A machine learning approach to predict mortality due to immune-mediated thrombotic thrombocytopenic purpura.

Background: Mortality due to immune-mediated thrombotic thrombocytopenic purpura (iTTP) remains significant. Predicting mortality risk may potentially help individualize treatment. The French Thrombotic Microangiopathy (TMA) Reference Score has not been externally validated in the United States. Recent advances in machine learning technology can help analyze large numbers of variables with complex interactions for the development of prediction models. Objectives: To validate the French TMA Reference Score in the United States Thrombotic Microangiopathy (USTMA) iTTP database and subsequently develop a novel mortality prediction tool, the USTMA TTP Mortality Index. Methods: We analyzed variables available at the time of initial presentation, including demographics, symptoms, and laboratory findings. We developed our model using gradient boosting machine, a machine learning ensemble method based on classification trees, implemented in the R package gbm. Results: In our cohort (n = 419), the French score predicted mortality with an area under the receiver operating characteristic curve of 0.63 (95% CI: 0.50-0.77), sensitivity of 0.35, and specificity of 0.84. Our gradient boosting machine model selected 8 variables to predict acute mortality with a cross-validated area under the receiver operating characteristic curve of 0.77 (95% CI: 0.71-0.82). The 2 cutoffs corresponded to sensitivities of 0.64 and 0.50 and specificities of 0.76 and 0.87, respectively. Conclusion: The USTMA Mortality Index was acceptable for predicting mortality due to acute iTTP in the USTMA registry, but not sensitive enough to rule out death. Identifying patients at high risk of iTTP-related mortality may help individualize care and ultimately improve iTTP survival outcomes. Further studies are needed to provide external validation. Our model is one of many recent examples where machine learning models may show promise in clinical prediction tools in healthcare.

Race, rituximab, and relapse in TTP.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by recurring episodes of thrombotic microangiopathy, causing ischemic organ impairment. Black patients are overrepresented in iTTP cohorts in the United States, but racial disparities in iTTP outcome and response to therapy have not been studied. Using the United States Thrombotic Microangiopathies Consortium iTTP Registry, we evaluated the impact of race on mortality and relapse-free survival (RFS) in confirmed iTTP in the United States from 1995 to 2020. We separately examined the impact of rituximab therapy and presentation with newly diagnosed (de novo) or relapsed iTTP on RFS by race. A total of 645 participants with 1308 iTTP episodes were available for analysis. Acute iTTP mortality did not differ by race. When all episodes of iTTP were included, Black race was associated with shorter RFS (hazard ratio [HR], 1.60; 95% CI, 1.16-2.21); the addition of rituximab to corticosteroids improved RFS in White (HR, 0.37; 95% CI, 0.18-0.73) but not Black patients (HR, 0.96; 95% CI, 0.71-1.31). In de novo iTTP, rituximab delayed relapse, but Black patients had shorter RFS than White patients, regardless of treatment. In relapsed iTTP, rituximab significantly improved RFS in White but not Black patients. Race affects overall relapse risk and response to rituximab in iTTP. Black patients may require closer monitoring, earlier retreatment, and alternative immunosuppression after rituximab treatment. How race, racism, and social determinants of health contribute to the disparity in relapse risk in iTTP deserves further study.

Novel venous thromboembolism mouse model to evaluate the role of complete and partial factor XIII deficiency in pulmonary embolism risk.

BACKGROUND: Venous thrombosis (VT) and pulmonary embolism (PE), collectively venous thromboembolism (VTE), cause high mortality and morbidity. Factor XIII (FXIII) crosslinks fibrin to enhance thrombus stability and consequently may influence PE risk. Elucidating mechanisms contributing to PE is limited by a lack of models that recapitulate human PE characteristics. OBJECTIVE: We aimed to develop a mouse model that permits embolization of red blood cell (RBC)- and fibrin-rich VT and determine the contribution of FXIII to PE risk. METHODS AND RESULTS: In a thrombin-infusion PE model, F13a+/+ , F13a+/- , and F13a-/-  mice had similar incidence of microthrombi in the lungs; however, thrombi were small, with low RBC content (≤7%), unlike human PEs (~70%). To identify a model producing PE consistent with histological characteristics of human PE, we compared mouse femoral vein electrolytic injury, femoral vein FeCl3 injury, and infrarenal vena cava (IVC) stasis models of VT. Electrolytic and FeCl3  models produced small thrombi with few RBCs (5% and 4%, respectively), whereas IVC stasis produced large thrombi with higher RBC content (68%) that was similar to human PEs. After IVC stasis and ligature removal (de-ligation) to permit thrombus embolization, compared to F13a+/+ mice, F13a+/-  and F13a-/-  mice had similar and increased PE incidence, respectively. CONCLUSIONS: Compared to thrombin infusion-, electrolytic injury-, and FeCl3 -based models, IVC stasis produces thrombi that are more histologically similar to human thrombi. IVC stasis followed by de-ligation permits embolization of existing RBC- and fibrin-rich thrombi. Complete FXIII deficiency increases PE incidence, but partial deficiency does not.

Spinal Anesthetic in a Patient With a Platelet Count of 7000 × 109/L and Undiagnosed Thrombotic Thrombocytopenic Purpura: A Case Report.

A parturient with unknown thrombotic thrombocytopenic purpura (TTP) received spinal anesthesia for cesarean delivery with subsequent discovery of a platelet count of 7000 × 10/L. Neurologic recovery was normal. Limited data exist to determine the risk of spinal epidural hematoma (SEH) in severely thrombocytopenic patients because they often receive alternate labor analgesia or general anesthesia during cesarean delivery. There is reporting bias in the literature toward cases in which severely thrombocytopenic patients sustain complications after regional anesthesia. It is important to report all cases of neuraxial anesthesia in severely thrombocytopenic patients, including those such as ours, wherein patients recover normally.

Upregulation of autophagy genes and the unfolded protein response in human heart failure.

The cellular environment of the mammalian heart constantly is challenged with environmental and intrinsic pathological insults, which affect the proper folding of proteins in heart failure. The effects of damaged or misfolded proteins on the cell can be profound and result in a process termed "proteotoxicity". While proteotoxicity is best known for its role in mediating the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, its role in human heart failure also has been recognized. The UPR involves three branches, including PERK, ATF6, and IRE1. In the presence of a misfolded protein, the GRP78 molecular chaperone that normally interacts with the receptors PERK, ATF6, and IRE-1 in the endoplasmic reticulum detaches to attempt to stabilize the protein. Mouse models of cardiac hypertrophy, ischemia, and heart failure demonstrate increases in activity of all three branches after removing GRP78 from these internal receptors. Recent studies have linked elevated PERK and CHOP in vitro with regulation of ion channels linked with human systolic heart failure. With this in mind, we specifically investigated ventricular myocardium from 10 patients with a history of conduction system defects or arrhythmias for expression of UPR and autophagy genes compared to myocardium from non-failing controls. We identified elevated Chop, Atf3, and Grp78 mRNA, along with XBP-1-regulated Cebpa mRNA, indicative of activation of the UPR in human heart failure with arrhythmias.

Chemotherapy enriches for an invasive triple-negative breast tumor cell subpopulation expressing a precursor form of N-cadherin on the cell surface.

BACKGROUND: Although most triple-negative breast cancer (TNBC) patients initially respond to chemotherapy, residual tumor cells frequently persist and drive recurrent tumor growth. Previous studies from our laboratory and others' indicate that TNBC is heterogeneous, being composed of chemo-sensitive and chemo-resistant tumor cell subpopulations. In the current work, we studied the invasive behaviors of chemo-resistant TNBC, and sought to identify markers of invasion in chemo-residual TNBC. METHODS: The invasive behavior of TNBC tumor cells surviving short-term chemotherapy treatment in vitro was studied using transwell invasion assays and an experimental metastasis model. mRNA expression levels of neural cadherin (N-cadherin), an adhesion molecule that promotes invasion, was assessed by PCR. Expression of N-cadherin and its precursor form (pro-N-cadherin) was assessed by immunoblotting and flow cytometry. Pro-N-cadherin immunohistochemistry was performed on tumors obtained from patients pre- and post- neoadjuvant chemotherapy treatment. RESULTS: TNBC cells surviving short-term chemotherapy treatment exhibited increased invasive behavior and capacity to colonize metastatic sites compared to untreated tumor cells. The invasive behavior of chemo-resistant cells was associated with their increased cell surface expression of precursor N-cadherin (pro-N-cadherin). An antibody specific for the precursor domain of N-cadherin inhibited invasion of chemo-resistant TNBC cells. To begin to validate our findings in humans, we showed that the percent cell surface pro-N-cadherin (+) tumor cells increased in patients post- chemotherapy treatment. CONCLUSIONS: TNBC cells surviving short-term chemotherapy treatment are more invasive than bulk tumor cells. Cell surface pro-N-cadherin expression is associated with the invasive and chemo-resistant behaviors of this tumor cell subset. Our findings indicate the importance of future studies determining the value of cell surface pro-N-cadherin as: 1) a biomarker for TNBC recurrence and 2) a therapeutic target for eliminating chemo-residual disease.

Asbestos content of lung tissue in patients with malignant peritoneal mesothelioma: A study of 42 cases.

Lung tissue from 42 peritoneal mesothelioma cases was analyzed by light microscopy and scanning electron microscopy/energy dispersive spectrometry. There were 34 men and 8 women with a mean age of 61 ± 10 years. Also, 17% of cases had histologically confirmed asbestosis, and 26% had only parietal pleural plaques. The asbestos body count exceeded our normal range in 22 of 42 cases (52%). Cases with asbestos-related pulmonary disease had higher fiber burdens than those without. The vast majority of fibers were commercial amphiboles (amosite with lesser amounts of crocidolite). These findings concur with previously published epidemiological observations.

BRG1 and BRM SWI/SNF ATPases redundantly maintain cardiomyocyte homeostasis by regulating cardiomyocyte mitophagy and mitochondrial dynamics in vivo.

There has been an increasing recognition that mitochondrial perturbations play a central role in human heart failure. Mitochondrial networks, whose function is to maintain the regulation of mitochondrial biogenesis, autophagy ('mitophagy') and mitochondrial fusion/fission, are new potential therapeutic targets. Yet our understanding of the molecular underpinning of these processes is just emerging. We recently identified a role of the SWI/SNF ATP-dependent chromatin remodeling complexes in the metabolic homeostasis of the adult cardiomyocyte using cardiomyocyte-specific and inducible deletion of the SWI/SNF ATPases BRG1 and BRM in adult mice (Brg1/Brm double mutant mice). To build upon these observations in early altered metabolism, the present study looks at the subsequent alterations in mitochondrial quality control mechanisms in the impaired adult cardiomyocyte. We identified that Brg1/Brm double-mutant mice exhibited increased mitochondrial biogenesis, increases in 'mitophagy', and alterations in mitochondrial fission and fusion that led to small, fragmented mitochondria. Mechanistically, increases in the autophagy and mitophagy-regulated proteins Beclin1 and Bnip3 were identified, paralleling changes seen in human heart failure. Evidence for perturbed cardiac mitochondrial dynamics included decreased mitochondria size, reduced numbers of mitochondria, and an altered expression of genes regulating fusion (Mfn1, Opa1) and fission (Drp1). We also identified cardiac protein amyloid accumulation (aggregated fibrils) during disease progression along with an increase in pre-amyloid oligomers and an upregulated unfolded protein response including increased GRP78, CHOP, and IRE-1 signaling. Together, these findings described a role for BRG1 and BRM in mitochondrial quality control, by regulating mitochondrial number, mitophagy, and mitochondrial dynamics not previously recognized in the adult cardiomyocyte. As critical to the pathogenesis of heart failure, epigenetic mechanisms like SWI/SNF chromatin remodeling seem more intimately linked to cardiac function and mitochondrial quality control mechanisms than previously realized.

Malignant mesothelioma not related to asbestos exposure: Analytical scanning electron microscopic analysis of 83 cases and comparison with 442 asbestos-related cases.

Epidemiological studies indicate that 80-90% of mesotheliomas are asbestos related. This suggests that 10-20% are not. Lung fiber burden analysis provides objective information about past exposures to asbestos. We have performed lung fiber burden analysis on a large cohort of mesothelioma cases and compared the findings with a reference population. Herein we report our findings along with demographic and exposure data.

Persistent thrombocytopaenia in a young man with splenomegaly, rebound thrombocytosis after splenectomy and subsequent pulmonary embolism: splenic littoral cell angioma and associated events.

Littoral cell angioma (LCA) is a rare endothelial cell neoplasm in the spleen. Although many cases of LCA are asymptomatic, some present with signs and symptoms related to splenomegaly, whereas others manifest with haematological abnormalities, including anaemia and/or thrombocytopaenia (ie, hypersplenism). We report a case of LCA presenting with chronic thrombocytopaenia, probably due to splenic sequestration of platelets or phagocytosis of platelets by neoplastic cells. Following therapeutic splenectomy, the patient suffered from a marked rebound thrombocytosis and subsequently developed pulmonary embolisms. He was treated with anticoagulant therapy combined with antiplatelet therapy, and his symptoms were quickly resolved. This case emphasises an exclusion of primary splenic disorders in patients with chronic thrombocytopaenia, especially in those with splenomegaly and the contemplation of thromboembolism prophylaxis postsplenectomy.

Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

INTRODUCTION: Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)]. METHODS: Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy. RESULTS: TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells. CONCLUSION: These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

Comparison of Acute Histologic and Biomechanical Effects of Radiofrequency Ablation and Cryoablation on Periarticular Structures in a Swine Model.

PURPOSE: To compare the acute effects of radiofrequency (RF) ablation and cryoablation on the structural integrity of nontarget periarticular tissues that may be placed at risk during percutaneous bone ablation. MATERIALS AND METHODS: RF ablation and cryoablation were separately performed on tendon, articular cartilage, and ligament in an ex vivo porcine model by using standard bone ablation protocols. Gross and histopathologic analysis was performed on cartilage and tendon (n = 6 for each treatment group, n = 5 controls). Tendon lengths were measured before and after ablation. Biomechanical tensile testing was performed on each ligament sample after ablation, with quantification of ultimate load at failure and linear stiffness (n = 7 ligaments in treatment and control groups). RESULTS: RF ablation and cryoablation injured chondrocytes within the ablation zones but caused minimal effects on gross and histologic cartilage architecture. Cryoablation resulted in minimal gross and histologic effects on tendon whereas RF ablation resulted in marked disruption of collagen fibers and significant longitudinal shortening (P = .002). Similarly, cryoablation did not alter ligament strength or stiffness compared with control, whereas RF ablation resulted in a significant decrease in tensile strength and stiffness compared with control and cryoablation samples (P < .001). CONCLUSIONS: Neither RF ablation nor cryoablation resulted in significant acute changes in cartilage architecture. However, RF ablation resulted in marked disruption of tendon architecture, tendon shortening, ligament weakening, and loss of ligament stiffness, whereas cryoablation had no significant effect on any of these parameters. These findings suggest that cryoablation may have fewer negative acute effects than RF ablation, although long-term outcomes are currently unknown.

Binding of tissue-type plasminogen activator to the glucose-regulated protein 78 (GRP78) modulates plasminogen activation and promotes human neuroblastoma cell proliferation in vitro.

The glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) receptor on the cell surface. In this study, we demonstrate that GRP78 also binds the tissue-type plasminogen activator (t-PA), which results in a decrease in K(m) and an increase in the V(max) for both its amidolytic activity and activation of its substrate, Pg. This results in accelerated Pg activation when GRP78, t-PA, and Pg are bound together. The increase in t-PA activity is the result of a mechanism involving a t-PA lysine-dependent binding site in the GRP78 amino acid sequence (98)LIGRTWNDPSVQQDIKFL(115). We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is also a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface. Furthermore, Pg binding to the COOH-terminal region of GRP78 stimulates cell proliferation via its microplasminogen domain. This study confirms previous findings from our laboratory showing that GRP78 acts as a growth factor-like receptor and that its association with t-PA, Pg, and VDAC on the cell surface may be part of a system controlling cell growth.

L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in ß-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial role for LTCCs in regulation of expression, activity and stability of aquaporin-0, connexins, cytoskeletal proteins, and the mechanical properties of lens, all of which have a vital role in maintaining lens function and cytoarchitecture.

The Escherichia coli subtilase cytotoxin A subunit specifically cleaves cell-surface GRP78 protein and abolishes COOH-terminal-dependent signaling.

GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH(2)-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78(low)) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH(2)-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.

A murine monoclonal antibody directed against the carboxyl-terminal domain of GRP78 suppresses melanoma growth in mice.

The HSP70 family member GRP78 is a selective tumor marker upregulated on the surface of many tumor cell types, including melanoma, where it acts as a growth factor receptor-like protein. Receptor-recognized forms of the proteinase inhibitor α2-macroglobulin (α2M*) are the best-characterized ligands for GRP78, but in melanoma and other cancer patients, autoantibodies arise against the NH2-terminal domain of GRP78 that react with tumor cell-surface GRP78. This causes the activation of signaling cascades that are proproliferative and antiapoptotic. Antibodies directed against the COOH-terminal domain of GRP78, however, upregulate p53-mediated proapoptotic signaling, leading to cell death. Here, we describe the binding characteristics, cell signaling properties, and downstream cellular effects of three novel murine monoclonal antibodies. The NH2-terminal domain-reactive antibody, N88, mimics α2M* as a ligand and drives PI 3-kinase-dependent activation of Akt and the subsequent stimulation of cellular proliferation in vitro. The COOH-terminal domain-reactive antibody, C38, acts as an antagonist of both α2M* and N88, whereas another, C107, directly induces apoptosis in vitro. In a murine B16F1 melanoma flank tumor model, we demonstrate the acceleration of tumor growth by treatment with N88, whereas C107 significantly slowed tumor growth whether administered before (P<0.005) or after (P<0.05) tumor implantation.

Changes in oligosaccharide chains of autoantibodies to GRP78 expressed during progression of malignant melanoma stimulate melanoma cell growth and survival.

A correlation between expression of the glucose-regulated protein of 78 kDa (GRP78) in malignant melanoma tumors and poor patient survival is well established. In this study, in addition to demonstrating the expression of GRP78 in tumor tissue, we investigated the immune response against GRP78 in a group of patients with different progression stages of malignant melanoma. Furthermore, we analyzed the glycosylation status of GRP78 immunoglobulin (Ig) G autoantibodies at these stages and evaluated their capacities to affect the protein B-dependent protein kinase signaling pathway and unfolded protein response signaling mechanisms, all known to promote malignant melanoma cell proliferation and survival. We found that progression of disease correlates not only with enhanced expression of GRP78 in the tumor but also with an increase in GRP78 autoantibody serum titers in these patients. We also found that the glycosylation status of anti-GRP78 IgG changes as the disease progresses. The anti-GRP78 IgG is abnormally glycosylated in the Fc region and asymmetrically glycosylated in the Fab region. We demonstrate that hyperglycosylated anti-GRP78 IgGs stimulate cell proliferation through protein B-dependent protein kinase signaling pathways. They also mimic the effects of α2-macroglobulin on the upregulation of GRP78 and X-box binding protein 1, activating transcription factor 6 α, and serine/threonine-protein kinase/endoribonuclease precursor α as endoplasmic reticulum stress biomarkers and show no effect on expression or activation of caspases 3, 9, or 12. In conclusion, the anti-GRP78 IgG autoantibodies downregulate apoptosis and activate unfolded protein response mechanisms, which are essential to promote melanoma cell growth and survival.

Modulation of the unfolded protein response by GRP78 in prostate cancer.

The unfolded protein response (UPR) is an adaptive survival mechanism through which cells can weather the stress of misfolded protein accumulation induced by a wide variety of pathophysiologic and pharmacologic insults. The ER chaperone GRP78 is a central modulator of the UPR both through its protein-binding capacity and its direct regulation of the UPR signaling molecules IRE1α, PERK, and ATF6. Recent reports have revealed the presence of GRP78 on the surface of cancer cells. Biological roles for cell-surface GRP78 include competing NH(2)-domain and COOH-domain agonist receptor activities that induce opposite effects on proliferation and apoptosis. Modulation of the UPR impacts both of these processes directly and indirectly. Here, we outline methods that we use to investigate UPR modulation via direct ligation of cell-surface GRP78. Specifically, we review methods of cell culture, cell-signaling analysis with emphasis on UPR components, and ultimately, the impact that these have on cell proliferation, survival, and apoptosis.

Autoantibodies against cell surface GRP78 promote tumor growth in a murine model of melanoma.

Autoantibodies that react with GRP78 expressed on the cell-surface of many tumor cell lines occur in the sera of patients with prostate cancer, melanoma, and ovarian cancer. These autoantibodies are a negative prognostic factor in prostate cancer and, when purified, stimulate tumor cell proliferation in vitro. It is unclear, however, whether these immunoglobulin Gs are merely a biomarker, or whether they actually promote the tumor growth in vivo. We immunized C57Bl/6 mice with recombinant GRP78 and then implanted the B16F1 murine melanoma cell line as flank tumors. We used the antisera from these mice for in-vitro cell signaling and proliferation assays. The immunodominant epitope in patients with cancer was well represented in the antibody repertoire of these immunized mice. We observed significantly accelerated tumor growth, and shortened survival in GRP78-immunized mice compared with controls. Furthermore, antisera from these mice, and purified anti-GRP78 immunoglobulin G from similarly immunized mice, stimulate Akt phosphorylation and proliferation in B16F1 and human DM6 melanoma cells in culture. These studies show a causal link between a humoral response to GRP78 and the progression of cancer in a murine melanoma model. They support the hypothesis that such autoantibodies are involved in the progression of human cancers and are not simply a biomarker. As GRP78 is present on the surface of many types of cancer cells, this hypothesis has broad clinical and therapeutic implications.