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Review GRP78 Induction in Cancer: Therapeutic and Prognostic Implications Amy S. Lee Department of Biochemistry and Molecular Biology, University of Southern California/Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California Abstract Cancer cells adapt to chronic stress in the tumor microenvironment by inducing the expression of GRP78/BiP, a major endoplasmic reticulum chaperone with Ca2+-binding and antiapoptotic properties. GRP78 promotes tumor proliferation, survival, metastasis, and resistance to a wide variety of therapies. Thus, GRP78 expression may serve as a biomarker for tumor behavior and treatment response. Combination therapy suppressing GRP78 expression may represent a novel approach toward eradication of residual tumors. Furthermore, the recent discovery of GRP78 on the cell surface of cancer cells but not in normal tissues suggests that targeted therapy against cancer via surface GRP78 may be feasible. [Cancer Res 2007;67(8):3496–9] Endoplasmic Reticulum Stress and Cancer The endoplasmic reticulum (ER) is an essential perinuclear organelle for the synthesis and folding of secretory and membrane proteins, which accounts for about one third of the cell’s proteins. When the protein load exceeds the folding capacity of the ER, the cells trigger the unfolded protein response (UPR), which activates the PERK, IRE1/X-box binding protein-1 (XBP-1), and activating transcription factor-6 (ATF6) signaling pathways as protective measures, resulting in general translational attenuation, up-regulation of chaperones and folding enzymes, and enhanced ER-associated degradation of malfolded proteins (1, 2). Depending on the severity of ER stress, the UPR can result in cell death through the activation of apoptotic pathways mediated specifically by the ER, as well as coupling with the mitochondrial pathways (2, 3). ER stress also induces autophagy, a cellular degradation process implicated in both cell death and survival (4). Cancer cells are subject to ER stress because of both intrinsic and extrinsic factors (5). Cancer cells exhibit elevated glucose metabolism with increased glycolytic activity, and solid tumors often grow faster than their blood supply. The latter creates a tumor microenvironment characterized by glucose deprivation, acidosis, and severe hypoxia. These combined factors leads to the accumulation of underglycosylated and misfolded proteins in the ER, triggering the UPR (Fig. 1). In xenograft models, XBP-1 is required for survival under hypoxic conditions and tumor growth, whereas PERK confers advantage for tumor growth (6, 7). Another major UPR adaptive survival response is the induction of ER Requests for reprints: Amy S. Lee, Department of Biochemistry & Molecular Biology and the University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176. Phone: 323-865-0507; Fax: 323-865-0094; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-0325 Cancer Res 2007; 67: (8). April 15, 2007 chaperone GRP78 in the tumor microenvironment (Fig. 1), which is the focus of this review. GRP78 Is a Key Survival Factor in Development and Cancer The glucose-regulated protein GRP78, also referred to as BiP (immunoglobulin heavy-chain binding protein), was discovered in the late 1970s together with GRP94 and GRP58 as cellular proteins induced by glucose starvation (1). Residing primarily in the ER, GRP78 belongs to the HSP70 protein family, which plays critical roles in the stress of oncogenesis. In addition to facilitating proper protein folding, preventing intermediates from aggregating, and targeting misfolded protein for proteasome degradation, GRP78 also binds Ca2+ and serves as an ER stress signaling regulator (1, 8). In nonstressed cells, GRP78 binds to ER transmembrane sensor proteins PERK, IRE1, and ATF6 and maintains them in an inactive form. When unfolded proteins pull GRP78 away from them, these pathways are activated, sending signals to the nucleus to trigger the UPR. GRP78 is induced by physiologic stress that perturbs ER function and homeostasis, protecting against tissue or organ damage under pathologic conditions such as neurotoxic stress, myocardial infarction, and arteriosclerosis (1). During mouse development, homozygous disruption of the Grp78 allele results in early embryonic lethality (9). Transcription of Grp78 is detectable as early as the two-cell stage and is required for both proliferation and survival for the embryonic inner cell mass, which are precursors of the pluripotent stem cells. Through synergistic interaction with cardiac specific transcription factor, Grp78 transcription is strongly induced in early embryonic heart, which uses glucose as the major energy source (10). Although GRP78 expression is maintained at low basal level in major adult organs such as the brain, lung, and heart, it is strongly induced in tumors (3, 11). In support of the notion that GRP78 is more critically needed for the survival of stressed cells such as cancer, heterozygous GRP78 mice with half of wild-type (WT) GRP78 level are comparable to WT siblings in growth and development. However, tumor progression was significantly impeded in these mice as exemplified by a longer latency period, reduced tumor size, and increased tumor apoptosis.1 This is consistent with earlier studies that GRP78 conferred lysis resistance to cytotoxic T cells and tumor necrosis factor a, and that reduction of GRP78 in xenografts inhibited tumor formation and growth. GRP78 may also be (12) important for tumor metastasis because it is elevated in metastatic cancer cell lines, lymph node metastasis, and knockdown of GRP78 inhibits tumor cell invasion in vitro and growth and metastasis in xenograft models (13, 14). The mechanism whereby GRP78 promotes growth and metastasis is just emerging. In addition to stress tolerance mediated by ER lumen GRP78, it has been reported that 3496 1 A. S. Lee, manuscript in preparation. www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 2007 American Association for Cancer Research. GRP78: Therapeutic and Prognostic Implications GRP78 is detected on the surface of highly metastatic prostate cancer cells and may mediate signal transduction pathways that induce proliferation and invasion (15). Therapeutic Implications of GRP78 Induction in Cancer Although therapeutic agents that target the tumor vasculature starve the tumors of vital nutrients, it has the unintended consequence of GRP78 induction. In xenograft models treated with antivascular and antiangiogenesis agents, GRP78 induction, which is hypoxia-inducible factor independent, is most intense in the viable tumor cells bordering necrotic regions created by the therapy (16). These cells are highly chemoresistant, correlating with GRP78 overexpression and inhibition of apoptosis. How might GRP78 suppress apoptosis? As a molecular chaperone, it prevents the formation of malfolded proteins. As a Ca2+-binding protein, it preserves ER calcium homeostasis. Interestingly, whereas the majority of GRP78 resides in the ER lumen, a fraction of GRP78 exists as an ER transmembrane protein, with its N-amino portion in the cytosol (17). This could provide an explanation why GRP78 may be able to directly inhibit the activity of proapoptotic effectors localized to the ER. For example, GRP78 binds and inhibits the activation of caspase-7, an executor caspase activated by both ER stress and genotoxic drugs (5, 17–19). GRP78 also binds and suppresses the activation of the BH-3 only proapoptotic protein BIK, its downstream target BAX, and prevents cytochrome c release from the mitochondria (20, 21). Furthermore, GRP78 in complex with other ER transmembrane proteins may also indirectly modulate the activity of these and other proapoptotic components. Thus, in multiple tumor types including lung, bladder, stomach, breast, gastric, and epidermoid carcinoma, GRP78 overexpression confers resistance to a wide variety of chemotherapeutic agents, and knockdown of GRP78 sensitizes the tumor cells to drug treatment (5). In malignant glioma, which represents a most malignant and resistant form of cancer, GRP78 is highly elevated, and knockdown of GRP78 enhances the efficacy of Temozolomide, the current standard of care for such cancer.2 GRP78-mediated drug resistance is not limited to proliferating tumor cells. Knockdown of GRP78 induces strong killing of growtharrested, dormant cancer cells treated with Adriamycin/doxorubicin, a topoisomerase inhibitor, suggesting that dormant cells rely on GRP78, rather than cell cycle arrest, a commonly held concept, for drug resistance (20). Similarly, quiescent tumor-associated endothelial cells also depend on GRP78 for resistance because knockdown of GRP78 greatly enhances its drug sensitivity.3 These new findings imply that drugs against GRP78 will be particularly potent to eradicate residual tumor because they can overcome drug resistance not only in proliferating cancer cells, but also in dormant cancer cells, as well as in nontumor cells supporting tumor growth (Fig. 1). GRP78 as a Therapeutic Target and Mediator of Cancer-Specific Therapy Given the importance of GRP78 in cancer cell survival, it represents a prime target for anticancer agents. Interestingly, several 2 P. Pyrko, A.H. Schönthal, F.M. Hofman, T.C. Chen, A.S. Lee. The unfolded protein response regulator GRP78 as a novel target for increasing chemosensitivity in malignant gliomas, submitted for publication. 3 F.M. Hofman, J. Virrey, D. Dong, et al. GRP78 confers chemoresistance to gliomaassociated brain endothelial cells, submitted for publication. www.aacrjournals.org naturally occurring compounds with putative anticancer activity inhibit either GRP78 expression or its activity at pharmacologic concentrations (Fig. 1). This includes genistein, an active ingredient of soy, ( )-epigallocatechin gallate (EGCG), a green tea component, and salicyclic acid from plants (18, 22, 23). Nonetheless, these compounds act on many cellular targets and pathways in addition to GRP78. In contrast, the highly lethal bacterial AB5 subtilase cytoxin specifically cleaves GRP78 at a single amino acid, raising the interesting idea of exploiting this for anticancer therapy (24). Furthermore, GRP78 is an intracellular target for the melanoma differentiation–associated gene-7/interleukin 24 (MDA7/IL-24) produced by immune cells, which induces cancer-selective growth suppression and apoptosis in a wide range of human cancers (25). Screening of compounds from microbes that specifically suppress the activation of the GRP78 promoter identified versipelostatin, which has no effect on basal GRP78 expression but inhibits transcriptional activation of the Grp78 gene by glucose starvation (26). This macrocyclic compound, which also disrupts some components of the UPR, selectively kills glucose-deprived cancer cells and acts synergistically with cisplatin in inhibiting tumor growth in xenografts. These observations, although preliminary and require more work on drug development, provide the proof of principle that inhibitors of GRP78 can be used in combination with standard therapeutic agents to enhance drug efficacy and possibly eliminate residual resistant tumor. The recent discovery that GRP78 can be found on the cell surface of tumors but not in normal organs opens up an exciting opportunity of targeting cell surface GRP78 function as well as using it as a cancer-targeting marker (Fig. 1). How GRP78 escapes to the cell surface in tumor cells is not well understood, but it may involve oversaturation of the ER retention system, cotrafficking with cell surface client proteins, ER transmembrane GRP78 cycling to the cell surface, as well as specific mechanisms adapted by tumor cells. Cell surface GRP78, as a high-affinity receptor for activated a2-macroglobulin, is postulated to promote proliferation, survival, and metastasis of prostate cancer cells (15). As proof of principle that cell surface GRP78 can serve as a conduit for cancerspecific delivery of cytotoxic agents, systemic administration of synthetic chimeric peptides with GRP78 binding motifs fused to proapoptotic sequence–suppressed tumor growth without affecting normal tissues (27). Similarly, other GRP78 targeting peptides, when linked to taxol, induced apoptosis in the targeted cancer cells (28). Another major advance is that cell surface GRP78 is the receptor for the angiogenesis inhibitor Kringle 5 (K5) of human plasminogen (19). Following internalization, K5 blocked the antiapoptotic activity of GRP78 and induced cell death. Although the primary target for K5 is likely to be growth-stimulated endothelial cells supporting tumor progression, recombinant K5 also induced apoptosis in stressed fibrosarcoma cells where surface GRP78 was also detected (19). Therefore, in principle, this class of drugs, which are currently tested in clinical trials, should have dual antiangiogenic and antitumor activities while sparing normal organs and tissues. GRP78 as a Novel Biomarker for Tumor Behavior and Responsiveness to Therapy In human cancers, elevated GRP78 level generally correlates with higher pathologic grade, recurrence, and poor patient survival in breast, liver, prostate, colon, and gastric cancers; however, there are differing reports on lung cancer and an apparent exception for 3497 Cancer Res 2007; 67: (8). April 15, 2007 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 2007 American Association for Cancer Research. Cancer Research Figure 1. ER stress induction of GRP78 in the tumor microenvironment. Both intrinsic and extrinsic factors lead to the up-regulation of GRP78 (also referred to as BiP) and its cell surface expression in tumor cells. Through inhibition of apoptosis, GRP78 facilitates tumor progression, immune resistance, metastasis, and drug resistance. Dormant tumor cells, as well as quiescent tumor endothelial cells, also rely on GRP78 to escape chemotherapy. Anticancer compounds that either inhibit the stress induction of GRP78 or suppress its catalytic function have been identified from multiple sources. When used in combination therapy, they should enhance drug efficacy, lower resistance and assist in eradicating residual tumor. GRP78 has also been identified as a cell surface receptor for Kringle 5 of human plasminogen and the activated form of the a2-macroglobulin. Because cell surface GRP78 is not detected in normal organs, it can serve as a conduit for cancer-specific delivery of cytotoxic agents via GRP78 binding peptides. Autoantibody levels against GRP78 in patient serum, as well as expression levels of GRP78 in biopsies, may represent novel biomarkers in stratifying patients for tumor behavior and responsiveness to treatment. Abbreviations: N, nucleus; ER, endoplasmic reticulum; C, cytoplasm. neuroblastoma that requires further investigation (13, 14). Autoantibodies against GRP78 at high levels in prostate cancer patients have also been reported, associating with aggressive tumor behavior (29, 30). Furthermore, expression of GRP78 was recently examined retrospectively in prostate cancer patients during the development of castration resistance (31). GRP78 is strongly upregulated during the transition from localized prostate cancer to metastatic castration and may serve as a novel prognostic indicator of recurrence in untreated patients with localized tumor. In the management of breast cancer patients, there are at present only two biomarkers that are used to predict potential benefits of adjuvant therapy for the disease, hormone receptor status, and Her2/neu status. The utility of these biomarkers to eliminate ineffective treatment cannot be underestimated. For women with hormone receptor–negative tumors, adjuvant hormonal therapy will not reduce the risk of recurrence. Therefore, such women may be spared the toxicities of such agents. Similarly, patients whose tumors do not overexpress Her2/neu may be spared treatment with Herceptin. Unfortunately, similar tests that would predict benefit from adjuvant systemic chemotherapy Cancer Res 2007; 67: (8). April 15, 2007 agents do not exist. Such tests would be useful in avoiding the toxicity associated with chemotherapy in patients who would not benefit from treatment with these agents. Based on preclinical studies strongly suggesting that GRP78-positive tumors may be resistant to topoisomerase inhibitors (5, 16–20), a retrospective study was conducted to evaluate the value of GRP78 as a biomarker for treatment response. The study revealed that twothirds of breast cancer patients show moderate to high levels of GRP78 in biopsies before treatment, and that in patients who received adjuvant systemic chemotherapy with Adriamycin-based regimens, GRP78 positivity indicated a higher risk of recurrence (32). Thus, upon validation, GRP78 positivity might identify patients who could be spared the toxicities of Adriamycin-based adjuvant chemotherapy. Another observation that warrants additional investigation is whether GRP78 positivity might also identify patients who are more likely to benefit from treatment with Adriamycin followed by taxanes (32). In preclinical studies with estrogen-positive human breast cancer cells, GRP78 confers resistance to estrogen starvation–induced apoptosis through suppressing the activity of BIK (21). Thus, GRP78 level may also 3498 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 2007 American Association for Cancer Research. GRP78: Therapeutic and Prognostic Implications provide guidance on selection of patients who will benefit from antiestrogen and aromatase inhibitor therapy. Although it should be emphasized that most human cancers are relatively heterogeneous diseases and many factors need to be considered when building models to predict outcome, if our findings on GRP78 are confirmed, the integration of testing GRP78 expression level into the management strategies of breast cancer patients would substantially enhance treatment by reducing exposure to agents that are not likely to be beneficial to the patient (Fig. 1). This approach should also be applicable to other forms of cancer. Concluding Thoughts and Future Directions Elimination of residual tumor remains a major challenge for cancer therapy. The recent explosion in new knowledge on GRP78 suggests that it may represent a unifying mechanism for tumor resistance and a novel predictive biomarker to guide patient treatment, and this warrants vigorous testing in preclinical and clinical settings. The exciting recent discoveries also raise new important questions. How does GRP78 regulate tumor growth as References 1. Lee AS. The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci 2001; 26:504–10. 2. Ma Y, Hendershot LM. The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer 2004;4:966–77. 3. Li J, Lee B, Lee AS. Endoplasmic reticulum stressinduced apoptosis: multiple pathways and activation of p53–up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem 2006;281:7260–70. 4. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005;5:726–34. 5. Li J, Lee AS. Stress induction of GRP78/BiP and its role in cancer. Curr Mol Med 2006;6:45–54. 6. Romero-Ramirez L, Cao H, Nelson D, et al. XBP1 is essential for survival under hypoxic conditions and is required for tumor growth. Cancer Res 2004;64:5943–7. 7. Fels DR, Koumenis C. The PERK/eIF2a/ATF4 module of the UPR in hypoxia resistance and tumor growth. Cancer Biol Ther 2006;5:723–8. 8. Hendershot LM. The ER function BiP is a master regulator of ER function. Mt Sinai J Med 2004;71:289–97. 9. Luo S, Mao C, Lee B, Lee AS. GRP78/BiP is required for cell proliferation and protecting the inner cell mass from apoptosis during early mouse embryonic development. Mol Cell Biol 2006;26:5688–97. 10. Mao C, Tai WC, Bai Y, Poizat C, Lee AS. In vivo regulation of Grp78/BiP transcription in the embryonic heart: role of the endoplasmic reticulum stress response element and GATA-4. J Biol Chem 2006;281:8877–87. 11. Dong D, Dubeau L, Bading J, et al. Spontaneous and controllable activation of suicide gene expression driven by the stress-inducible grp78 promoter resulting in eradication of sizable human tumors. Hum Gene Ther 2004;15:553–61. 12. Jamora C, Dennert G, Lee AS. Inhibition of tumor progression by suppression of stress protein GRP78/BiP induction in fibrosarcoma B/C10ME. Proc Natl Acad Sci U S A 1996;93:7690–4. www.aacrjournals.org well as invasion and metastasis? Does GRP78 also play important roles in the survival and function of nontumor cells supporting cancer growth and recurrence? Are there specific adaptive mechanisms for GRP78 cell surface expression in certain cancer cells? Finally, to test the therapeutic value of GRP78, it is critical to identify, through targeted screen or rational design, small-molecule inhibitors that are potent and specific modulators of GRP78 expression and/or activity. One approach is to target the catalytic activity of GRP78, which is required for its function. The recent establishment of conditional knock-out models of GRP78 will also provide further insight into the regulation and function of GRP78 in various types of cancers and other human diseases. Acknowledgments Received 1/24/2007; revised 2/28/2007; accepted 3/2/2007. Grant support: National Cancer Institute grants CA027607 and CA111700, the Susan G. Komen Foundation, and the L.K. Whittier Foundation. I thank Darcy Spicer, Susan Groshen, Peter Baumeister, Miao Wang, and members of the Lee laboratory for helpful discussions and assistance. Due to space limitations, I apologize that many important primary articles cannot be cited. 13. Fu Y, Lee AS. Glucose regulated proteins in cancer progression, drug resistance and immunotherapy. Cancer Biol Ther 2006;5:741–4. 14. Zhang J, Jiang Y, Jia Z, et al. Association of elevated GRP78 expression with increased lymph node metastasis and poor prognosis in patients with gastric cancer. Clin Exp Metastasis 2006;23:401–10. 15. Misra UK, Deedwania R, Pizzo SV. Activation and cross-talk between Akt, NF-nB, and unfolded protein response signaling in 1-LN prostate cancer cells consequent to ligation of cell surface-associated GRP78. 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Functional coupling of p38-induced upregulation of BiP and activation of RNA-dependent protein kinase-like endoplasmic reticulum kinase to drug resistance of dormant carcinoma cells. Cancer Res 2006;66:1702–11. 21. Fu Y, Li J, Lee AS. GRP78/BiP inhibits endoplasmic reticulum BIK and protects human breast cancer cells against estrogen-starvation induced apoptosis. Cancer Res 2007;3734–40. 3499 22. Zhou Y, Lee AS. Mechanism for the suppression of the mammalian stress response by genistein, an anticancer phytoestrogen from soy. J Natl Cancer Inst 1998;90:381–8. 23. Deng WG, Ruan KH, Du M, Saunders MA, Wu KK. Aspirin and salicylate bind to immunoglobulin heavy chain binding protein (BiP) and inhibit its ATPase activity in human fibroblasts. FASEB J 2001;15:2463–70. 24. Montecucco C, Molinari M. Microbiology: death of a chaperone. Nature 2006;443:511–2. 25. Dent P, Yacoub A, Grant S, Curiel DT, Fisher PB. MDA-7/IL-24 regulates proliferation, invasion and tumor cell radiosensitivity: a new cancer therapy? J Cell Biochem 2005;95:712–9. 26. Park HR, Tomida A, Sato S, et al. Effect on tumor cells of blocking survival response to glucose deprivation. J Natl Cancer Inst 2004;96:1300–10. 27. Arap MA, Lahdenranta J, Mintz PJ, et al. Cell surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands. Cancer Cell 2004;6:275–84. 28. Kim Y, Lillo AM, Steiniger SC, et al. Targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand. Biochemistry 2006;45:9434–44. 29. Mintz PJ, Kim J, Do KA, et al. Fingerprinting the circulating repertoire of antibodies from cancer patients. Nat Biotechnol 2003;21:57–63. 30. Gonzalez-Gronow M, Cuchacovich M, Llanos C, Urzua C, Gawdi G, Pizzo SV. Prostate cancer cell proliferation in vitro is modulated by antibodies against glucose-regulated protein 78 isolated from patient serum. Cancer Res 2006;66:11424–31. 31. Pootrakul L, Datar RH, Shi SR, et al. Expression of stress response protein Grp78 is associated with the development of castration-resistant prostate cancer. Clin Cancer Res 2006;12:5987–93. 32. Lee E, Nichols P, Spicer D, Groshen S, Yu MC, Lee AS. GRP78 as a novel predictor of responsiveness to chemotherapy in breast cancer. Cancer Res 2006;66: 7849–53. Cancer Res 2007; 67: (8). April 15, 2007 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 2007 American Association for Cancer Research. GRP78 Induction in Cancer: Therapeutic and Prognostic Implications Amy S. Lee Cancer Res 2007;67:3496-3499. Updated version Cited articles Citing articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/67/8/3496 This article cites 31 articles, 17 of which you can access for free at: http://cancerres.aacrjournals.org/content/67/8/3496.full.html#ref-list-1 This article has been cited by 77 HighWire-hosted articles. Access the articles at: /content/67/8/3496.full.html#related-urls Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 2007 American Association for Cancer Research.