Multiple myeloma (MM) is a plasma cell malignancy. UPR plays a critical role in plasma cell differentiation and myeloma pathogenesis (67, 68). UPR is an evolutionally conserved mechanism that maintains protein quality control in the secretory pathway. Accumulation of misfolded proteins in the ER triggers the activation of three well-known pathways: activating transcription factor 6 (ATF6), the double-stranded RNA-activated protein kinase-like ER kinase (PERK), and the spliced form of X-box binding protein 1 (XBP1s). XBP1s and downstream ER chaperones are consistently upregulated in myeloma cells (69). Malignant plasma cells characteristically produce large amounts of proteins in the form of immunoglobulins that require ER chaperones such as GRP94 to prevent ER stress-induced cell death. Hua et al. found that the persistence of plasma cells and the development of myeloma in XBP1s-transgenic mice are critically dependent on GRP94. The addiction of myeloma cells to GRP94 was also demonstrated genetically and pharmacologically using multiple human myeloma cell lines (10). Mechanically, GRP94 is a critical chaperone for LRP6 and is essential for canonical Wnt signaling. LRP6 is required for the release of β-catenin from its destruction complex, accumulation of nuclear β-catenin, and upregulation of Wnt targets including survivin. Deletion of GRP94 compromises survivin expression, leading to its failure to safeguard mitotic spindles and the initiation of apoptosis (10, 70) through the activation of proapoptotic molecule CHOP, which causes downstream activation of the JNK and intrinsic caspase pathways that finally lead to apoptosis of tumor cells (71). Also, Chhabra et al. found that GRP94 is highly expressed in malignant plasma cells in MM. The higher level of GRP94 is significantly associated with a worse clinical stage in MM (34). These studies uncover the critical roles of GRP94 in the initiation and progression of MM, suggesting that blockade of GRP94 is a novel therapeutic strategy against this disease.

An early study showed that GRP94 is highly expressed in breast carcinoma cells but not in normal mammary tissue (26). CD44^hi/CD24^lo breast cancer stem cells represent the main driving factor in breast cancer initiation, growth, metastasis, and poor responses to anti-cancer agents. Nami et al. found that CD44^hi/CD24^lo cells exhibited higher expression of GRP94 at both mRNA and protein levels compared to their original bulk cells (72). Dejeans et al. found that overexpression of GRP94 in breast cancers is associated with resistance to oxidative stress and the promotion of cancer cell proliferation and migration (4). The expression level of GRP94 was higher in recurrent human breast cancers than in their paired primary tumor (29). Recent studies also showed that GRP94 overexpression is associated with brain metastasis and poor survival in breast cancer patients (73, 74). Upregulation of the GRP94 is associated with triple-negative breast cancer brain metastasis through the Wnt-β-catenin signaling pathway (75). A recent study showed that GRP94 promoted brain metastasis by engaging pro-survival autophagy (76). Patients with infiltrated axillary lymph nodes also displayed increased expression of GRP94 protein.

Recently, Hou et al. found that GRP94 also regulated ER-α36 expression and signaling on the cell membrane of breast cancer. ER-α36 is a variant of human ERα that is overexpressed in breast cancer and involved in tamoxifen resistance. Targeting GRP94 with siRNA or monoclonal antibody blocked the GRP94-ER-α36 interaction and inhibited breast cancer growth and invasion (57). Also, GRP94 can control the stability of the nascent and mature forms of HER2, an RTK, which leads to the upregulation of numerous cancer-driving signaling pathways (77). GRP94 specific inhibitors provide evidence for the role of GRP94 in maintaining the architecture of high-density HER2 formations at the plasma membrane, which is vital for proper HER2 functioning in breast cancers (56). Targeting GRP94 with a specific monoclonal antibody or peptide-based inhibitor p37 disrupted HER2 dimerization and led to HER2 degradation, which subsequently decreased tumor cell growth and increased apoptosis (57, 78). These results provide insights into the dependence of breast cancer cells on GRP94 for survival and suggest that GRP94 could be a potential therapeutic target for HER2 or ER-α36-overexpressing breast cancer.

The intestinal epithelial cells are continually replenished through the proliferation and differentiation of intestinal stem cells within the intestinal crypts (79). The interplay between ER stress and inflammation contributes to the pathogenesis of inflammatory bowel diseases and inflammation-associated colon cancer (80). Canonical Wnt signaling through the surface receptor Frizzled and its coreceptors LRP5 or LRP6 is essential for the homeostatic proliferation of epithelial cells in the gut. Unsurprisingly aberrant Wnt/β-catenin signaling also plays a role in the initiation and progression of colorectal cancer, as well as its invasion and metastasis (81). Epithelial-specific GRP94 deficiency resulted in the loss of intestinal barrier function in mice dependent on canonical LRP6/Wnt-signaling (9). This study uncovered the role of GRP94 in chaperoning LRP6-MesD to coordinate intestinal homeostasis, placing the canonical Wnt-signaling pathway under the direct regulation of the general protein quality control machinery in the ER (9).

Macrophages are important drivers in the development of inflammation-associated colon cancers. Using a unique macrophage-specific GRP94 deficient mouse model, Morales et al. found that macrophages promoted colitis and colitis-associated colon tumorigenesis in a GRP94-dependent manner. Strikingly, they also discovered that deletion of GRP94 in macrophages attenuated colon tumor initiation, which was correlated with reduced mutation rates of β-catenin, reduced activation of the canonical Wnt signaling, and increased efficiency of the DNA repair machinery as well as reduced expression of pro-inflammatory cytokines, including IL-17 and IL-23 in the tumor microenvironment (23). These studies reveal that GRP94 is a strategically important ER chaperone that integrates stress and innate immunity and plays a pivotal role in macrophage biology and tumor oncogenesis. Besides, GRP94 is upregulated in human metastatic colorectal cancer (28). Therefore, targeting GRP94 in macrophages may prove to be an attractive strategy for the treatment of colon cancer by regulating the Wnt and inflammatory signaling in the tumor microenvironment.

The liver is an exocrine and endocrine organ involved in the synthesis of bioactive molecules and proteins, detoxification, and metabolism, resulting in a particularly high need for adequate ER maintenance and stress-coping mechanisms. Hence, the loss of GRP94 in hepatocytes would drastically affect normal physiological functions. Chen et al. demonstrated that conditional deletion of GRP94 and PTEN from the mouse liver increased liver tumorigenesis (20). However, the GRP94 status of these tumors was not reported, leaving open a possibility of GRP94 being either a tumor suppressor or a pro-oncogenic chaperone (21). Using a similar strategy, Rachidi et al. (22) found that GRP94 maintains liver development and hepatocyte function in vivo. Deletion of GRP94 in hepatocytes promotes adaptive accumulation of long-chain ceramides, accompanied by steatosis and regeneration of residual GRP94⁺ hepatocytes. The need for compensatory expansion of GRP94⁺ cells in the GRP94^- background predisposes mice to develop carcinogen-induced hepatic hyperplasia and cancer from GRP94⁺ but not GRP94^- hepatocytes. They also demonstrated that both genetic and pharmacological inhibitions of GRP94 in human hepatocellular carcinoma cells perturbed multiple growth signals and attenuated their proliferation and expansion. The development of GRP94⁺ but not GRP94^- tumors in the same hosts indicated that GRP94 played tumor-promoting rather than a tumor-suppressive role in liver cancer (21). Similar results were found in another study. Deletion of GRP94 resulted in liver injury, activation of oncogenic signaling, repopulation of GRP94⁺ hepatocytes, and spontaneous development of hepatocellular carcinoma in aged mice (82).

Additionally, Lim et al. showed that the expression of GRP94 is up-regulated in hepatitis B virus-related hepatocellular carcinoma in humans, and is strongly correlated with vascular invasion and intrahepatic metastasis (83). Wei et al. also found that knockdown of GRP94 inhibited the growth, invasion, and metastasis of hepatocellular carcinoma cells in vivo and in vitro. The high expression of GRP94 in tumors indicated poor survival (84, 85). These studies show the oncologenic role of GRP94 and its potential as a prognostic indicator of liver cancer.

Cigarette smoking is the most relevant environmental risk factor associated with chronic obstructive pulmonary disease (COPD) and lung malignancies. It is well established that cigarette smoke induces ER stress, which activates UPR, and COPD subjects have intense ER stress evidenced by high expression of ER stress markers in fully differentiated human bronchial epithelial cells (86). Chronic ER stress upon exposure to cigarette smoking or other causative agents may play a pivotal role in the etiology or progression of lung cancers (87). UPR activation and enhanced ER chaperon translation, including GRP94 may promote lung cancer progression.

Wang et al. revealed that GRP94 is overexpressed in lung cancer at the mRNA and protein level and correlated with poor epithelial differentiation and tumor progression (88). Lee et al. showed that 98% of small cell lung cancer patients were GRP94 positive with moderate to high expression (27). GRP94 was also highly expressed in non-small cell lung cancer patients with brain metastasis (74). Furthermore, the upregulation of GRP94 by the interference of calcium stores can confer drug resistance to lung cancer cells against the chemotherapy agent etoposide (89). A recent study demonstrated that GRP94 is highly expressed in lung adenocarcinoma and is associated with an advanced stage of the disease as well as poor survival. Also, the GRP94 expression level was positively correlated with FoxP3⁺ regulatory T cells in tumors (90). Hence, targeting GRP94 will provide a new therapeutic approach to the clinical management of lung cancer with chemo-resistance.

Elevated expression of GRP94 correlates with the aggressiveness of numerous other tumors, including esophageal (91), gastric (92), and pancreatic cancers (93) as well as oral carcinoma (94) and glioblastoma (95), indicating that GRP94 plays a tumor-promoting role in many different cancers. Shen et al. generated a uterus-specific GRP94 knockout mouse model and discovered that GRP94 suppressed PTEN-null driven endometrioid adenocarcinoma. Deletion of GRP94 reduced cellular proliferation through attenuating β-catenin signaling and decreasing AKT/S6 activation (25).

Tramentozzi et al. found that GRP94 was highly expressed in both gastrointestinal tumor tissues and tumor-infiltrating lymphocytes regardless of stage or anatomical location. GRP94 was also found in the plasma in stable complexes with Immunoglobulin G (IgG). The study showed that GRP94-IgG complexes are significantly increased in cancer patients compared to healthy control subjects, suggesting its potential as a diagnostic biomarker. GRP94 is over-expressed in a wide variety of both solid and hematological tumors and correlates with poor outcomes, which indicates that GRP94 is a potential diagnostic and prognostic biomarker.

Mounting evidence indicates that the surface expression of GRP94/GP96 plays an integral role in shaping the immune landscape of tumors. Melendez et al. found that surface expression of GRP94 in malignant breast cells correlates with NK-mediated cytotoxicity, and the use of a GRP94 blocking antibody protected tumor cells from NK cytotoxicity (96). Zheng et al. found that overexpressed surface GRP94 on colon cancer and fibrosarcoma cells are capable of inducing maturation of dendritic cells leading to the release of proinflammatory cytokines and upregulation of antigen presentation machinery (64). Similarly, another study found that the administration of a GRP94 and Her2/neu DNA vaccine to HER2⁺ breast cancer-bearing mice led to an increased immune response against the tumors evidenced by increased IFN-γ/IL-4 levels and decreased Tregs at the tumor site (97). These studies reveal the ability of extra-cellular GRP94 to induce anti-tumor immune responses, and highlighting its potential to be used as an antigen for tumor vaccines ( Table 1 ).

Immunization of tumor-bearing mice with GRP94-peptide complexes has been shown to generate effective immune responses in numerous pre-clinical and clinical settings (99, 106). Autologous tumor-derived GRP94/GP96 significantly attenuated tumor growth and improved survival in various spontaneous and carcinogen-induced cancer models. The effectiveness of the treatment was dependent on the presence of CD4⁺ and CD8⁺ T cells and NK cells (106). Similarly, placental derived GRP94 was found to bind to tumor-associated antigens such as HER2 and MUC1 and induce tumor-specific T cell responses (98). The therapeutic effectiveness of GRP94 complexes likely results from their ability to improve the uptake and processing of tumor antigens. Consequently, Shinagawa et al. found that delivering GRP94 complexes directly to dendritic cells ex vivo before adoptive transfer of DCs into tumor-bearing mice led to improved tumor control compared to treatment with DCs or tumor-derived GRP94 complexes alone (102). Fromm et al. reported that the secreted form of GRP94 (GRP94-Ig) and costimulator combination cellular vaccine increased antigen-specific CD8 and memory T cells, delayed melanoma tumor growth, and prolonged overall survival (101) ( Table 1 ).

GRP94 is expressed in most human cells. Therefore, specificity for cancer cells is a major concern when designing small molecular inhibitors. Identification of geldanamycin (GM) revealed the possibility of selectively targeting HSP90 within tumor cells. A more recent study showed that the purine scaffold small molecule DN401 simultaneously inhibited all HSP90 paralogs, including HSP90, TRAP1, and GRP94, and synergized the anti-tumor effects with another TRAP1 inhibitor, gamitrnib (107). However, pan HSP90 inhibitors inactivate all HSP90 isoforms, including GRP94, and display severe toxicity. To improve selectivity and further reduce toxicity, Patel et al. developed novel purine-based ligands that are greater than 100-fold more selective for GRP94 over HSP90α/β (56). Based on a strategy that combined library screening of purine-scaffold compounds and structural studies, GRP94-specific inhibitors, PU-WS13, PU-H39, and PU-H54, were developed, and these inhibitors significantly induced apoptosis of human HER2 positive breast cancer cells (56). Also, Hua et al. found that PU-WS13 significantly inhibited the growth of multiple human MM cells, including those that are resistant to cytosolic HSP90 inhibitor PU-H71, doxorubicin, and proteasome inhibitor bortezomib in vitro (10). Recently, based on an extensive Structure–Activity relationship study, Patel et al. designed a new GRP94-specific inhibitor, compound 18c. Specifically, 18c inhibited the post-ER expression of TLR9, and also reduced the steady-state levels of HER2 kinase in HER2-overexpressing breast cancer cells. When administered into tumor-bearing mice, compound 18c was cleared rapidly from normal tissue while being selectively retained in the tumor (108). Crowley et al. developed another series of GRP94-selective inhibitors (103). These small molecular inhibitors reduced the migratory capabilities of metastatic breast and prostate cancer cells through the degradation of integrin α2 and with better selectivity than their previous reported compounds (109–112) ( Table 1 ). Recently, the Xu group developed a GRP94 selective inhibitor, Compound 54, with an IC50 of 2 nM and over 1,000-fold selectivity for GRP94 versus HSP90α. They found that this compound has anti-inflammation efficacy in the DSS-induced colitis mouse model (113). However, Compound 54 hasn’t been tested in the treatment of cancers.

The therapeutic targeting of surface GRP94 in tumors has emerged with the recent development of specific monoclonal antibodies that have potent GRP94 inhibitory effects and consequent anti-tumor activities (114). Li et al. developed a GRP94 monoclonal antibody that interfered with GRP94-dependent HER2 dimerization and phosphorylation in breast cancer and suppressed the HER2⁺ breast cancer cell growth in vitro and in vivo (57, 78). Moreover, Sabbatino et al. developed a novel grp94-antagonizing monoclonal antibody, W9 mAb, which selectively targets the extracellular epitope of GRP94 in malignant cells but is not detectable on normal cells. W9 mAb increased the sensitivity of human BRAF^V600E melanoma cells to BRAF inhibitors (104). Using a phage display approach, Jeoung et al. isolated an antibody binding to the cell surface of human colon cancer cells. Furthermore, they demonstrated that this antibody specifically targeted GRP94 and inhibited the growth of Cetuximab-resistant colorectal cancer (105) ( Table 1 ). These studies lay a strong foundation for developing GRP94-targeted therapeutics for cancers in the future.