Study Design. A dissection-based study of 6 embalmed cadavers.Objective. To identify and describe the extraforaminal ligaments(EFLs) in relation to the area of the cervical intervertebral foramina (IVF) and to evaluate their clinical significance.Summary of Background Data. EFLs between the lumbar spinal nerves and the tissues surrounding the intervertebral foramens have been well established. However, research work has been undertaken to describe the local anatomy of the extraforaminal part of the cervical spine, detailed anatomic studies of the EFLs of cervical nerves have not been performed.Methods. One hundred ninety-six cervical IVFs from 6 adult embalmed cadavers were studied, and the existence and type of the EFLs were identified. The morphology, quantity, origin, insertion, and the spatial orientation of the EFLs in the cervical region were observed, and the length, width, or diameter and thickness of the ligaments were measured with a vernier caliper.Results. The EFLs could be found from the second cervical to the first thoracic spinal nerve. These ligaments could be divided into 2 types: Radiating ligaments, which connected the nerve root sleeves radiated to the transverse processes, the wall of the IVF and even the adjacent nerve root through the small transverse foramen; Transforaminal ligaments(TFLs), which originated from the anteroinferior margin of cranial transverse process and inserts in the superior margin of the anterior tubercle of caudal transverse process crossing the spinal nerve ventrally.Conclusion. Between the cervical spinal nerves and nearby structures, there are two types of the EFLs. The radiating ligaments may serve as a protective mechanism against traction and play an important role in the positioning of the nerves in the intervertebral foramen. However, in all probability, the transforaminal ligaments may be the underlying cause of the cervical radiculopathy.
Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum (ER) of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER-associated degradation quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins. To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full-length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal mannose recognition homology lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains "mammalian-specific insets" that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate and it additionally hints at a plausible biological role for the Grp94/OS-9 complex.
As an adaptive response to the overloading with misfolded proteins in the endoplasmic reticulum (ER), ER stress plays critical roles in maintaining protein homeostasis in the secretory pathway to avoid damage to the host. Such a conserved mechanism is accomplished through three well-orchestrated pathways known collectively as unfolded protein response (UPR). Persistent and pathological ER stress has been implicated in a variety of diseases in metabolic, inflammatory, and malignant conditions. Furthermore, ER stress is directly linked with inflammation through UPR pathways, which modulate transcriptional programs to induce the expression of inflammatory genes. Importantly, the inflammation induced by ER stress is directly responsible for the pathogenesis of metabolic and inflammatory diseases. In this review, we will discuss the potential signaling pathways connecting ER stress with inflammation. We will also depict the interplay between ER stress and inflammation in the pathogenesis of hepatic steatosis, inflammatory bowel diseases and colitis-associated colon cancer.
The effects of heightened microbial translocation on B cells during HIV infection are unknown. We examined the in vitro effects of HIV and lipopolysaccharide (LPS) on apoptosis of CD27+ IgD- memory B (mB) cells from healthy controls. In vivo analysis was conducted on a cohort of 82 HIV+ donors and 60 healthy controls. In vitro exposure of peripheral blood mononuclear cells (PBMCs) to LPS and HIV led to mB cell death via the Fas/Fas ligand (FasL) pathway. Plasmacytoid dendritic cells (pDCs) produced FasL in response to HIV via binding to CD4 and chemokine coreceptors. HIV and LPS increased Fas expression on mB cells in PBMCs, which was dependent on the presence of pDCs and monocytes. Furthermore, mB cells purified from PBMCs and pretreated with both HIV and LPS were more sensitive to apoptosis when cocultured with HIV-treated pDCs. Blocking the interferon receptor (IFNR) prevented HIV-stimulated FasL production in pDCs, HIV-plus-LPS-induced Fas expression, and apoptosis of mB cells. In vivo or ex vivo, HIV+ donors have higher levels of plasma LPS, Fas expression on mB cells, and mB cell apoptosis than controls. Correspondingly, in HIV+ donors, but not in controls, a positive correlation was found between plasma FasL and HIV RNA levels and between Fas expression on mB cells and plasma LPS levels. This work reveals a novel mechanism of mB cell apoptosis mediated by LPS and HIV through the Fas/FasL pathway, with key involvement of pDCs and type I IFN, suggesting a role for microbial translocation in HIV pathogenesis.
BackgroundHead and neck squamous cell carcinoma (HNSCC) mortality rates have not shown significant reduction in decades. Platelets are being implicated in having cancer-promoting roles, an observation supported by the adverse outcomes in many malignancies associated with thrombocytosis. However, the prognostic significance of platelet counts in HNSCC is unknown. Here, we comprehensively investigate the predictive value of platelet counts at diagnosis and post-diagnosis antiplatelet treatment in the overall survival of HNSCC patients.MethodsThe study population consists of 1051 pathologically confirmed HNSCC cases diagnosed between years 2000 and 2012 in a tertiary medical center. Platelet count was investigated as a predictor of survival by fitting Cox Proportional Hazards (CPH) regression models to generate Hazard Ratios (HR) and 95% confidence intervals (CI), while adjusting for age, sex, race, stage, treatment and smoking status. Finally, we evaluated the association between overall survival and antiplatelet medication intake after diagnosis.ResultsMultivariable analysis showed an increased death rate in patients with thromobocytosis [HR 2.37, 95% CI 1.60-3.50)] and high normal platelet counts [HR 2.20, 95% CI 1.58-3.05] compared to the reference middle normal group. Post-diagnosis treatment with antiplatelet medications was inversely associated with death rate [HR 0.76, 95% CI 0.58-0.99].ConclusionsHigher platelet counts were associated with poorer prognosis in HNSCC patients, whereas antiplatelet agents were associated with better prognosis. Antiplatelet agents warrant evaluation in preclinical and clinical settings as a way to improve survival in HNSCC.
CD24 binds to and suppresses inflammation triggered by danger-associated molecular patterns such as heat shock proteins (HSPs) and high-mobility group box 1. Paradoxically, CD24 has been shown to enhance autoimmune disease. In this study, we attempt to reconcile this paradox by deletion of CD24 (24KO) in a lupus-like disease model driven by forced expression of HSP gp96 at the cell surface (transgenic mice [tm]). As expected, tm24KO mice showed increased CD11c(+) dendritic cell activation coupled to a significant increase in dendritic cell-specific IL-12 production compared with tm mice. However, tm24KO mice showed less CD4 T cell activation and peripheral inflammatory cytokine production in comparison with tm mice. We characterized an enhanced immune suppressive milieu in tm24KO mice distinguished by increased TGF-? and greater regulatory T cell-suppressive capacity. We found greater absolute numbers of myeloid-derived suppressor cells (MDSCs) in tm24KO mice and showed that the Ly6C(+) MDSC subset had greater suppressive capacity from tm24KO mice. Deletion of CD24 in tm mice led to diminished lupus-like pathology as evidenced by anti-nuclear Ab deposition and glomerulonephritis. Finally, we show that expanded MDSC populations were mediated by increased free high-mobility group box 1 in tm24KO mice. Thus, the deletion of CD24 in an HSP-driven model of autoimmunity led to the unexpected development of regulatory T cell and MDSC populations that augmented immune tolerance. Further study of these populations as possible negative regulators of inflammation in the context of autoimmunity is warranted.
With the development of natural orifice trans-luminal endoscopic surgery, studies on transoral video-assisted thyroidectomy in preclinical experiments (e.g., human anatomy and animal trials) were progressing gradually. From 2009 to 2011, embalmed human cadavers were dissected to define the anatomical location, surgical planes, and related neural and vascular structures to create a safe transoral access to the front cervical spaces. Recently, experimental transoral endoscopic thyroidectomy was performed to verify the feasibility of this approach on 15 fresh specimens.
The dural sac is anchored within the vertebral canal by connective tissue called meningovertebral ligaments in the epidural space. During flavectomy and laminectomy, inadvertent disruption of the dorsal meningovertebral ligaments may lead to dura laceration and cerebrospinal fluid (CSF) leaks. All the described dorsal meningovertebral ligaments were located in the lumbar region. A rare study is available about dorsal meningovertebral ligaments of the cervical spinal dura to the adjacent vertebrae.
The purpose of this study was to discuss the distribution characteristics and the anatomical angioarchitecture of cutaneous branches arising from the second dorsal metacarpal artery for the repair of small tissue defects in the hand or fingers using the second dorsal metacarpal artery chain-link flap.
It is unclear how tumor-associated macrophages (TAMs) contribute to the initiation of oncogenesis and how they are regulated at the molecular level. By using a lineage-specific deletion strategy, we found that heat shock protein 90kDa ? (Grp94), member 1 (HSP90B1), a master chaperone for Toll-like receptors and integrins also known as GP96, critically endows TAMs with the ability to promote genotoxic stress and colitis-associated colon cancer.
T helper 17 (Th17) cells play a complex and controversial role in tumor immunity and have been found to exhibit a fluctuating identity within the context of cancer. The recent, expanding literature on these cells attests to their puzzling nature, either promoting or suppressing tumor growth depending on the malignancy and course of therapeutic intervention investigated. This review addresses several newly appreciated factors that may help delineate Th17 cells' immunological properties in the context of cancer. Several reports suggest that inflammatory signals induced in the tumor milieu regulate the functional fate and antitumor activity of Th17 cells. Recent findings also point to significant alterations in Th17 cells due to their interplay with regulatory T lymphocytes and cytotoxic CD8(+) T cells within the tumor microenvironment. Finally, an appreciation for the stem cell-like properties of Th17 cells that augment their persistence and activity emerges from recent reports. The impact of these factors on Th17 cells' antitumor efficacy and how these factors may be exploited to improve cancer therapies will be discussed.
Macrophages are important drivers in the development of inflammation-associated colon cancers, but the mechanistic underpinnings for their contributions are not fully understood. Further, Toll-like receptors (TLR) have been implicated in colon cancer, but their relevant cellular sites of action are obscure. In this study, we show that the endoplasmic reticulum chaperone gp96 is essential in tumor-associated macrophages (TAM) to license their contributions to inflammatory colon tumorigenesis. Mice where gp96 was genetically deleted in a macrophage-specific manner exhibited reduced colitis and inflammation-associated colon tumorigenesis. Attenuation of colon cancer in these mice correlated strikingly with reduced mutation rates of ?-catenin, increased efficiency of the DNA repair machinery and reduced expression of pro-inflammatory cytokines, including IL-17 and IL-23 in the tumor microenvironment. The genotoxic nature of TAM-associated inflammation was evident by increased expression of genes in the DNA repair pathway. Our work deepens understanding of how TAM promote oncogenesis by altering the molecular oncogenic program within epithelial cells, and it identifies gp96 as a lynchpin chaperone needed in TAM to license their function and impact on expression of critical inflammatory cytokines in colon tumorigenesis. -
Multiple myeloma (MM) is a debilitating disease of proliferating and malignant plasma cells that is currently incurable. The ability of monoclonal recurrence of disease suggests it might arise from a stem cell-like population capable of self-renewal. The difficulty to isolate the cancer stem-like cell in MM has introduced confusion toward this hypothesis. However, recent evidence has suggested that MM originates from the B cell lineage with memory-B cell like features, allowing for self-renewal of the progenitor-like status and differentiation to a monoclonal plasma cell population. Furthermore, this tumor-initiating cell uses signaling pathways and microenvironment similar to the hematopoietic stem cell, though hijacking these mechanisms to create and favor a more tumorigenic environment. The bone marrow niche allows for pertinent evasion, either through avoiding immunosurveillance or through direct interaction with the stroma, inducing quiescence and thus drug resistance. Understanding the interaction of the MM stem cell to the microenvironment and the mechanisms utilized by various stem cell-like populations to allow persistence and therapy-resistance can enable for better targeting of this cell population and potential eradication of the disease.
The immune system is the built-in host defense mechanism against infectious agents as well as cancer. Protective immunity against cancer was convincingly demonstrated in the 1940s with syngeneic animal models (JNCI 18:769-778, 1976; Cancer Immun 1:6, 2001). Since then, the last centurys dream has been to effectively prevent and cure cancers by immunological means. This dream has slowly but surely become a reality (Nature 480:480-489, 2011). The successful examples of immunoprophylaxis and therapy against cancers include: (i) targeted therapy using monoclonal antibodies (Nat Rev Cancer 12:278-287, 2012); (ii) allogeneic hematopoietic stem cell transplantion to elicit graft-versus-cancer effect against a variety of hematopoietic malignancies (Blood 112:4371-4383, 2008); (iii) vaccination for preventing cancers with clear viral etiology such as hepatocellular carcinoma and cervical cancer (Cancer J Clin 57:7-28, 2007; NEJM 336:1855-1859, 1997); (iv) T cell checkpoint blockade against inhibitory pathways including targeting CTLA-4 and PD-1 inhibitory molecules for the treatment of melanoma and other solid tumors (NEJM 363:711-723, 2010; NEJM 366:2443-2454, 2012; NEJM 369:122-133, 2013; NEJM 366:2455-2465, 2012); (v) antigen-pulsed autologous dendritic cell vaccination against prostate cancer (NEJM 363:411-422, 2010); and (vi) the transfer of T cells including those genetically engineered with chimeric antigen receptors allowing targeting of B cell neoplasms (NEJM 365:725-733, 2011; NEJM 368:1509-1518, 2013; Blood 118:4817-4828, 2013; Sci Transl Med 5:177ra138, 2013).This article provides an overview on the exciting and expanding immunological arsenals against cancer, and discusses critical remaining unanswered questions of cancer immunology. The inherent specificity and memory of the adaptive immune response towards cancer will undoubtedly propel cancer immunotherapy to the forefront of cancer treatment in the immediate near future. Study of the fundamental mechanisms of the immune evasion of cancer shall also advance the field of immunology towards the development of effective immunotherapeutics against a wide spectrum of human diseases.
gp96 (grp94) is a key downstream chaperone in the endoplasmic reticulum (ER) to mediate unfolded protein response (UPR) and the pathogenesis of multiple myeloma is closely linked to dysregulated UPR. In this study, we aimed to determine the roles of gp96 in the initiation and progression of multiple myeloma in vivo and in vitro.
Through a process known as melanogenesis, melanocyte produces melanin in specialized organelles termed melanosomes, which regulates pigmentation of the skin, eyes, and hair. Gp96 is a constitutively expressed heat shock protein in the endoplasmic reticulum whose expression is further upregulated upon ultraviolet irradiation. However, the roles and mechanisms of this chaperone in pigmentation biology are unknown. In this study, we found that knockdown of gp96 by RNA interference significantly perturbed melanin synthesis and blocked late melanosome maturation. Gp96 knockdown did not impair the expression of tyrosinase, an essential enzyme in melanin synthesis, but compromised its catalytic activity and melanosome translocation. Further, mice with melanocyte-specific deletion of gp96 displayed decreased pigmentation. A mechanistic study revealed that the defect in melanogenesis can be rescued by activation of the canonical Wnt pathway, consistent with the critical roles of gp96 in chaperoning Wnt-coreceptor LRP6. Thus, this work uncovered the essential role of gp96 in regulating melanogenesis.
Although percutaneous posterior-ring tension-band metallic plate and percutaneous iliosacral screws are used to fix unstable posterior pelvic ring fractures, the biomechanical stability and compatibility of both internal fixation techniques for the treatment of Denis I, II and III type vertical sacral fractures remain unclear.
Integrins play important roles in regulating a diverse array of cellular functions crucial to the initiation, progression, and metastasis of tumors. Previous studies have shown that a majority of integrins are folded by the endoplasmic reticulum chaperone gp96. Here, we demonstrate that the dimerization of integrin ?L and ?2 is highly dependent on gp96. The ?I domain (AID), a ligand binding domain shared by seven integrin ?-subunits, is a critical region for integrin binding to gp96. Deletion of AID significantly reduced the interaction between integrin ?L and gp96. Overexpression of AID intracellularly decreased surface expression of gp96 clients (integrins and Toll-like receptors) and cancer cell invasion. The ?7 helix region is crucial for AID binding to gp96. A cell-permeable ?7 helix peptide competitively inhibited the interaction between gp96 and integrins and blocked cell invasion. Thus, targeting the binding site of ?7 helix of AID on gp96 is potentially a new strategy for treatment of cancer metastasis.
Increasing evidence points to a role for the protein quality control in the endoplasmic reticulum (ER) in maintaining intestinal homeostasis. However, the specific role for general ER chaperones in this process remains unknown. Herein, we report that a major ER heat shock protein grp94 interacts with MesD, a critical chaperone for the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6). Without grp94, LRP6 fails to export from the ER to the cell surface, resulting in a profound loss of canonical Wnt signaling. The significance of this finding is demonstrated in vivo in that grp94 loss causes a rapid and profound compromise in intestinal homeostasis with gut-intrinsic defect in the proliferation of intestinal crypts, compromise of nuclear ?-catenin translocation, loss of crypt-villus structure, and impaired barrier function. Taken together, our work has uncovered the role of grp94 in chaperoning LRP6-MesD in coordinating intestinal homeostasis, placing canonical Wnt-signaling pathway under the direct regulation of the general protein quality control machinery in the ER.
The activating receptor NK cell group 2 member D (NKG2D) mediates antitumor immunity in experimental animal models. However, whether NKG2D ligands contribute to tumor suppression or progression clinically remains controversial. Here, we have described 2 novel lines of "humanized" bi-transgenic (bi-Tg) mice in which native human NKG2D ligand MHC class I polypeptide-related sequence B (MICB) or the engineered membrane-restricted MICB (MICB.A2) was expressed in the prostate of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous carcinogenesis. Bi-Tg TRAMP/MICB mice exhibited a markedly increased incidence of progressed carcinomas and metastasis, whereas TRAMP/MICB.A2 mice enjoyed long-term tumor-free survival conferred by sustained NKG2D-mediated antitumor immunity. Mechanistically, we found that cancer progression in TRAMP/MICB mice was associated with loss of the peripheral NK cell pool owing to high serum levels of tumor-derived soluble MICB (sMICB). Prostate cancer patients also displayed reduction of peripheral NK cells and high sMIC levels. Our study has not only provided direct evidence in "humanized" mouse models that soluble and membrane-restricted NKG2D ligands pose opposite impacts on cancer progression, but also uncovered a mechanism of sMIC-induced impairment of NK cell antitumor immunity. Our findings suggest that the impact of soluble NKG2D ligands should be considered in NK cell-based cancer immunotherapy and that our unique mouse models should be valuable for therapy optimization.
Immune evasion is one of the recognized hallmarks of cancer. Inflammatory responses to cancer can also contribute directly to oncogenesis. Since the immune system is hardwired to protect the host, there is a possibility that cancers, regardless of their histological origins, endow themselves with a common and shared inflammatory cancer-associated molecular pattern (iCAMP) to promote oncoinflammation. However, the definition of iCAMP has not been conceptually and experimentally investigated.
The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) are not completely understood. In this study, we investigated the roles of serine/threonine Pim kinases in hematopoiesis in mice. We generated PIM1 transgenic mice (Pim1-Tx) overexpressing human PIM1 driven by vav hematopoietic promoter/regulatory elements. Compared to wild-type littermates, Pim1-Tx mice showed enhanced hematopoiesis as demonstrated by increased numbers of Lin(-) Sca-1 (+) c-Kit (+) (LSK) hematopoietic stem/progenitor cells and cobblestone area forming cells, higher BrdU incorporation in long-term HSC population, and a better ability to reconstitute lethally irradiated mice. We then extended our study using Pim1(-/-), Pim2(-/-), Pim3(-/-) single knockout (KO) mice. HSCs from Pim1(-/-) KO mice showed impaired long-term hematopoietic repopulating capacity in secondary and competitive transplantations. Interestingly, these defects were not observed in HSCs from Pim2(-/-) or Pim3(-/-) KO mice. Limiting dilution competitive transplantation assay estimated that the frequency of LSKCD34(-) HSCs was reduced by approximately 28-fold in Pim1(-/-) KO mice compared to wild-type littermates. Mechanistic studies demonstrated an important role of Pim1 kinase in regulating HSC cell proliferation and survival. Finally, our polymerase chain reaction (PCR) array and confirmatory real-time PCR (RT-PCR) studies identified several genes including Lef-1, Pax5, and Gata1 in HSCs that were affected by Pim1 deletion. Our data provide the first direct evidence for the important role of Pim1 kinase in the regulation of HSCs. Our study also dissects out the relative role of individual Pim kinase in HSC functions and regulation.
Monoclonal antibody (mAb) has fulfilled the promise of being the "Magic Bullet" in oncology with the clinical success of mAbs against CD20, Her-2/neu, epidermal growth factor receptor, vascular endothelial cell growth factor and others in a variety of cancers. Most manufacturers of mouse-human chimeric antibodies (and most immunologists) have treated the constant region of human immunoglobulin (Ig) as if it were naturally monomorphic and therefore not immunogenic in humans. In fact, the constant region of Ig heavy and light chain is highly polymorphic, and yet Ig haplotypes are usually not defined by genome-wide association studies nor are they considered to be important for optimizing mAb therapy. We hereby summarize evidence that Ig allotypes are important and biologically relevant in that they contribute to the etiopathogenesis of many malignant, infectious, and autoimmune diseases. Because Ig allotypes differ from each other in engaging Fc receptor, we argue that future development of effective mAb therapy for cancer should take a patient-specific approach by using the correct allotype for each patient to maximize the efficacy of this therapy.
Both passive flexion-active extension and active rehabilitation have shown advantages and disadvantages in tendon healing. The purpose of this study was to measure the effect of a combination of these 2 rehabilitation protocols.
The platelet glycoprotein Ib-IX-V complex (GPIb-IX-IV) is the receptor for VWF and is responsible for VWF-mediated platelet activation and aggregation. Loss of the GPIb-IX-V complex is pathogenic for Bernard-soulier Syndrome (BSS), which is characterized by macrothrombocytopenia and impaired platelet function. It remains unclear how the GPIb-IX-V complex is assembled and whether there is a role for a specific molecular chaperone in the process. In the present study, we report that the assembly of the GPIb-IX-V complex depends critically on a molecular chaperone in the endoplasmic reticulum (ER): gp96 (also known as grp94 and HSP90b1). gp96/grp94 deletion in the murine hematopoietic system results in thrombocytopenia, prolonged bleeding time, and giant platelets that are clinically indistinguishable from human BSS. Loss of gp96/grp94 in vivo and in vitro leads to the concomitant reduction in GPIb-IX complex expression due to ER-associated degradation. We further demonstrate that gp96/grp94 binds selectively to the GPIX subunit, but not to gpIb? or gpIb?. Therefore, we identify the platelet GPIX subunit of the GPIb-IX-V complex as an obligate and novel client of gp96/grp94.
Hsp90b1 is an endoplasmic reticulum (ER) chaperone (also named Grp94, ERp99, gp96,Targ2, Tra-1, Tra1, Hspc4) (MGI:98817) contributing with Hspa5 (also named Grp78, BIP) (MGI:95835) to protein folding in ER compartment. Besides its high protein expression in mouse oocytes, little is known about Hsp90b1 during the transition from oocyte-to-embryo. Because the constitutive knockout of Hsp90b1 is responsible for peri-implantation embryonic lethality, it was not yet known whether Hsp90b1 is a functionally important maternal factor.
The unfolded protein response (UPR) is a cascade of intracellular stress signaling events in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER). Cancer cells are often exposed to hypoxia, nutrient starvation, oxidative stress and other metabolic dysregulation that cause ER stress and activation of the UPR. Depending on the duration and degree of ER stress, the UPR can provide either survival signals by activating adaptive and antiapoptotic pathways, or death signals by inducing cell death programs. Sustained induction or repression of UPR pharmacologically may thus have beneficial and therapeutic effects against cancer. In this review, we discuss the basic mechanisms of UPR and highlight the importance of UPR in cancer biology. We also update the UPR-targeted cancer therapeutics currently in clinical trials.
Heat shock protein 90 (HSP90) inhibitors have emerged as a promising class of anti-cancer drugs in both solid and hematologic malignancies. The HSP90 family includes the cytosolic HSP90 (HSP90AA1), the ER paralogue gp96 (HSP90B1) and the mitochondrial member TRAP1 (HSP90L). We evaluated the in vitro anti-tumor activity and mechanism of action of PU-H71, a novel purine scaffold HSP90 inhibitor in human multiple myeloma cell lines.
A new head and neck location frame for positron emission tomography (PET) and computed tomography (CT) cross-modality medical image fusion in radiotherapy was developed. The solid mark bars of N form in the old designed CT location frame were replaced by closed hollow pipes which could be filled with different developer solutions before CT or PET scan, respectively. Nine points of external marker used for registration and fusion were obtained in CT images and PET images, respectively. The locations of the two sets of nine points showed the method of registration to be effective and accurate in achieving the PET and CT image fusion. This method, based on its characteristics of simple structure and easy-to-use, can be of wide application in clinical setting.
Cytosolic HSP90 requires multiple cochaperones in folding client proteins. However, the function of gp96 (HSP90b1, grp94), an HSP90 paralogue in the endoplasmic reticulum (ER), is believed to be independent of cochaperones. Here, we demonstrate that gp96 chaperones multiple Toll-like receptors (TLRs), but not TLR3, in a manner that is dependent on another ER luminal protein, CNPY3. gp96 directly interacts with CNPY3, and the complex dissociates in the presence of adenosine triphosphate (ATP). Genetic disruption of gp96-CNPY3 interaction completely abolishes their TLR chaperone function. Moreover, we demonstrate that TLR9 forms a multimolecular complex with gp96 and CNPY3, and the binding of TLR9 to either molecule requires the presence of the other. We suggest that CNPY3 interacts with the ATP-sensitive conformation of gp96 to promote substrate loading. Our study has thus established CNPY3 as a TLR-specific cochaperone for gp96.
Due to the low survival rates from invasive ovarian cancer, new effective treatment modalities are urgently needed. Compelling evidence indicates that the immune response against ovarian cancer may play an important role in controlling this disease. We herein summarize multiple immune-based strategies that have been proposed and tested for potential therapeutic benefit against advanced stage ovarian cancer. We will examine the evidence for the premise that an effective therapeutic vaccine against ovarian cancer is useful not only for inducing remission of the disease but also for preventing disease relapse. We will also highlight the questions and challenges in the development of ovarian cancer vaccines, and critically discuss the limitations of some of the existing immunotherapeutic strategies. Finally, we will summarize our own experience on the use of patient-specific tumor-derived heat shock protein-peptide complex for the treatment of advanced ovarian cancer.
Heat shock proteins have been implicated as endogenous activators for dendritic cells (DCs). Chronic expression of heat shock protein gp96 on cell surfaces induces significant DC activations and systemic lupus erythematosus (SLE)-like phenotypes in mice. However, its potential as a therapeutic target against SLE remains to be evaluated. In this work, we conducted chemical approach to determine whether SLE-like phenotypes can be compromised by controlling surface translocation of gp96. From screening of chemical library, we identified a compound that binds and suppresses surface presentation of gp96 by facilitating its oligomerization and retrograde transport to endoplasmic reticulum. In vivo administration of this compound reduced maturation of DCs, populations of antigen presenting cells, and activated B and T cells. The chemical treatment also alleviated the SLE-associated symptoms such as glomerulonephritis, proteinuria, and accumulation of anti-nuclear and -DNA antibodies in the SLE model mice resulting from chronic surface exposure of gp96. These results suggest that surface translocation of gp96 can be chemically controlled and gp96 as a potential therapeutic target to treat autoimmune disease like SLE.
Lipopolysaccharide (LPS) is a potent natural adjuvant, commonly used to amplify Th1 responses. Here, we report that systemic immunization using LPS generates large numbers of specific Th17 cells in murine small intestinal lamina propria. The priming of these Th17 cells required IL-23p19 production by bone marrow-derived cells. In contrast, IL-23 had no impact on Th1 differentiation or overall numbers of Ag-specific regulatory T cells. Experiments using T-cell adoptive transfers revealed a previously unappreciated mechanism for how Th17 responses are amplified in vivo: stimulation through LPS expanded precommitted Th17 cells rather than causing Th17 differentiation. Second, LPS drove Th17 cell expansion independently of IL-23, demonstrating that this cytokine is not necessary for expansion and possibly functions at an earlier stage in Th17 priming. Our data provide an impetus for using LPS-based peripheral vaccination to augment specific T-cell-mediated immunity in the gut mucosa.
Integrins contribute to lymphopoiesis, whereas Toll-like receptors (TLRs) facilitate the myeloid replenishment during inflammation. The combined role of TLRs and integrin on hematopoiesis remains unclear. gp96 (grp94, HSP90b1) is an endoplasmic reticulum master chaperone for multiple TLRs. We report herein that gp96 is also essential for expression of 14 hematopoietic system-specific integrins. Genetic deletion of gp96 thus enables us to determine the collective roles of gp96, integrins, and TLRs in hematopoiesis. We found that gp96-null hematopoietic stem cells could support long-term myelopoiesis. B- and T-cell development, however, was severely compromised with transitional block from pro-B to pre-B cells and the inability of thymocytes to develop beyond the CD4(-)CD8(-) stage. These defects were cell-intrinsic and could be recapitulated on bone marrow stromal cell culture. Furthermore, defective lymphopoiesis correlated strongly with failure of hematopoietic progenitors to form close contact with stromal cell niche and was not the result of the defect in the assembly of antigen receptor or interleukin-7 signaling. These findings define gp96 as the only known molecular chaperone to specifically regulate T- and B-cell development.
The history of immunizing with embryonic materials to generate an antitumor immune response dates back to a century ago. The premise is that cancer cells share the expression of oncofetal antigens with embryonic materials and that the immune response against these antigens in the embryonic tissues is cross-protective against cancer. However, such a practice has never advanced beyond experimental animal settings, because of lack of uniformed source tissues and ethical challenges. With the availability of well-characterized human pluripotent stem cells, it is now possible to ask whether tumor protective immunity could indeed be elicited with stem cells. Herein, we investigated whether vaccination with defined human embryonic stem cells (hESCs) or induced pluripotent stem (iPS) cells was effective against a colon carcinoma. We discovered that vaccination of mice with hESC line H9 generated consistent cellular and humoral immune responses against CT26 colon carcinoma. Protection correlated strongly with the expansion of tumor-responsive and interferon-gamma-producing cells and the profound loss of CD11b(+)Gr-1(+) myeloid-derived suppressor cells in the spleen. No evidence of autoimmunity was observed. We also compared the immunogenicity against colon cancer between a hESC line CT2 and an iPS cell line TZ1 that were generated in the same stem cell facility. We found that the iPS cell line was inferior to the hESC line in conferring tumor protection, suggesting that there is heterogeneity of expression of oncofetal antigens by hESCs and iPS cells. We conclude that the hESC-based vaccine is a promising modality for immunotherapy of cancer.
Mammalian heat shock protein gp96 is an obligate chaperone for multiple integrins and TLRs, the mechanism of which is largely unknown. We have identified gp93 in Drosophila having high sequence homology to gp96. However, no functions were previously attributed to gp93. To determine whether gp93 and gp96 are functionally conserved, we have expressed gp93 in gp96-deficient mouse cells. Remarkably, the Drosophila gp93 is able to chaperone multiple murine gp96 clients including integrins alpha(4), alpha(L), and beta(2) and TLR2 and TLR9. This observation has led us to examine the structural basis of the chaperone function of gp96 by a close comparison between gp96 and gp93. We report that whereas gp96 undergoes intermolecular disulfide bond formation via Cys(138), gp93 is unable to do so due to the absence of a cysteine near the same region. However, abrogation of disulfide bond formation by substituting C with A (C138A) in gp96 via site-directed mutagenesis did not compromise its chaperone function. Likewise, gp93 chaperone ability could not be improved by forcing intermolecular bond formation between gp93 N termini. We conclude that gp93 is the Drosophila ortholog of gp96 and that the chaperone function of the two molecules is conserved. Moreover, gp96 N-terminal disulfide bond formation is not critical for its function, underscoring the importance of N-terminal dimerization via non-disulfide bond-mediated interactions in client protein folding by gp96. Further study of gp96 from an evolutionary angle shall be informative to uncover the detailed mechanism of its chaperone function of client proteins in the secretory pathway.
Heat shock protein 90 (HSP90) family of proteins are ubiquitous molecular chaperones that are involved in folding, activation, maturation and assembly of many proteins that include essential mediators of signal transduction and cell cycle progression. They are abundant in eukaryotic cells and localized to the cytoplasm, mitochondria as well as the endoplasmic reticulum under normal conditions, making up 1-2% of all cellular proteins. HSP90 proteins have increased expression in a number of malignancies. A large number of HSP90 client proteins have been shown to be necessary for the development, proliferation and survival of specific types of cancers. HSP90 inhibition can affect multiple oncogenic pathways and involved proteins, therefore make it an attractive target for drug development. This article serves as an overview of the pre-clinical data and clinical trial data on HSP90 inhibitor 17-AAG in different malignancies. 17-AAG has shown significant anti-tumor activity against a spectrum of cancers in the pre-clinical studies and information from various phases of clinical trials is growing. The potential indication of 17-AGG for the treatment of refractory multiple myeloma now awaits for the results of two phase III studies. More work needs to be done before the broader oncological use of HSP90 inhibitors in the area of defining HSP90 client proteins, understanding the mechanism of HSP90 actions, identifying reliable surrogate markers for HSP90 inhibition in vivo and optimizing drug delivery and efficacy.
Inflammation is a necessary albeit insufficient component of tumorigenesis in some cancers. Infectious agents directly implicated in tumorigenesis have been shown to induce inflammation. This process involves both the innate and adaptive components of the immune system which contribute to tumor angiogenesis, tumor tolerance and metastatic properties of neoplasms. Recently, heat-shock proteins have been identified as mediators of this inflammatory process and thus may provide a link between infection-mediated inflammation and subsequent cancer development. In this review, the role of heat-shock proteins in infection-induced inflammation and carcinogenesis will be discussed.
Toll-like receptors (TLRs) are critical sensors for microbial products and are important in initiating both innate and adaptive immune defenses against pathogens. Emerging evidence suggests that TLRs are also expressed by regulatory T cells (Treg) that constitute an important immune suppressive cellular mechanism to curtail TLR hyperactivity to avoid sepsis and autoimmune diseases. This review brings up to date on the expression of functional TLRs on Treg and the functional impact of TLR activation on Treg biology. We argue that the suppressive function of Treg can be augmented or attenuated depending on the nature of TLR stimulations.
As a scientific discipline, medicine can only be advanced by experimentation. Experimentation could either validate or refute a hypothesis. Unfortunately, todays publication climate strongly favors publication of positive research findings, especially with clinical trials. Experimental Hematology & Oncology (eHO) is a new open access online journal that emphasizes preclinical, patient-oriented and translational aspects of research. The journal differentiates from others in the field by making a deliberate effort in publishing clinical trials with "negative" results and basic science studies with provocative findings. The focus of the peer-review mechanism for eHO will be on the technical merit of the study and not on demanding a long list of additional experiments that hinders rapid information dissemination.
Basophil has been implicated in anti-parasite defense, allergy and in polarizing T(H)2 response. Mouse model has been commonly used to study basophil function although the difference between human and mouse basophils is underappreciated. As an essential chaperone for multiple Toll-like receptors and integrins in the endoplasmic reticulum, gp96 also participates in general protein homeostasis and in the ER unfolded protein response to ensure cell survival during stress. The roles of gp96 in basophil development are unknown.
Low-dose endotoxemia is prevalent in humans with adverse health conditions, and it correlates with the pathogenesis of chronic inflammatory diseases such as atherosclerosis, diabetes, and neurologic inflammation. However, the underlying molecular mechanisms are poorly understood. In this study, we demonstrate that subclinical low-dose LPS skews macrophages into a mild proinflammatory state, through cell surface TLR4, IL-1R-associated kinase-1, and the Toll-interacting protein. Unlike high-dose LPS, low-dose LPS does not induce robust activation of NF-?B, MAPKs, PI3K, or anti-inflammatory mediators. Instead, low-dose LPS induces activating transcription factor 2 through Toll-interacting protein-mediated generation of mitochondrial reactive oxygen species, allowing mild induction of proinflammatory mediators. Low-dose LPS also suppresses PI3K and related negative regulators of inflammatory genes. Our data reveal novel mechanisms responsible for skewed and persistent low-grade inflammation, a cardinal feature of chronic inflammatory diseases.
Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were ?30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.
HSP90 chaperones a large number of proteins, and it plays essential roles in multiple signaling pathways to maintain protein homeostasis in the cytosol. In addition, HSP90 has been implicated in mediating recognition of lipopolysaccharide (LPS). However, no pharmacologic agents have been developed to interrogate this pathway. Herein we demonstrate that a peptide-based inhibitor that was previously reported to inhibit the master Toll-like receptor-chaperone gp96, an endoplasmic reticulum paralog of HSP90, in fact blocks HSP90-LPS interaction. It inhibited the binding of LPS to the cell surface of both wild type and gp96-null cells and thereby abrogated the cellular response to LPS but not to other Toll-like receptor ligands. We also generated a series of peptide derivatives (named peptide inhibitors of endotoxin responsiveness (PIERs)) from the N-terminal helix structure of HSP90 and demonstrated their effectiveness in blocking LPS activity. PIER inhibition of LPS signaling was partially reversed by CD14 expression. Moreover, we found that a cell-permeable PIER abrogated HSP90 function and caused degradation of multiple known HSP90 client proteins in cancer cells. Thus, targeting HSP90 is a promising modality for treatment of both LPS-mediated pathology and cancer.
The structural basis for molecular chaperones to discern misfolded proteins has long been an enigma. As the endoplasmic reticulum paralogue of the cytosolic HSP90, gp96 (GRP94, HSP90b1) is an essential molecular chaperone for Toll-like receptors (TLRs) and integrins. However, little is known about its client-binding domain (CBD). Herein, we provide genetic and biochemical evidence to definitively demonstrate that a C-terminal loop structure, formed by residues 652-678, is the critical region of CBD for both TLRs and integrins. Deletion of this region affects neither the intrinsic ATPase activity nor the overall conformation of gp96. However, without it, the chaperoning function of gp96 collapses. We also find a critical Met pair (Met(658)-Met(662)) for the folding of integrins but not TLRs. Moreover, we find that the TLR binding to gp96 is also dependent on the C-terminal dimerization domain but not the N-terminal ATP-binding pocket of gp96. Our study has unveiled surprisingly the exquisite specificity of gp96 in substrate binding and suggests a manipulation of its CBD as an alternative strategy for targeted therapy of a variety of diseases.
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