A novel CSN5/CRT O-GlcNAc/ER stress regulatory axis in platinum resistance of epithelial ovarian cancer.

Background: High levels of COP9 signalosome subunit 5 (CSN5) in epithelial ovarian cancer (EOC) are associated with poor prognosis and are implicated in mediating platinum resistance in EOC cells. The underlying mechanisms, however, remained undefined. This study aimed to elucidate the molecular process and identify potential therapeutic targets. Methods: RNA-sequencing was used to investigate differentially expressed genes between platinum-resistant EOC cells with CSN5 knockdown and controls. O-GlcNAc proteomics were employed to identify critical modulators downstream of CSN5. The omics findings were confirmed through qRT-PCR and immunoblotting. In vitro and in vivo experiments assessed the sensitivity of resistant EOCs to platinum. Results: We demonstrated an involvement of aberrant O-GlcNAc and endoplasmic reticulum (ER) stress disequilibrium in CSN5-mediated platinum resistance of EOC. Genetic or pharmacologic inhibition of CSN5 led to tumor regression and surmounted the intrinsic EOC resistance to platinum both in vitro and in vivo. Integration of RNA-sequencing and O-GlcNAc proteomics pinpointed calreticulin (CRT) as a potential target of aberrant O-GlcNAc modification. CSN5 upregulated O-GlcNAc-CRT at T346 to inhibit ER stress-induced cell death. Blocking T346 O-GlcNAc-CRT through CSN5 deficiency or T346A mutation resulted in Ca2+ disturbances, followed by ER stress overactivation, mitochondrial dysfunction, and ultimately cell apoptosis. Conclusion: This study reveals that CSN5-mediated aberrant O-GlcNAc-CRT acts as a crucial ER stress checkpoint, governing cell fate response to stress, and emphasizes an unrecognized role for the CSN5/CRT O-GlcNAc/ER stress axis in platinum resistance of EOC.

Impact of increased plasma levels of calreticulin on prognosis of patients with advanced lung cancer undergoing combination treatment of chemotherapy and immune checkpoint inhibitors.

BACKGROUND: Damage-associated molecular pattern (DAMP)-related immunogenic cell death triggers secondary adaptive immune responses. The relationship between DAMP levels and prognosis in patients with non-small cell lung cancer (NSCLC) who undergo a combination therapy of immune checkpoint inhibitors (ICI) and chemotherapy remains unclear. METHODS: Serial plasma samples were prospectively collected from 45 patients treated with ICI combination therapy for advanced NSCLC. Plasma concentrations of high-mobility group box 1 (HMGB1), calreticulin (CRT), annexin A1, and heat shock protein 70 were measured. Associations between increases in plasma DAMP levels and the efficacy of the ICI combination therapy were evaluated. RESULTS: The maximum fold changes in plasma levels differed across individuals but demonstrated a marked increase, especially for CRT (mean ± SEM, 11.61 ± 46.15). Increased plasma DAMP levels were not clearly associated with clinical responses. There was a significant correlation between the maximum fold change in CRT levels and progression-free survival (PFS; r = 0.49, P < 0.001). Median PFS and overall survival (OS) rates were higher in patients with a ≥ 2-fold increase in plasma CRT levels than in those with a < 2-fold increase (PFS, 14.9 versus 6.0 months, hazard ratio (HR), 0.58; P = 0.17; OS, not reached versus 21.6 months, HR, 0.31, P = 0.02). CONCLUSIONS: Plasma CRT level monitoring has the potential to predict the efficacy of ICI combination therapy and shed light on the mechanisms underlying DAMP-related immunogenic cell death.

Normalization of Tumor Vessels by Lenvatinib-Based Metallo-Nanodrugs Alleviates Hypoxia and Enhances Calreticulin-Mediated Immune Responses in Orthotopic HCC and Organoids.

Immunocheckpoint inhibitors combined with Lenvatinib is the first line treatment for hepatocellular carcinoma (HCC), but their potency is hampered by the low response rate and adverse events. Herein, a targeted therapeutic strategy through the coassembly of Lenvatinib, Adriamycin, Fe3+ ion, and a natural polyphenol (metallo-nanodrugs) is presented by coordination effect for potentiating tumor vascular normalization and systematic chemo-immunotherapy to effectively inhibit the progression of HCC in both orthotopic model and patients-derived organoids. In mice with orthotopic HCC, the obtained metallo-nanodrugs efficiently increase the drug accumulation in orthotopic tumors and can respond to acidic tumor environment. The promotion of tumor vascular normalization by metallo-nanodrugs is observed, which enhances the infiltrating T lymphocytes in tumor, and reinforces the calreticulin-mediated antitumor immunity through alleviating hypoxia, reducing regulatory T cells, and down-regulating PDL1 expression of tumors. The excellent therapeutic efficiency with complete remission of orthotopic tumors (3/6) and long-term survival of mice (4/6, 42 days) are also achieved. Furthermore, the excellent therapeutic effect of metallo-nanodrugs is also validated in 5 patient-derived organoids, and hence can provide a marvelous systemic chemo-immunotherapy strategy for enhancing HCC treatment.

The anticancer effect of PASylated calreticulin-targeting L-ASNase in solid tumor bearing mice with immunogenic cell death-inducing chemotherapy.

L-Asparaginase (L-ASNase), a bacterial enzyme that degrades asparagine, has been commonly used in combination with several chemical drugs to treat malignant hematopoietic cancers such as acute lymphoblastic leukemia (ALL). In contrast, the enzyme was known to inhibit the growth of solid tumor cells in vitro, but not to be effective in vivo. We previously reported that two novel monobodies (CRT3 and CRT4) bound specifically with calreticulin (CRT) exposed on tumor cells and tissues during immunogenic cell death (ICD). Here, we engineered L-ASNases conjugated with monobodies at the N-termini and PAS200 tags at the C-termini (CRT3LP and CRT4LP). These proteins were expected to possess four monobody and PAS200 tag moieties, which did not disrupt the L-ASNase conformation. These proteins were expressed 3.8-fold more highly in E. coli than those without PASylation. The purified proteins were highly soluble, with much greater apparent molecular weights than expected ones. Their affinity (Kd) against CRT was about 2 nM, 4-fold higher than that of monobodies. Their enzyme activity (∼6.5 IU/nmol) was similar to that of L-ASNase (∼7.2 IU/nmol), and their thermal stability was significantly increased at 55 °C. Their half-life times were > 9 h in mouse sera, about 5-fold longer than that of L-ASNase (∼1.8 h). Moreover, CRT3LP and CRT4LP bound specifically with CRT exposed on tumor cells in vitro, and additively suppressed the tumor growth in CT-26 and MC-38 tumor-bearing mice treated with ICD-inducing drugs (doxorubicin and mitoxantrone) but not with a non-ICD-inducing drug (gemcitabine). All data indicated that PASylated CRT-targeted L-ASNases enhanced the anticancer efficacy of ICD-inducing chemotherapy. Taken together, L-ASNase would be a potential anticancer drug for treating solid tumors.

Interference of immunogenic chemotherapy by artificially controlled calreticulin secretion from tumor cells.

The exposure of calreticulin (CALR) on the cell surface of apoptotic cancer cells is an important "eat-me" signal that stimulates the engulfment by antigen presenting cells (APCs). When cells are exposed to immunogenic cell death (ICD) inducers, CALR translocates from the lumen of the endoplasmic reticulum (ER) to the cell surface, where it serves as a ligand for LDL-receptor-related protein 1 (LRP1, also known as CD91) expressed by dendritic cells (DCs). Surface-exposed CALR facilitates tumor antigen transfer to DCs and in turn antigen cross-presentation to cytotoxic T cells, altogether culminating in the activation of adaptive immune responses. Consistent with its role as an apical signaling event in anticancer immunity, blocking or neutralizing CALR abolishes the immune-dependent anticancer efficacy of a variety of ICD inducing anticancer agents. Recently we showed that saturating CALR receptors on DCs with abundant recombinant CALR protein, or soluble CALR secreted from cancer cells decreases the potency of ICD-mediated antitumor immune responses. Here we detail how to harness an artificially inducible release of soluble CALR from engineered cancer cells, which can blind DCs from recognizing immunogenic cancer cells, resulting in reduced anticancer immunity. This system offers precise control over the release of immunosuppressive soluble CALR, thus yielding a useful tool for the validation of ICD-inducing immunotherapies.

Camptothesome elicits immunogenic cell death to boost colorectal cancer immune checkpoint blockade.

Camptothesome is an innovative nanovesicle therapeutic comprising the sphingomyelin-derived camptothecin (CPT) lipid bilayer. In this work, we deciphered that Camptothesome was taken up by colorectal cancer (CRC) cells through primarily the clathrin-mediated endocytotic pathway and displayed the potential of eliciting robust immunogenic cancer cell death (ICD) via upregulating calreticulin, high mobility group box 1 protein (HMGB-1), and adenosine triphosphate (ATP), three hallmarks involved in the induction of ICD. In addition, use of dying MC38 tumor cells treated with Camptothesome as vaccine prevented tumor growth in 60% mice that received subsequent injection of live MC38 cells on the contralateral flank, validating Camptothesome was a legitimate ICD inducer in vivo. Camptothesome markedly reduced the acute bone marrow toxicity and gastrointestinal mucositis associated with free CPT and beat free CPT and Onivyde on anti-CRC efficacy and immune responses in a partially interferon gamma (IFN-γ)-dependent manner. Furthermore, Camptothesome enhanced the efficacy of immune checkpoint inhibitors to shrink late-stage orthotopic MC38 CRC tumors with diminished tumor metastasis and markedly prolonged mice survival.

Two myeloproliferative diseases in one patient - co-occurence of clones / Két myeloproliferativ betegség egy betegben - klónok együttélése és vetélkedése

Classical myeloproliferative diseases can be divided into Philadelphia chromosome-positive chronic myeloid leukemia and Philadelphia chromosome-negative myeloproliferative neoplasm. The driver mutations of the latter occur in the Janus kinase 2 or calreticulin genes. The coincidence of Philadelphia chromosome-negative and -positive myeloproliferative neoplasms in the same patient is exceptionally rare in the literature. During the long-term follow-up of our 120 patients with chronic myeloid leukemia, we investigated the clinical data of patients in whom Philadelphia chromosome-negative myeloproliferative disease was also confirmed. Philadelphia chromosome was detected by classical cytogenetic methods and/or fluorescence in situ hybridization. The amount of BCR-ABLI fusion RNA was monitored by quantitative real-time polymerase chain reaction. Mutations in the Janus kinase 2 and calreticulin genes were detected by quantitative allele-specific polymerase chain reaction and fragment analysis. The dynamics of disease development were inferred from the change in the amount of mutant clones over time and from the clinical data. We identified four cases carrying both Philadelphia chromosome and Janus kinase 2/calreticulin gene mutation. In some cases, competition between the clones, in other cases their co-occurrence in a common clone was observed. Isolated thrombocytosis at the time of diagnosis or persisting thrombocytosis during targeted therapy with good molecular response may call attention to the possibility of the co-occurrence of the two diseases. Co-occurrence of Philadelphia chromosome-positive and -negative myeloproliferative neoplasms is more frequent than the literature suggests. If the disease has an unusual appearance, the association of the two myeloproliferative dieseases may be suspected.

Immunogenic analysis of epitope-based vaccine candidate induced by photodynamic therapy in MDA-MB-231 triple-negative breast cancer cells.

BACKGROUND: Photodynamic therapy (PDT) is used to treat tumors through selective cytotoxic effects. PDT induces damage-associated molecular patterns (DAMPs) expression, which can cause an immunogenic death cell (IDC). In this study we identified potential immunogenic epitopes generated by PDT on triple-negative breast cancer cell line (MDA-MB-231). METHODS: MDA-MB-231 cells were exposed to PDT using ALA (160 µg/mL)/630 nm at 8 J/cm2. Membrane proteins were extracted and separated by 2D PAGE. Proteins overexpressed were identified by LC-MS/MS and analyzed in silico through a peptide-HLA docking in order to identify the epitopes with more immunogenicity and antigenicity properties, as well as lower allergenicity and toxicity activity. The selected peptides were evaluated in response to macrophage activation and cytokine release by flow cytometry. RESULTS: Differential proteins were overexpressed in the cells treated with PDT. A group of 16 peptides were identified from them, established in a rigorous selection by measuring antigenicity, immunogenicity, allergenicity, and toxicity in silico. The final selection was based on molecular dynamics, where 2 peptides showed the highest stability regarding to the RMSD value. These peptides were obtained from the proteins calreticulin and HSP90. The cytokine analysis evidenced macrophage activation by the releasing of TNF. CONCLUSION: Two peptides were identified from calreticulin and HSP90; proteins induced by PDT in MDA-MB-231 cells. Both epitopes showed immunogenic potential as a peptide-based vaccine for triple-negative breast cancer.

CALR-ETdb, the database of calreticulin variants diversity in essential thrombocythemia.

Essential thrombocythemia (ET) is a blood cancer defined by a strong increase of platelet numbers. A quarter of patients suffering from ET show mutations in the last exon of calreticulin (CALR) gene. Two variants named type 1 and type 2 represent 85% of these patients. However, a large number of other variants have been determined. In this study, we have compiled variants taken from COSMIC database and literature leading to 155 different variants. This large number of variants allowed redefining 5 new classes extending the classification of type 1-like and type 2-like to a finer description. These analyses showed that last class, named E, corresponding to more than 10% of CALR variants seemed not attached to ET. Structural properties analyzed showed that CALR variants associated to ET have common features. All the compiled and refined information had been included into a freely dedicated database CALR-ETdb (https://www.dsimb.inserm.fr/CALR-ET).

Cytotoxic chemotherapeutic agents and the EGFR-TKI osimertinib induce calreticulin exposure in non-small cell lung cancer.

OBJECTIVES: Synergistic anticancer efficacy of combination treatment with immune checkpoint inhibitors (ICIs) and platinum-based chemotherapy in patients with advanced non-small cell lung cancer (NSCLC) may be attributable in part to the phenomenon of immunogenic cell death (ICD), which is characterized by the release of damage-associated molecular patterns (DAMPs) from dying tumor cells. The ability of cytotoxic chemotherapeutic agents and molecularly targeted drugs such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) to induce DAMPs during the treatment of NSCLC has remained unclear, however. MATERIALS AND METHODS: We investigated the ability of seven cytotoxic chemotherapeutic agents and the third-generation EGFR-TKI osimertinib to induce translocation of the DAMP calreticulin to the cell surface in multiple NSCLC cell lines. The plasma concentration of soluble CRT in advanced NSCLC patients treated with cytotoxic chemotherapy or osimertinib was measured. RESULTS: Antimetabolites and microtubule inhibitors induced expression of CRT at the cell surface (ecto-CRT) to a greater extent than did platinum agents in six NSCLC cell lines, exhibiting higher up-regulation of phosphorylation of eukaryotic initiation factor-2α (eIF2α). Ecto-CRT expression was positively correlated with apoptosis induction in NSCLC cells treated with these various chemotherapeutic agents. The drug-induced up-regulation of ecto-CRT in NSCLC cells was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Osimertinib similarly increased ecto-CRT expression in association with apoptosis induction in five EGFR-mutated NSCLC cell lines. Furthermore, the plasma concentration of soluble CRT in 16 NSCLC patients treated with single-agent pemetrexed or docetaxel and in nine EGFR-mutated NSCLC patients treated with osimertinib was increased after treatment onset. CONCLUSION: Our findings indicate that antimetabolites, microtubule inhibitors, and osimertinib are effective inducers both of CRT exposure in NSCLC cell lines and of soluble CRT release in patients with advanced NSCLC, suggesting that these agents might prove effective for promotion of antitumor immunity in combination immunotherapy.

Antitumor effect of adenoviruses expressing mutant non-oncogenic E7 versions from HPV-16 fused to calreticulin.

PURPOSE: To compare the antitumor effect of adenoviruses that express mutant variants of the protein E7 from HPV-16 fused to calreticulin. METHODS: Recombinant adenoviruses were generated to express calreticulin fused to mutant versions of E7 (CRT/E7m and CRT/E7dm). Western blot and immunofluorescence assays were made to demonstrate protein expression. Antitumor assays were performed in C57BL6 mice injected with TC-1 cell line. RESULTS: When HEK293 cells were infected with these adenoviruses, we detected that all the recombinant proteins were expressed at endoplasmic reticulum, as expected. Next, the antitumor effect was tested on a murine tumor model established by inoculation of TC-1 cell line. We detected that both Ad CRT/E7m and Ad CRT/E7dm were capable of reducing the antitumor volume when compared to Ad LacZ, which was used as negative control. No significant difference was observed when compared to Ad CRT/E7, a positive control. CONCLUSIONS: Here we demonstrated that the mutant versions of E7 HPV-16 fused to calreticulin generate similar antitumor effect than the wild type version.

Novel calreticulin-nanoparticle in combination with focused ultrasound induces immunogenic cell death in melanoma to enhance antitumor immunity.

Rationale: Some studies have shown that the local activation of immunogenic cell death (ICD) by upregulating calreticulin (CRT) expression in solid tumors can improve antitumor effects. Although a promising approach, a key current challenge in ICD tumor therapy is the absence of a clinically translatable method for reproducibly inducing the CRT expression. Herein, we report a novel calreticulin-nanoparticle (CRT-NP) that enhances ICD and synergizes with focused ultrasound (FUS) to achieve local and systemic antitumor effects. Methods: Full-length clone DNA of calreticulin was encapsulated in NPs made from DOTAP and cholesterol. Three CRT-NP intratumoral injections of 20 µg each were given 2 days apart, and FUS heating (42-45°C, ~15min) was applied sequentially 24h after each injection to induce ICD. To investigate ICD specific immune effect, the splenocytes of mice vaccinated with CRT-NP (± FUS) treated B16F10 cells were evaluated ex-vivo for TRP-2 antigen specific immunity. Additionally, the long-term protection was evaluated by re-challenging with the melanoma cells in the flank regions of tumor bearing mice. Results: CRT-NP plus FUS (CFUS) upregulated CRT expression, expanded the population of melanoma TRP-2 specific functional CD4+ and CD8+ T cells and tumor-suppressing M1 phenotype, and increased PD-1 and PD-L1 marker expression in the T cells. Therapeutically, CFUS suppressed B16 melanoma growth by >85% vs. that seen in untreated controls, and >~50% vs. CRT-NP or FUS alone, and prevented tumor growth in distal untreated sites. Conclusions: CRT-NP amplifies the FUS and ICD therapeutic outcomes against melanoma, suggesting that the proposed combinatorial methodology may be clinically translatable.

In Vitro Employment of Recombinant Taenia solium Calreticulin as a Novel Strategy Against Breast and Ovarian Cancer Stem-like Cells.

BACKGROUND AND AIMS: Calreticulin is a chaperone and master regulator of intracellular calcium homeostasis. Several additional functions have been discovered. Human and parasite calreticulin have been shown to suppress mammary tumor growth in vivo. Here, we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modulate cancer cell growth in vitro. METHODS: We used different concentrations of rTsCRT to treat cancer cell lines and analyzed viability and colony formation capacity. We also tested the combination of the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A was employed. The effect of the drug combinations was also assessed in cancer stem-like cells. Additionally, scavenger receptor ligands were employed to identify the role of this receptor in the rTsCRT anti-tumoral effect. RESULTS: rTsCRT has a dose-dependent in vitro anti-tumoral effect, being SKOV3 the most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used, MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher reduction in cell viability, while those from SKOV3 were more sensitive to colony disaggregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the reduction in viability induced by rTsCRT in both the parental and stem-like cells. CONCLUSION: Our data suggest that rTsCRT alone or in combination with 5-fluorouracil inhibits the growth of breast and ovarian cancer cell lines through its interaction with scavenger receptors.

Postconditioning with Calreticulin Attenuates Myocardial Ischemia/Reperfusion Injury and Improves Autophagic Flux.

BACKGROUND: Impaired autophagic flux contributes to cardiomyocyte death in ischemia/reperfusion (I/R) injury. Restoring the impaired autophagic flux by using agents may be a promising strategy that alleviates myocardial I/R injury. The present study aimed to evaluate the effect of exogenous calreticulin (CRT) postconditioning on impaired autophagic flux induced by hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: Rat myocardial I/R injury model was prepared. CRT postconditionging was fulfilled by an intraperitoneal injection of CRT (0.5 mg/kg body weight) 5 min before reperfusion. Hemodynamics, serum lactate dehydrogenase (LDH) activity and Cardiac troponin T (TnT) content, and infarct size were measured. The H/R injury model of H9c2 cells was prepared. CRT postconditioning was performed by adding 25 pg/mL CRT to the medium at the onset of reoxygenation. Cell death rate, lactate dehydrogenase (LDH) leakage, intracellular reactive oxygen species (ROS), and malondialdehyde (MDA) were assessed. Autophagic flux was monitored by mRFP-GFP-LC3 adenovirus infection. The number of autophagosomes and autolysosomes in cells were determined by counting the fluorescence dots. Western blot assay was used to determine the expression of autophagy-related proteins. RESULTS: CRT postconditionging improved cardiac function, reduced serum LDH activity and TnT content, and limited myocardial infarct size after myocardial I/R injury in rat. H/R induced H9c2 cells injury and autophagosomes accumulation in cells. CRT postconditioning attenuated H/R-induced cell death, LDH leakage, and the increase of intracellular ROS and MDA. Meanwhile, CRT postconditioning suppressed H/R-induced excessive formation of autophagosomes, as shown by a decrease of autophagosomes and the ratio of LC3-II/LC3-I, LC3-II, and Beclin1. It also improved H/R-induced impaired autophagosomes clearance, as shown by an increase of autolysosomes and the level of LAMP-2, and a decrease of the level of p62. CONCLUSION: These findings suggested that CRT postconditioning reduced myocardial I/R injury. CRT postconditioning also inhibited the excessive formation of autophagosomes, promoted the clearance of autophagosomes, and resorted the autophagic flux, consequently reduced the H/R injury in H9c2 cells.

Estimation of diagnosis and prognosis in ET by assessment of CALR and JAK2V617F mutations and laboratory findings: a meta-analysis

Background. Essential thrombocythemia (ET) is a benign disease with slow progress in which thrombosis is a cause of mortality. JAK2V617F and calreticulin (CALR) are the most frequent mutations in this disease. In this systematic review and meta-analysis, we compared the prevalence of JAK2V617F and CALR mutations in ET and examined the incidence of thrombosis and other hematologic indices. Methods. After choosing MeSH keywords, including essential thrombocythemia, JAK2V617F, calreticulin, prognosis, and diagnosis, as well as searching Medline/PubMed and Scopus, 12 papers were selected. Data were pooled, and summary prevalence and OR were estimated using either a random-effects model or a fixed-effects model. Results. The frequency of JAK2V617F and CALR shows heterogeneity in Caucasian population [JAK2V617I2% = 84.3, P < 0.001, 95% CI 0.56 (0.51-0.61)], [CALR I2% = 96.1, P < 0.001, 95% CI 0.23 (0.15-0.31)]. The prevalence of JAK2V617F and CALR was 0.57 (95% CI 0.53-0.61), I2% = 79.3 and 0.22 (95% CI 0.16-0.27), I2% = 94, respectively. JAK2V617F positive ET was associated with increasing odds of thrombosis [OR 2.35 (95% CI 1.83-3.02), P < 0.001]. The incidence of splenomegaly was not statistically different between these two mutations. Hemoglobin, platelet, and WBC count did not affect the risk of thrombosis. Conclusions. Detection of CALR mutation is helpful for molecular diagnosis of ET patients as well as JAK2V617F. Due to reduction of thrombosis in CALR-positive patients, it can be stated that such patients have less thrombotic disorders and better prognosis relative to patients bearing JAK2V617F mutation. Therefore, detection of mutation in CALR and JAK2V617F may contribute to diagnosis and prognosis of ET patients (AU)

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Calreticulin inhibits inflammation-induced osteoclastogenesis and bone resorption.

Osteoclasts play key roles in bone remodeling and pathologic osteolytic disorders such as inflammation, infection, bone implant loosening, rheumatoid arthritis, metastatic bone cancers, and pathological fractures. Osteoclasts are formed by the fusion of monocytes in response to receptor activators of NF-κB-ligand (RANKL) and macrophage colony stimulating factor 1 (M-CSF). Calreticulin (CRT), a commonly known intracellular protein as a calcium-binding chaperone, has an unexpectedly robust anti-osteoclastogenic effect when its recombinant form is applied to osteoclast precursors in vitro or at the site of bone inflammation externally in vivo. Externally applied Calreticulin was internalized inside the cells. It inhibited key pro-osteoclastogenic transcription factors such as c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)-in osteoclast precursor cells that were treated with RANKL in vitro. Recombinant human Calreticulin (rhCRT) inhibited lipopolysaccharide (LPS)-induced inflammatory osteoclastogenesis in the mouse calvarial bone in vivo. Cathepsin K molecular imaging verified decreased Cathepsin K activity when rhCalreticulin was applied at the site of LPS application in vivo. Recombinant forms of intracellular proteins or their derivatives may act as novel extracellular therapeutic agents. We anticipate our findings to be a starting point in unraveling hidden extracellular functions of other intracellular proteins in different cell types of many organs for new therapeutic opportunities. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2658-2666, 2017.

Gene Delivery of Calreticulin Anti-Angiogenic Domain Attenuates the Development of Choroidal Neovascularization in Rats.

Choroidal neovascularization (CNV) is a common pathological feature in neovascular age-related macular degeneration, which is the leading cause of vision loss among elderly populations in developed countries. This study evaluated the effect of a novel endogenous inhibitor of angiogenesis, calreticulin anti-angiogenic domain (CAD), subconjunctivally delivered by an adenoviral vector (Ad-CAD) in a rat model of laser-induced CNV. CAD was expressed in Ad-CAD-infected cells and inhibited the angiogenic activity in human umbilical vein endothelial cells in vitro. CAD expression was also found in various ocular tissues after in vivo subconjunctival Ad-CAD injection. Via bioluminescence imaging it is shown that a single subconjunctival injection of Ad-luciferase induced the expression of the transgene in the injected eyes within 24 h, which lasted for at least 112 days. Forty-two days after subconjunctival injection of Ad-CAD, retinal structure and function were unaffected, as measured using optical coherence tomography and electroretinography, respectively. After laser injury, subconjunctival Ad-CAD gene delivery significantly inhibited CNV lesions as measured via choroid flat-mounts (51% reduction at 21 days; p < 0.001), as well as by fundus fluorescein angiography (19.3%, 28.2%, 31%, and 27.5% reductions at days 21, 28, 35, and 42, respectively; p < 0.05) in rats. The data suggest that subconjunctival Ad-CAD gene therapy could effectively inhibit laser-induced CNV and might be an attractive therapeutic approach for the management of choroidal neovascularization.

Calreticulin: a new horizon for the testing and treatment of myeloproliferative neoplasms.

The recent discovery of mutations of the gene calreticulin has allowed raising the proportion of patients with essential thrombocythemia and primary myelofibrosis with known mutational abnormality up to 85-90%. Knowledge of the mechanisms by which mutated calreticulin underlie a myeloproliferative neoplasm as well as the clinical and therapeutic implications is just at the very beginning, and exciting times await research in this field.

Human survivin and Trypanosoma cruzi calreticulin act in synergy against a murine melanoma in vivo.

Immune-based anti-tumor or anti-angiogenic therapies hold considerable promise for the treatment of cancer. The first approach seeks to activate tumor antigen-specific T lymphocytes while, the second, delays tumor growth by interfering with blood supply. Tumor Associated Antigens are often employed to target tumors with therapeutic drugs, but some are also essential for tumor viability. Survivin (Surv) is a member of the inhibitor of apoptosis protein family that is considered a Tumor Associated Antigen important for cancer cell viability and proliferation. On the other hand, Trypanosoma cruzi (the agent of Chagas' disease) calreticulin (TcCRT) displays remarkable anti-angiogenic properties. Because these molecules are associated with different tumor targets, we reasoned that immunization with a Surv-encoding plasmid (pSurv) and concomitant TcCRT administration should generate a stronger anti-tumor response than application of either treatment separately. To evaluate this possibility, C57BL/6 mice were immunized with pSurv and challenged with an isogenic melanoma cell line that had been pre-incubated with recombinant TcCRT (rTcCRT). Following tumor cell inoculation, mice were injected with additional doses of rTcCRT. For the combined regimen we observed in mice that: i). Tumor growth was impaired, ii). Humoral anti-rTcCRT immunity was induced and, iii). In vitro rTcCRT bound to melanocytes, thereby promoting the incorporation of human C1q and subsequent macrophage phagocytosis of tumor cells. These observations are interpreted to reflect the consequence of the following sequence of events: rTcCRT anti-angiogenic activity leads to stress in tumor cells. Murine CRT is then translocated to the external membrane where, together with rTcCRT, complement C1 is captured, thus promoting tumor phagocytosis. Presentation of the Tumor Associated Antigen Surv induces the adaptive anti-tumor immunity and, independently, mediates anti-endothelial cell immunity leading to an important delay in tumor growth.

Herbal compound triptolide synergistically enhanced antitumor activity of vasostatin120-180.

Angiogenesis is essential for the survival and growth of most tumors. As such, targeting the tumor neovasculature is an attractive strategy for effective cancer therapy. Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression. The functional domain within amino acid residues 120-180 of vasostatin (VAS) has been confirmed to be effective in inhibiting the proliferation, migration, and invasiveness of cancer cells by its suppressive capacity against angiogenesis. Triptolide (TPL) is an active component extracted from the traditional Chinese herbal medicine Tripterygium wilfordii Hook F that has shown antitumor activities in various cancer cell types. However, its therapeutic application is limited by its toxicity in normal tissues and complications caused in patients. In this study, we attempted to investigate the synergistic antitumor activity of TPL and VAS in solid tumor cells. Our results showed that the sensitivity of combined therapy using TPL and VAS was higher than that of monotherapy using TPL or VAS. Apoptosis induced by the combined treatment was accompanied by activation of caspase-9, caspase-8, and caspase-3. Upregulation of proapoptotic protein (Bax, Bak, and Bad) expression and suppression of NF-κB transcriptional activity and its targeting antiapoptotic genes (c-FLIP, cIAP, Bcl-2, Bcl-xl, and Mcl-1) may contribute to the synergistic effects of this combination therapy. Further, using a mouse xenograft model, we demonstrated that combined treatment completely suppressed tumor growth as compared with treatment with TPL or VAS alone. These results suggest that the combination of TPL and VAS at lower concentrations may produce a synergistic antitumor effect that warrants further investigation for its potential clinical applications.