Biquan Luo

The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies

Cancer progression is characterized by rapidly proliferating cancer cells that are in need of increased protein synthesis. Therefore, enhanced endoplasmic reticulum (ER) activity is required to facilitate the folding, assembly and transportation of membrane and secretory proteins. These functions are carried out by ER chaperones. It is now becoming clear that the ER chaperones have critical functions outside of simply facilitating protein folding. For example, cancer progression requires glucose regulated protein (GRP) 78 for cancer cell survival and proliferation, as well as angiogenesis in the microenvironment. GRP78 can translocate to the cell surface acting as a receptor regulating oncogenic signaling and cell viability. Calreticulin, another ER chaperone, can translocate to the cell surface of apoptotic cancer cells and induce immunogenic cancer cell death and antitumor responses in vivo. Tumor-secreted GRP94 has been shown to elicit antitumor immune responses when used as antitumor vaccines. Protein disulfide isomerase is another ER chaperone that demonstrates pro-oncogenic and pro-survival functions. Because of intrinsic alterations of cellular metabolism and extrinsic factors in the tumor microenvironment, cancer cells are under ER stress, and they respond to this stress by activating the unfolded protein response (UPR). Depending on the severity and duration of ER stress, the signaling branches of the UPR can activate adaptive and pro-survival signals, or induce apoptotic cell death. The protein kinase RNA-like ER kinase signaling branch of the UPR has a dual role in cancer proliferation and survival, and is also required for ER stress-induced autophagy. The activation of the inositol-requiring kinase 1α branch promotes tumorigenesis, cancer cell survival and regulates tumor invasion. In summary, perturbance of ER homeostasis has critical roles in tumorigenesis, and therapeutic modulation of ER chaperones and/or UPR components presents potential antitumor treatments.

Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis

Neurodegenerative diseases are often associated with dysfunction in protein quality control. The endoplasmic reticulum (ER), a key site for protein synthesis, senses stressful conditions by activating the unfolded protein response (UPR). In this study we report the creation of a novel mouse model in which GRP78/BiP, a major ER chaperone and master regulator of UPR, is specifically eliminated in Purkinje cells (PCs). GRP78-depleted PCs activate UPR including the induction of GRP94, PDI, CHOP and GADD34, feedback suppression of eIF2α phosphorylation and apoptotic cell death. In contrast to current models of protein misfolding in which an abnormal accumulation of ubiquitinated protein is prominent, cytosolic ubiquitin staining is dramatically reduced in GRP78-null PCs. Ultrastructural evaluation reveals that the ER shows prominent dilatation with focal accumulation of electron-dense material within the ER. The mice show retarded growth and severe motor coordination defect by week 5 and cerebellar atrophy by week 13. Our studies uncover a novel link between GRP78 depletion and reduction in cytosolic ubiquitination and establish a novel mouse model of accelerated cerebellar degeneration with basic and clinical applications.

A Critical Role for GRP78/BiP in the Tumor Microenvironment for Neovascularization during Tumor Growth and Metastasis

Glucose-regulated protein 78 (GRP78)/BiP is a multifunctional protein which plays a major role in endoplasmic reticulum (ER) protein processing, protein quality control, maintaining ER homeostasis, and controlling cell signaling and viability. Previously, using a transgene-induced mammary tumor model, we showed that Grp78 heterozygosity impeded cancer growth through suppression of tumor cell proliferation and promotion of apoptosis and the Grp78(+/-) mice exhibited dramatic reduction (70%) in the microvessel density (MVD) of the endogenous mammary tumors, while having no effect on the MVD of normal organs. This observation suggests that GRP78 may critically regulate the function of the host vasculature within the tumor microenvironment. In this article, we interrogated the role of GRP78 in the tumor microenvironment. In mouse tumor models in which wild-type (WT), syngeneic mammary tumor cells were injected into the host, we showed that Grp78(+/-) mice suppressed tumor growth and angiogenesis during the early phase but not during the late phase of tumor growth. Growth of metastatic lesions of WT, syngeneic melanoma cells in the Grp78(+/-) mice was potently suppressed. We created conditional heterozygous knockout of GRP78 in the host endothelial cells and showed severe reduction of tumor angiogenesis and metastatic growth, with minimal effect on normal tissue MVD. Furthermore, knockdown of GRP78 expression in immortalized human endothelial cells showed that GRP78 is a critical mediator of angiogenesis by regulating cell proliferation, survival, and migration. Our findings suggest that concomitant use of current chemotherapeutic agents and novel therapies against GRP78 may offer a powerful dual approach to arrest cancer initiation, progression, and metastasis.

Inducible knockout of GRP78/BiP in the hematopoietic system suppresses Pten-null leukemogenesis and AKT oncogenic signaling

Traditionally, GRP78 is regarded as protective against hypoxia and nutrient starvation prevalent in the microenvironment of solid tumors; thus, its role in the development of hematologic malignancies remains to be determined. To directly elucidate the requirement of GRP78 in leukemogenesis, we created a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient to restore the hematopoietic stem cell population back to the normal percentage and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null BM cells is potently inhibited by Grp78 heterozygosity, corresponding with suppression of the PI3K/AKT pathway by GRP78 knockdown in leukemia cell lines. This is the first demonstration that GRP78 is a critical effector of leukemia progression, at least in part through regulation of oncogenic PI3K/AKT signaling. In agreement with PI3K/AKT as an effector for cytosine arabinoside resistance in acute myeloid leukemia, overexpression of GRP78 renders human leukemic cells more resistant to cytosine arabinoside-induced apoptosis, whereas knockdown of GRP78 sensitizes them. These, coupled with the emerging association of elevated GRP78 expression in leukemic blasts of adult patients and early relapse in childhood leukemia, suggest that GRP78 is a novel therapeutic target for leukemia.

Targeted Mutation of the Mouse Grp94 Gene Disrupts Development and Perturbs Endoplasmic Reticulum Stress Signaling

Glucose-regulated protein 94 (GRP94) is one of the most abundant endoplasmic reticulum (ER) resident proteins and is the ER counterpart of the cytoplasmic heat shock protein 90 (HSP90). GRP94, a component of the GRP78 chaperone system in protein processing, has pro-survival properties with implicated function in cancer progression and autoimmune disease. Previous studies on the loss of GRP94 function showed that it is required for embryonic development, regulation of toll-like receptors and innate immunity of macrophages. Here we report the creation of mouse models targeting exon 2 of the Grp94 allele that allows both traditional and conditional knockout (KO) of Grp94. In this study, we utilized the viable Grp94+/+ and +/− mice, as well as primary mouse embryonic fibroblasts generated from them as experimental tools to study its role in ER chaperone balance and ER stress signaling. Our studies reveal that while Grp94 heterozygosity reduces GRP94 level it does not alter ER chaperone levels or the ER stress response. To study the effect of complete loss of GRP94 function, since homozygous GRP94 KO leads to embryonic lethality, we generated Grp94−/− embryonic stem cells. In contrast to Grp94 heterozygosity, complete knockout of GRP94 leads to compensatory upregulation of the ER chaperones GRP78, calnexin and calreticulin but not protein disulphide isomerase. Unexpectedly, loss of GRP94 leads to significant decrease in the level of ER-stress induced spliced form of XBP-1 protein, a downstream target of the IRE1 signaling pathway. Furthermore, from analysis of microarray database and immunohistochemical staining, we present predictions where GRP94 may play an important role in specific adult organ homeostasis and function.

Acute Inducible Ablation of GRP78 Reveals Its Role in Hematopoietic Stem Cell Survival, Lymphogenesis and Regulation of Stress Signaling

GRP78, a master regulator of the unfolded protein response (UPR) and cell signaling, is required for inner cell mass survival during early embryonic development. However, little is known about its role in adult hematopoietic stem cells (HSCs) and hematopoiesis. Here we generated a conditional knockout mouse model that acutely deletes Grp78 in the adult hematopoietic system. Acute GRP78 ablation resulted in a significant reduction of HSCs, common lymphoid and myeloid progenitors, and lymphoid cell populations in the mutant mice. The GRP78-null induced reduction of the HSC pool could be attributed to increased apoptosis. Chimeric mice with Grp78 deletion only in the hematopoietic cells also showed a loss of HSCs and lymphopenia, suggesting a cell intrinsic effect. Analysis of GRP78 deficient bone marrow (BM) cells showed constitutive activation of all the major UPR signaling pathways, including activation of eIF2α, ATF6, xbp-1 splicing, as well as caspase activation. A multiplex cytokine assay further revealed alteration in select cytokine and chemokine serum levels in the mutant mice. Collectively, these studies demonstrate that GRP78 plays a pleiotropic role in BM cells and contributes to HSC survival and the maintenance of the lymphoid lineage.

Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis

Liver cancer is one of the most common solid tumors, with poor prognosis and high mortality. Mutation or deletion of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is strongly correlated with human liver cancer. Glucose‐regulated protein 94 (GRP94) is a major endoplasmic reticulum (ER) chaperone protein, but its in vivo function is still emerging. To study the role of GRP94 in maintaining liver homeostasis and tumor development, we created two liver‐specific knockout mouse models with the deletion of Grp94 alone, or in combination with Pten, using the albumin‐cre system. We demonstrated that while deletion of GRP94 in the liver led to hyperproliferation of liver progenitor cells, deletion of both GRP94 and PTEN accelerated development of liver tumors, including both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), suggestive of progenitor cell origin. Furthermore, at the premalignant stage we observed disturbance of cell adhesion proteins and minor liver injury. When GRP94 was deleted in PTEN‐null livers, ERK was selectively activated. Conclusion: GRP94 is a novel regulator of cell adhesion, liver homeostasis, and tumorigenesis. (Hepatology 2014;59:947–957)

The Endoplasmic Reticulum Chaperone Protein GRP94 Is Required for Maintaining Hematopoietic Stem Cell Interactions with the Adult Bone Marrow Niche

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology.

Deficiency of GRP94 in the Hematopoietic System Alters Proliferation Regulators in Hematopoietic Stem Cells

We have previously reported that acute inducible knockout of the endoplasmic reticulum chaperone GRP94 led to an expansion of the hematopoietic stem and progenitor cell pool. Here, we investigated the effectors and mechanisms for this phenomenon. We observed an increase in AKT activation in freshly isolated GRP94-null HSC-enriched Lin(-) Sca-1(+) c-Kit(+) (LSK) cells, corresponding with higher production of PI(3,4,5)P3, indicative of PI3K activation. Treatment of GRP94-null LSK cells with the AKT inhibitor MK2206 compromised cell expansion, suggesting a causal relationship between elevated AKT activation and increased proliferation in GRP94-null HSCs. Microarray analysis demonstrated a 97% reduction in the expression of the hematopoietic cell cycle regulator Ms4a3 in the GRP94-null LSK cells, and real-time quantitative PCR confirmed this down-regulation in the LSK cells but not in the total bone marrow (BM). A further examination comparing freshly isolated BM LSK cells with spleen LSK cells, as well as BM LSK cells cultured in vitro, revealed specific down-regulation of Ms4a3 in freshly isolated BM GRP94-null LSK cells. On examining cell surface proteins that are known to regulate stem cell proliferation, we observed a reduced expression of cell surface connexin 32 (Cx32) plaques in GRP94-null LSK cells. However, suppression of Cx32 hemichannel activity in wild-type LSK cells through mimetic peptides did not lead to increased LSK cell proliferation in vitro. Two other important cell surface proteins that mediate HSC-niche interactions, specifically Tie2 and CXCR4, were not impaired by Grp94 deletion. Collectively, our study uncovers novel and unique roles of GRP94 in regulating HSC proliferation.

Abstract B09: Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis

Liver cancer is one of the most common solid tumors with poor prognosis and high mortality. Mutation or deletion of the tumor suppressor PTEN is strongly correlated with human liver cancer. Glucose-regulated protein 94 (GRP94) is a major endoplasmic reticulum (ER) chaperone protein, but its in vivo function is still emerging. To study the role of GRP94 in maintaining liver homeostasis and tumor development, we created two liver-specific knockout mouse models with the deletion of Grp94 alone, or in combination with Pten , using the albumin-cre system. We demonstrated that while deletion of GRP94 in the liver led to hyperproliferation of liver progenitor cells, deletion of both GRP94 and PTEN accelerated development of liver tumors, including both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) suggestive of progenitor cell origin. Furthermore, at the premalignant stage, we observed disturbance of cell adhesion proteins but no detectable overt liver injury. We further discovered that when GRP94 was deleted in PTEN-null livers, ERK was selectively activated, suggesting that this could be a potential molecular mechanism for enhanced liver tumorigenesis in this mouse model. Conclusion: We identify GRP94 as a novel regulator of cell adhesion, liver homeostasis, and tumorigenesis. Citation Format: Wan-Ting Chen, Chun-Chih Tseng, Kyle Pfaffenbach, Gary Kanel, Biquan Luo, Bangyan L. Stiles, Amy S. Lee. Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B09.