits in chemotherapy treatment.

3.3. Stemness

Stemness of cancer cells remains one of the great challenges in curing cancer. Therapeutic strategies need to include specific targeting of this subpopulation of cells because they remain resistant to current treatments. CsGRP78 is involved in stemness (Fig. 3), both owing to its expression in induced pluripotent stem cells and also its expression in breast cancer cells being associated with the expression of stemness- associated genes [77]. The expression of these genes characterize a pool of cells within the breast cancer cell population called “tumor initiating cells” because of their enhanced ability for metastasis in vivo [77]. An interactome study of head and neck cancer cell lines showed the involvement of csGRP78 in the expression of stemness-associated markers such as nanog, octamer-binding transcription factor 4, sex-determining region Y-box 2, telomeric repeat binding factor 1 and PR/SET domain 14 [78]. In these head and neck cancer cells, csGRP78 is associated with radiotherapy resistance, cell differentiation and self-renewal [79]. These properties may be attributed to cell-surface csGRP78 partners such as Cripto. A Cripto antagonist promoted differentiation of the mammary stem cells ex vivo, which suggests that Cripto plays a role in stemness maintenance [80]. The Cripto association with csGRP78 favors the proliferation of the metastatic subpopulation of prostate cancer cells by promoting the epithelial–mesenchymal transi- tion and invasiveness [81]. In addition, the Cripto–csGRP78 complex was found involved in hematopoietic stem cells maintenance by modulating the hypoxia-inducible factor 1 alpha pathway in the endosteal region of bones [82].

Fig. 3. Involvement of csGRP78 in cancer pathophysiology. CsGRP78 is involved in various oncogenic processes such as proliferation, angiogenesis, migration, invasion, and stemness. It also affects the tumor response to treatment. α2M: alpha 2 macroglobulin; Ab: anti-GRP78 antibody; Akt: Akt serine/threonine-protein kinase; C38: anti-GRP78 antibody clone C38; CD44v: CD44 spliced variant v; CLPTM1L: cleft lip and palate transmembrane 1-like protein; Cripto:

teratocarcinoma-derived growth factor 1; c-Src: proto-oncogene tyrosine-protein kinase Src; DLC1: deleted in liver cancer 1; EGFR: epidermal growth factor receptor;

FOXO: forkhead box protein O; GSK3β: glycogen synthase kinase-3 beta; IGF-1: insulin-like growth factor-1; GTP-Rac1: Rac family small GTPase 1; IGF-R1: insulin- like growth factor 1 receptor; MAPK: mitogen-activated protein kinase; MMP2: matrix metalloproteinase 2; mTORC1/C2: mammalian target of rapamycin complex 1/2; NF-κB: nuclear factor-kappa B; PI3K: phosphoinositide 3-kinase; Rho: Ras homologous protein; STAT3: signal transducer and activator of transcription 3; uPA/

uPAR system: urokinase plasminogen activator/urokinase plasminogen activator receptor; YAP/TAZ: Yes-associated protein/transcriptional coactivator with PDZ- binding motif.

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3.4. Angiogenesis

CsGRP78 on the surface of proliferating endothelial cells can bind to kringle 5, thereby inducing apoptosis by cell cycle arrest [83], which then leads to an antiangiogenic effect in the tumor environment (Fig. 3) [13]. Davidson and collaborators further hypothesized that the anti-oncogenic property of kringle 5 binding to csGRP78 occurs by a two-step mechanism. First, kringle 5 binds to the GRP78 expressed on the surface of proliferative endothelial cells inside or surrounding the tumor. This interaction leads to apoptosis, which then reduces the tumoral blood supply and establishes a hypoxic environment. The local hypoxia enhances csGRP78 expression in tumor cells. In turn, kringle 5 can bind to csGRP78 expressed on these malignant cells, thereby inducing apoptosis via caspase-7 activation [13].

3.5. Metabolic adaptation

Glucose starvation can induce the expression of csGRP78. In turn, csGRP78 promotes the translocation of glucose transporter type 1 to the cell membrane, thus decreasing the expression of pyruvate kinase M2 and increasing that of pyruvate dehydrogenase A and B in human colon carcinoma cell lines [84]. Also, csGRP78 interacting with α2M modu- lated aerobic glycolysis in 1-LN and DU145 prostate cancer cells, A375 melanoma cells and A172 glioma cells [66]. Thus, csGRP78 may be directly implicated in glucose metabolism of cancer cells.

CsGRP78 plays a key role in the proliferation and survival of tumor cells as well as the adaptation of the tumor-specific environment tran- sition (Fig. 3) and thus is a central actor in tumor cell fate. Hence, csGRP78 is an extremely valuable biomarker candidate as well as a therapeutic target for cancer. Not surprisingly, GRP78 antibodies have been evaluated for the past 20 years, revealing promising clinical applications.

4. Clinical value of GRP78 antibodies in cancer 4.1. GRP78 autoantibodies as prognostic markers

CsGRP78 is expressed in various cancer cells, so its extracellular exposure may lead to an immune response, which translates into the presence of autoantibodies directed against GRP78 in systemic circula- tion. Because csGRP78 is involved in multiple cellular responses in neoplastic cells, several studies have focused on the role and potential clinical application of GRP78 autoantibodies in cancer. The first description of circulating GRP78 autoantibodies in cancer was in prostate cancer, where their level is increased. The presence of these autoantibodies is negatively associated with patient survival; therefore, their detection may be predictive of aggressive tumor behavior [42].

Recently, levels of circulating GRP78 autoantibodies were also found increased in hepatocellular carcinoma, even at the early disease stage [60]. The GRP78 autoantibody level was associated with the clinical disease stage and metastasis, with a significant increase in serum level in stage IV cancer [60].

The prognostic value of quantifying circulating GRP78 autoantibodies in ovarian cancer is controversial. Taylor et al. evaluated GRP78 autoantibodies in serum of patients at various stages of ovarian cancer.

Autoantibody levels were correlated with disease stage [43]. The results raised great hope for developing a GRP78 prognostic biomarker for ovarian cancer. However, other studies showed that the anti-GRP78 autoantibody level in serum from women with ovarian cancer was slightly decreased or similar to the level in healthy patients [44,45].

These major differences in reported observations might result from the method used to evaluate the GRP78 antibody level. Taylor et al. used dot blot arrays with a purified exosomal GRP78 protein as an antigen, whereas the other studies used an ELISA method with recombinant GRP78 as an antigen. Furthermore, the GRP78 autoantibody population in serum of ovarian cancer patients is heterogenous and could have

different effects depending on targeted epitopes [44,45]. Indeed, autoantibodies recognizing the N-terminus of GRP78 seems to have pro-proliferative properties [42,68] whereas those recognizing the C-terminus induce apoptosis [45]. A specific ELISA assay that detects specific GRP78 autoantibodies, for example distinguishing those that bind to C-terminal or N-terminal epitopes, should be developed and tested in a large cohort of patients to confirm the biomarker potential.

Thus, the identification of pro-apoptotic GRP78 autoantibodies in ovarian cancer [45] highlights the potential use of GRP78 antibodies for therapeutic strategies.

4.2. GRP78 antibodies as therapeutic tools

To date, several strategies have been explored to design therapeutic tools based on csGRP78 targeting. The proof of concept was initially demonstrated in vitro and in vivo with commercial polyclonal GRP78 antibodies. Monoclonal antibodies were then isolated from mice and humans or designed to be used as therapeutic agents. One such antibody, PAT-SM6 was assessed in phase I clinical trials. More recently, a new tool involving a nanocarrier binding GRP78 was engineered, thus opening the door to more efficient anti-cancer therapeutic tools. These strategies are discussed in the following sections.

4.2.1. Polyclonal GRP78 antibodies

Several commercial polyclonal antibodies recognizing C-terminus part of GRP78 induce apoptosis or decrease proliferation of cancer cells.

These effects are summarized in Table 1. Using the commercial SPA-826 antibody, Misra et al. demonstrated its efficacy to trigger a pro-apoptotic signal by increasing p53 expression and promoting its nuclear translocation in 1-LN and DU-145 prostate cancer cells [30,85]. The SPA-826 antibody also leads to downregulation of the UPR partners IRE1-α, PERK and ATF-6α and upregulation of the expression of apoptosis-associated proteins such as poly [ADP ribose] polymerase 1, endoplasmic reticulum DNA J domain-containing protein 5 and growth arrest and DNA damage-inducible gene 153 [86]. The studies also detail its involvement in pro-apoptotic processes by inhibiting mitogen-activated protein kinase kinase 3/6 (MKK3/6), ERK1/2 and p38MAPK pathways, promoting MKK4 and subsequently c-Jun N-terminal kinase activation, and increasing the expression of pro-apoptotic proteins such as Bcl-2 associated agonist of cell death (BAD), Bcl-2 homologous antagonist/killer (BAK), Bcl-2 associated X (BAX) and cleaved caspases-3, -7 and -9 [87].

In 1-LN prostate cancer cell lines, SPA-826 has anti-proliferative properties by inhibiting the NF-κB pathway [85].

Antitumor activity of this polyclonal GRP78 antibody was also demonstrated in non-small cell lung cancer (NSCLC) and glioblastoma multiform cancer cell lines. Their treatment with a polyclonal GRP78 antibody suppressed PI3K/Akt/mTOR signaling and resulted in decreased cell proliferation and increased apoptosis [88]. This antibody also reduced tumor growth when associated with ionizing radiation treatment in in vivo models of NSCLC and glioblastoma multiform [88], giving hope for the development of new treatments targeting csGRP78 for patients with these two deadliest cancers.

4.2.2. Development of GRP78 antibodies with therapeutic potential With the evidence of the potential use of csGRP78 as a therapeutic target, interest is growing to develop monoclonal antibodies to GRP78 as stand-alone or combination treatment for cancer.

In 2012, De Ridder et al. developed two monoclonal antibodies targeting the GRP78 C-terminus, named C38 and C107, and demonstrated their anti-tumoral potential in vitro and in vivo. Indeed, treatment with C107 directly induced apoptosis of B16F1 murine melanoma cells and reduced tumor growth in a mouse xenograft model [89]. C38 treatment of irradiated pancreatic ductal adenocarcinoma cells over- came their resistance to radiotherapy. The antibody inhibited Rho-mediated motility and invasiveness of these cells and increased apoptosis [70]. C38 also acted as an antagonist of α2M in B16F1 cells

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Table 1

GRP78 antibodies with potential clinical interest against cancer. The part of GRP78 targeted is annotated when known. The experimental model, the effects and the mechanism involved in the response to anti-GRP78 antibody treatment and patent information are summarized. Ascending arrow (↗): increase, descending arrow (↘): decrease in level.

Antibody GRP78 targeted

part Effect on cells/

tumor Model Involved mechanism Patents

Antibodies with antitumoral properties in vitro and in vivo Antibodies with antitumoral properties in vitro and in vivo

Rabbit anti-GRP78 polyclonal Ab Cat# SPA-826 from Stressgen, Victoria, BC

C-terminus ↗ apoptosis 1-LN prostate

cancer cell line - ↘ Akt kinase activity [87]

- ↘phosphorylation of FOXO and GSK3β [87]

- ↘ERK1/2, p38 MAPK and MKK3/6 activation [87]

- ↗MAKK4 and JNK activity [87]

- ↘ expression of Bcl-2 [87]

- ↗ expression of BAD/BAX/BAK [87]

- ↗ expression of cleaved caspase

−3/-7/-8/-9 [87]

- ↗p53 expression and accumulation in cytosol and nucleus [30,85]

- Cyclin D1 degradation [85]

- ↗ p21WAF/CIP and p27kip expression [85]

–

- ↘ UPR related IRE1-α/PERK/

ATF6α-dependent pathways [86]

- ↗ GADD153 level [86]

- ↗ cleaved PARP-1 and Erdj5 [86]

DU-145 prostate cancer cell line

Anti-

proliferation 1-LN prostate

cancer cell line - ↘ NF-κB1/NF-κB2 and IKKα, IKKα/β, IκBα and IκBβ α₂M- induced activation [85]

Goat anti-GRP78 polyclonal Ab

from Santa Cruz Biotechnology Undisclosed Anti-

proliferation D54 and U251 human glioblastoma cell lines

A549 and H460 human non- small cell lung carcinoma cell lines

- Akt/mTOR signaling inhibition

[88] –

↗ apoptosis [88]

↗

radiosentitivity [88]

D54 and U251 human glioblastoma cell lines

A549 and H460 human non- small cell lung carcinoma cell lines

Unexplored

Tumor growth

inhibition A549 and U251 xenograft mice models

- - Akt/mTOR signaling inhibition [88]

↗

radiosensitivity [88]

Unexplored

Mouse C38 monoclonal IgG2b

antibody C-terminus Blocking of Akt

activation B16F1 murine melanoma cell line

- N88 and α₂M antagonist – steric interference [89]

–

(continued on next page)

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Table 1 (continued)

↗

radiosensitivity Pancreatic ductal adenocarcinoma cell line (PDAC)

- Akt/DLC1/Rho and Rho dependent YAP/TAZ activation [70]

Mouse C107 monoclonal IgG2b

antibody C-terminus ↗ apoptosis

[89] B16F1 murine

melanoma cell line

Unexplored –

Slowed tumor

growth [89] B16F1 murine melanoma tumor model

AEP5857 Undisclosed Anti-

proliferation DU145 prostate

cancer cell line - Antagonist of anti-GRP78 autoantibodies [90]

- Inhibits UPR-mediated survival and TF activity by blocking anti- GRP78 autoantibodies fixation [90]

–

2D6F3 monoclonal antibody Undisclosed ↗ radiation induced cytotoxicity

MDA-MB-231 breast adenocarcinoma cell line

Undisclosed Inventors: Hallahan Dennis E, Yan Heping Application dates and number:

2017-06-20 (US20170298142)

2015-01-28 (EP15743922/EP3099718/WO/2015/116653) 2014-01-28 (US14166251/US20140316186)

2012-07-30 (WO2013019730) NSCLC H460

non-small-cell lung carcinoma cell line GD-17 monoclonal IgG1

antibody 376-415 amino

acids sequence of GRP78

Tumor growth

reduction Undisclosed xenograft mouse model

Unexplored Inventors: Kimura Naoki

Application dates and numbers:

2009-09-24 (N5665/CHENP/2009)

2008-02-27 (EP08721318.7/EP2130552/US20100041074/

WO2008105560) Anti-

proliferation 22Rv1/LNCaP prostate cancer cell lines

Unexplored

The antibody is internalized MCF7 breast

cancer cell line BxPC3/PANC-1 pancreatic cancer cell lines SKOV3 ovarian cancer cell line Humanized Mab159 IgG

antibody undisclosed Anti-

proliferation MCF7 breast carcinoma cell lines HT29 colon cancer cell line

PI3K/Akt downregulation [91] Inventors: Gill Parkash, Liu Ren, Lee Amy Application dates and numbers:

2019-06-11 (US20200140541) 2017-05-09 (US20180094054)

2014-03-14 (EP14770483/EP2970443/US20160185853/

SG11201507563S/NZ713042/CA2906688/WO2014153056 AU2014236309/PH1/2015/502112/MYPI 2015002358) 2015-09-16 (IL241646)

Tumor development or growth inhibition [91]

Tumor xenograft model (HT29, A549 lung adenocarcinoma and H249 small cell lung carcinoma)

- ↘Ki67 expression [91]

- ↘ vessel density evaluated by CD31 staining [91]

- Inhibition of PI3K signaling [91]

PTEN knockout spontaneous tumor mice model

Unexplored

↗ apoptosis MCF7 breast carcinoma cell lines

Caspase 8 and 9 activation [91]

Hernandez and M. Cohen

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Table 1 (continued)

HT29 colon cancer cell line Tumor

regression Tumor xenograft model (HT29, A549 lung adenocarcinoma and H249 small cell lung carcinoma)

↗ apoptosis [91]

Inhibition of tumor metastasis [91]

Orthotopic tumor model of mouse breast adenocarcinoma cell 4T1 B16-Fluc-A1 melanoma cell line

PI3K signaling inhibition [91]

Antibodies evaluated in clinical

trials Human PAT-SM6 IgM antibody Undisclosed ↗ apoptosis Stomach

carcinoma cell line 23132/87 Pancreatic carcinoma cell line BXPC-3

- Lethal accumulation of oxidized lipoproteins [92]

- Cytochrome C release from mitochondria [92]

- Activation of caspase-8/9/3/6 [92]

Inventors: Ilag Leodevico I, Power Barbara, Udabage Lishanthi Application dates and numbers:

2010-02-09 (AU2010000128/WO2010088739)

BXPC-3 cell line

[93] Unexplored

Antibody specificity: IgM – O-linked carbohydrate structure

Primary human multiple myeloma cells [95]

MM1.S/OPM-2/

INA-6/U266 myeloma cell line [95]

CRL-1424 and HTB-69 malignant melanoma cell lines [94]

Accumulation of intracellular lipid [94]

Inhibition of tumor metastasis [94]

C8161 melanoma metastasis bearing mice model

Unexplored

RESULTS OF CLINICAL TRIALS/Proof of concept Phase of the clinical

trial Population Results Conclusion

Phase I Individual with

recurrent in- transit cutaneous melanoma with/without asymptomatic distant

- All clinical parameters assessed remained unchanged during the clinical trial [94]

- Presence of PAT-SM6 was detected in post- treatment biopsies from two of eight patients [94]

- Apoptosis was observed in biopsies from four patients [94]

PAT-SM6 was well tolerated: low/few treatment -emergent adverse events and no immunogenicity

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[89] (Table 1).

More recently, Al-Hashimi et al. synthesized a recombinant GRP78 antibody (AEP8587) that acts as a competitive antagonist of anti–N- terminus GRP78 autoantibodies. In vitro, AEP8587 competed with GRP78 autoantibody-driven induction of the UPR and tissue factor procoagulation activity associated with prostate cancer progression [90]. AEP8587 may represent a potential tool to manage prostate cancer evolution (Table 1).

Other GRP78 antibodies have been developed and patented for cancer therapy. GD-17 is a monoclonal antibody, specifically recognizing csGRP78, more specifically, the extracellular 376–415 amino acids of GRP78. Despite its rapid internalization, this antibody has interesting anti-oncogenic properties because of its cancer cell-specific cytotoxicity shown in vitro and in vivo (Table 1). 2D6F3 is another monoclonal antibody able to enhance radiation-induced cytotoxicity in breast adenocarcinoma and NSCLC cell lines (Table 1).

Mab159 is a monoclonal antibody that inhibits tumor progression in prostate, colon, lung cancer and leukemia models [91]. It blocks interaction between GRP78 and the regulatory subunits of PI3K p85, thus inhibiting the PI3K/AKT pathway and leading to decreased tumor proliferation, vessel density formation and metastasis [91]. Mab159 also decreases csGRP78 bioavailability by promoting its endocytosis, which activates caspase − 8 and − 9, thus resulting in cell death [91]. Despite pre-clinical evidence that humanized Mab159 has a favorable therapeutic window, owing to absence of toxicity in mice, stable antitumor activity and good pharmacokinetic parameters [91], clinical trials of MAb159 have not been reported.

To date, the only GRP78 antibody evaluated in clinical trials is PAT- SM6. Derived from a human IgM antibody named SAM-6, it was origi- nally isolated from a gastric cancer patient and described as part of the patient’s natural immunity [92]. The first in vitro studies using SAM-6 antibody were conducted on BXPC-3 pancreatic cancer cell lines and demonstrated that PAT-SM6 bound to low-density lipoprotein and that the pro-apoptotic response was caused by its endocytosis, thus leading to the accumulation of intracellular lipids, activation of caspases and release of cytochrome c by mitochondria [92,93] (Table 1). PAT-SM6 showed a strong anti-tumoral effect when initially tested in melanoma tumor-bearing mice [94]. Its safety and anti-tumor activity were then evaluated in a phase I clinical trial of a cohort of patients with malignant melanoma. Antibodies were detected in tumors of two out of eight patients, and apoptosis was seen in tumor biopsies from four patients;

however, these data were not sufficient to show a significant clinical effect [94].

Meanwhile, Rasche et al. described the effect of PAT-SM6 on multiple myeloma cell lines. Treatment with PAT-SM6 led to dose- dependent cell death, not only for these cell lines but also for primary multiple myeloma cells from patients, independent of disease stage (i.e., de novo or relapses) [95] (Table 1). The PAT-SM6 pro-apoptotic effect relied on a complement-dependent cytotoxicity mechanism and seemed to be tumor-specific because it did not affect healthy cells [95]. These results led to the initiation of a new phase I clinical trial of patients with relapsed multiple myeloma in 2012. The results showed a modest clinical effect, which suggested that combination with other agents may be necessary to obtain a more significant effect [96]. Further studies combined PAT-SM6 with other agents, dexamethasone and lenalidomide, and showed a synergistic cytotoxic activity in human myeloma cell lines. As proof of concept, PAT-SM6 was combined with bortezomib and lenalidomide in a 62-year-old man with triple-resistant multiple myeloma and led to partial remission of lesions [97] (Table 1). Never- theless, since 2012, there have been no reports of other clinical trials evaluating PAT-SM6 alone or combined with other treatments, most likely because of the lack of therapeutic effect of the antibody when used as a single agent as well as the short duration of clinical benefit, because progression-free survival was evaluated only at 2 months [97].

Table 1 (continued) Antibody GRP78 targeted part Effect on cells/ tumor Model Involved mechanism Patents metastatic disease

Phase I Individual with relapsed or refractory multiple myeloma Modest clinical activity in both groups of patients [96] PAT-SM6 was well tolerated. Further clinical trials required to evaluate its efficacy in combination with existing myeloma therapies Proof of concept

62-year-old patient with triple resistant

multiple myeloma. Use of combination of

PAT-SM6/ bortezomib/ lenalidomide Partial remission of intra and extra medullary lesions [97] Only 2-month-progression -free survival Other studies are required to identify effective doses and synergistic combination partners and to determine whether this strategy is limited to late-stage multiple myeloma

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4.2.3. GRP78 antibodies associated with nanocarriers or cytotoxic agents to specifically target cancer cells

To optimize the potential use of anti-GRP78 in cancer treatment, researchers turned to engineering, using GRP78 antibodies to target powerful cytotoxic agents to cancer cells. Semiconductor quantum dots (Qdots) have been explored for photodynamic therapy owing to their high stability and low toxic profile. As a payload, when delivered to a biological target, they are transformed into effective anti-cancer agents, inducing DNA damage and cell apoptosis. Qdot-GRP78 was engineered by conjugating Qdots to the ScFv fragment of a GRP78 antibody [98].

The resulting bioconjugate could not only target and penetrate into cancer cells but also decrease tumor growth by modulating the Akt pathway in mice [98].

A different approach was explored by developing paclitaxel-loaded nanoparticles coated with antibodies recognizing the C-terminus of GRP78. Particles functionalized with monoclonal antibodies showed higher binding to tumor cells and an enhanced effect of paclitaxel in ovarian and prostate cancer cells expressing GRP78 at their surface [99, 100]. The functionalization of nanoparticles was repeated with a polyclonal antibody also targeting the GRP78 C-terminus epitope and pre- viously characterized for its proapoptotic property. These nanoparticles significantly reduced tumor growth as compared with free paclitaxel or unfunctionalized paclitaxel-loaded nanoparticles in a chick chorioal- lantoic membrane model [45]. Thus, the use of anti-GRP78 antibody to functionalize a drug delivery system may enhance both the targeting and effect of paclitaxel in cancer cells.

Recently, the first nanobody directed against the C-terminus of GRP78 was designed to specifically bind cancer cells [101]. The antibody alone had no effect on cell viability; however, studies using nanobodies conjugated with cytotoxic agents [102] highlighted the potential therapeutic use of this nanobody directed against csGRP78. To date, clinical therapeutic applications for these compounds have not been reported.

5. Conclusion and future perspectives

Accumulating evidence has demonstrated the therapeutic potential of targeting csGRP78. Nevertheless, different strategies still need to be explored to take full advantage of the proapoptotic and antiproliferative properties of csGRP78. Recently, Gopal and Pizzo recommended that future research on csGRP78 biology emphasizes deciphering its impact on modulating downstream transcription factors [103] by using genomic analysis tools to understand the effect of csGRP78 on transcriptional control in malignant cells. Meanwhile, the design and development of csGRP78 targeting-based tools to offer a precise and highly controlled response remains essential. Several pharmacologic agents or functionalized peptides have been described, as reviewed here, but there have been only a few attempts to develop GRP78 monoclonal antibodies. Therapeutic antibodies have several advantages over small molecules or peptides, such as high affinity and specificity for the target and few drug–drug interactions and off-target adverse effects [104]. In addition, their half-life can reach several weeks as compared with smaller molecules, for which the half-life rarely exceeds 24 h [104].

Current bioengineering techniques allow for improving the properties of existing antibodies, and the development of bispecific antibodies pro- vides options to enhance their therapeutic potential [104]. At this time, GRP78 antibodies specifically designed to trigger the apoptotic and antiproliferative response in cancer cells represent the greatest potential for csGRP78-based therapies (Fig. 4).

Only one GRP78 antibody has been tested in human clinical trials (PAT-SM6). Two phase I clinical trials have involved patients with melanoma or multiple myeloma [94,96]. Unfortunately, the clinical results obtained are insufficient or have been overshadowed by the advent of more promising alternative therapies to justify further clinical studies. As demonstrated in this review, several types of solid tumor cells express GRP78 at their surface and could be the subject of future clinical trials. The clinical interest of using the GRP78 antibody in cancers for which other antibody-based therapy exists could be the development of tools allowing for targeting and drug delivery for personalized Fig. 4.Schematic representation of four different therapeutic strategies using GRP78 antibodies for treating cancer. 1) An antiproliferative or proapoptotic response is triggered when GRP78 antibodies bind to their target on the surface of tumor cells, 2) functionalized nanocarriers coated with GRP78 antibodies or antibody–drug conjugates are targeted specifically to tumor cells expressing csGRP78 for delivering their chemotherapy payload to malignant cells, 3) modulation of the activation of tumoral microenvironment macrophages by regulating csGRP78-mediated secreted factors and 4) modulation of the response to treatment by targeting csGRP78 on mononuclear cells surrounding the tumor. GRP78: glucose-related protein 78; PBMCs: peripheral blood mononuclear cells.