Sandro Jube

Programmed necrosis induced by asbestos in human mesothelial cells causes high-mobility group box 1 protein release and resultant inflammation.

Asbestos carcinogenesis has been linked to the release of cytokines and mutagenic reactive oxygen species (ROS) from inflammatory cells. Asbestos is cytotoxic to human mesothelial cells (HM), which appears counterintuitive for a carcinogen. We show that asbestos-induced HM cell death is a regulated form of necrosis that links to carcinogenesis. Asbestos-exposed HM activate poly(ADP-ribose) polymerase, secrete H2O2, deplete ATP, and translocate high-mobility group box 1 protein (HMGB1) from the nucleus to the cytoplasm, and into the extracellular space. The release of HMGB1 induces macrophages to secrete TNF-α, which protects HM from asbestos-induced cell death and triggers a chronic inflammatory response; both favor HM transformation. In both mice and hamsters injected with asbestos, HMGB1 was specifically detected in the nuclei, cytoplasm, and extracellular space of mesothelial and inflammatory cells around asbestos deposits. TNF-α was coexpressed in the same areas. HMGB1 levels in asbestos-exposed individuals were significantly higher than in nonexposed controls (P < 0.0001). Our findings identify the release of HMGB1 as a critical initial step in the pathogenesis of asbestos-related disease, and provide mechanistic links between asbestos-induced cell death, chronic inflammation, and carcinogenesis. Chemopreventive approaches aimed at inhibiting the chronic inflammatory response, and especially blocking HMGB1, may decrease the risk of malignant mesothelioma among asbestos-exposed cohorts.

Cancer Cell Secretion of the DAMP Protein HMGB1 Supports Progression in Malignant Mesothelioma

Human malignant mesothelioma is an aggressive and highly lethal cancer that is believed to be caused by chronic exposure to asbestos and erionite. Prognosis for this cancer is generally poor because of late-stage diagnosis and resistance to current conventional therapies. The damage-associated molecular pattern protein HMGB1 has been implicated previously in transformation of mesothelial cells. Here we show that HMGB1 establishes an autocrine circuit in malignant mesothelioma cells that influences their proliferation and survival. Malignant mesothelioma cells strongly expressed HMGB1 and secreted it at high levels in vitro. Accordingly, HMGB1 levels in malignant mesothelioma patient sera were higher than that found in healthy individuals. The motility, survival, and anchorage-independent growth of HMGB1-secreting malignant mesothelioma cells was inhibited in vitro by treatment with monoclonal antibodies directed against HMGB1 or against the receptor for advanced glycation end products, a putative HMGB1 receptor. HMGB1 inhibition in vivo reduced the growth of malignant mesothelioma xenografts in severe-combined immunodeficient mice and extended host survival. Taken together, our findings indicate that malignant mesothelioma cells rely on HMGB1, and they offer a preclinical proof-of-principle that antibody-mediated ablation of HMBG1 is sufficient to elicit therapeutic activity, suggesting a novel therapeutic approach for malignant mesothelioma treatment.

Erionite exposure in North Dakota and Turkish villages with mesothelioma

Exposure to erionite, an asbestos-like mineral, causes unprecedented rates of malignant mesothelioma (MM) mortality in some Turkish villages. Erionite deposits are present in at least 12 US states. We investigated whether increased urban development has led to erionite exposure in the United States and after preliminary exploration, focused our studies on Dunn County, North Dakota (ND). In Dunn County, ND, we discovered that over the past three decades, more than 300 miles of roads were surfaced with erionite-containing gravel. To determine potential health implications, we compared erionite from the Turkish villages to that from ND. Our study evaluated airborne point exposure concentrations, examined the physical and chemical properties of erionite, and examined the hallmarks of mesothelial cell transformation in vitro and in vivo. Airborne erionite concentrations measured in ND along roadsides, indoors, and inside vehicles, including school buses, equaled or exceeded concentrations in Boyali, where 6.25% of all deaths are caused by MM. With the exception of outdoor samples along roadsides, ND concentrations were lower than those measured in Turkish villages with MM mortality ranging from 20 to 50%. The physical and chemical properties of erionite from Turkey and ND are very similar and they showed identical biological activities. Considering the known 30- to 60-y latency for MM development, there is reason for concern for increased risk in ND in the future. Our findings indicate that implementation of novel preventive and early detection programs in ND and other erionite-rich areas of the United States, similar to efforts currently being undertaken in Turkey, is warranted.

CSPG4 as a Target of Antibody-Based Immunotherapy for Malignant Mesothelioma

Purpose: Malignant mesothelioma (MM) is an aggressive cancer, resistant to current therapies. Membrane chondroitin sulphate proteoglycan 4 (CSPG4), which has been successfully targeted in melanoma and breast cancer, was found highly expressed in MM, but not in normal mesothelium. Therefore, we explored CSPG4 as a suitable target for monoclonal antibody (mAb)–based immunotherapy for MM. Experimental design: We assayed adhesion, motility, invasiveness, wound-healing, apoptosis, and anchorage-independent growth of MM cells on cell cultures. CSPG4 expression and signaling was studied by immunoblotting. The growth of MM severe combined immunodeficient (SCID) mice xenografts induced by PPM-Mill cells, engineered to express the luciferase reporter gene, was monitored by imaging, upon treatment with CSPG4 mAb TP41.2. Animal toxicity and survival were assayed in both tumor inhibition and therapeutic experiments. Results: CSPG4 was expressed on 6 out of 8 MM cell lines and in 25 out of 41 MM biopsies, with minimal expression in surrounding healthy cells. MM cell adhesion was mediated by CSPG4-dependent engagement of ECM. Cell adhesion was inhibited by mAb TP41.2 resulting in decreased phosphorylation of focal adhesion kinase (FAK) and AKT, reduced expression of cyclin D1 and apoptosis. Moreover, mAb TP41.2 significantly reduced MM cell motility, migration, and invasiveness, and inhibited MM growth in soft agar. In vivo, treatment with mAb TP41.2 prevented or inhibited the growth of MM xenografts in SCID mice, with a significant increase in animal survival. Conclusion: These results establish the safety of CSPG4 mAb-based immunotherapy and suggest that CSPG4 mAb-based immunotherapy may represent a novel approach for the treatment of MM. Clin Cancer Res; 18(19); 5352–63. ©2012 AACR.

Continuous Exposure to Chrysotile Asbestos Can Cause Transformation of Human Mesothelial Cells via HMGB1 and TNF-α Signaling

Malignant mesothelioma is strongly associated with asbestos exposure. Among asbestos fibers, crocidolite is considered the most and chrysotile the least oncogenic. Chrysotile accounts for more than 90% of the asbestos used worldwide, but its capacity to induce malignant mesothelioma is still debated. We found that chrysotile and crocidolite exposures have similar effects on human mesothelial cells. Morphological and molecular alterations suggestive of epithelial-mesenchymal transition, such as E-cadherin down-regulation and β-catenin phosphorylation followed by nuclear translocation, were induced by both chrysotile and crocidolite. Gene expression profiling revealed high-mobility group box-1 protein (HMGB1) as a key regulator of the transcriptional alterations induced by both types of asbestos. Crocidolite and chrysotile induced differential expression of 438 out of 28,869 genes interrogated by oligonucleotide microarrays. Out of these 438 genes, 57 were associated with inflammatory and immune response and cancer, and 14 were HMGB1 targeted genes. Crocidolite-induced gene alterations were sustained, whereas chrysotile-induced gene alterations returned to background levels within 5 weeks. Similarly, HMGB1 release in vivo progressively increased for 10 or more weeks after crocidolite exposure, but returned to background levels within 8 weeks after chrysotile exposure. Continuous administration of chrysotile was required for sustained high serum levels of HMGB1. These data support the hypothesis that differences in biopersistence influence the biological activities of these two asbestos fibers.

Aspirin delays mesothelioma growth by inhibiting HMGB1-mediated tumor progression

High-mobility group box 1 (HMGB1) is an inflammatory molecule that has a critical role in the initiation and progression of malignant mesothelioma (MM). Aspirin (acetylsalicylic acid, ASA) is the most widely used nonsteroidal anti-inflammatory drug that reduces the incidence, metastatic potential and mortality of many inflammation-induced cancers. We hypothesized that ASA may exert anticancer properties in MM by abrogating the carcinogenic effects of HMGB1. Using HMGB1-secreting and -non-secreting human MM cell lines, we determined whether aspirin inhibited the hallmarks of HMGB1-induced MM cell growth in vitro and in vivo. Our data demonstrated that ASA and its metabolite, salicylic acid (SA), inhibit motility, migration, invasion and anchorage-independent colony formation of MM cells via a novel HMGB1-mediated mechanism. ASA/SA, at serum concentrations comparable to those achieved in humans taking therapeutic doses of aspirin, and BoxA, a specific inhibitor of HMGB1, markedly reduced MM growth in xenograft mice and significantly improved survival of treated animals. The effects of ASA and BoxA were cyclooxygenase-2 independent and were not additive, consistent with both acting via inhibition of HMGB1 activity. Our findings provide a rationale for the well documented, yet poorly understood antitumorigenic activity of aspirin, which we show proceeds via HMGB1 inhibition. Moreover, the use of BoxA appears to allow a more efficient HMGB1 targeting while eluding the known gastrointestinal side effects of ASA. Our findings are directly relevant to MM. Given the emerging importance of HMGB1 and its tumor-promoting functions in many cancer types, and of aspirin in cancer prevention and therapy, our investigation is poised to provide broadly applicable information.

Development of an Agrobacterium-mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos

The tree-legume Leucaena leucocephala (leucaena) is used as a perennial fodder because of its fast-growing foliage, which is high in protein content. The use of leucaena as a fodder is however restricted due to the presence of the toxin mimosine. Improvements in the nutritional contents as well as other agronomic traits of leucaena can be accomplished through genetic transformation. The objective of this research was to develop a transformation protocol for leucaena using phosphinothricin resistance as the plant selectable marker. Explants obtained from immature zygotic embryos infected with the Agrobacterium tumefaciens strain C58C1 containing the binary plasmid pCAMBIA3201 produced four putative transformed leucaena plants. Transformation was confirmed by PCR, RT-PCR, Southern blot, Western analyses, GUS-specific enzyme activity and herbicide leaf spraying assay. A transformation efficiency of 2% was established using this protocol.

HMGB1 targeting by ethyl pyruvate suppresses malignant phenotype of human mesothelioma

Human malignant mesothelioma (MM) is an aggressive cancer linked to asbestos and erionite exposure. We previously reported that High-Mobility Group Box-1 protein (HMGB1), a prototypic damage-associated molecular pattern, drives MM development and sustains MM progression. Moreover, we demonstrated that targeting HMGB1 inhibited MM cell growth and motility in vitro, reduced tumor growth in vivo, and prolonged survival of MM-bearing mice. Ethyl pyruvate (EP), the ethyl ester of pyruvic acid, has been shown to be an effective HMGB1 inhibitor in inflammation-related diseases and several cancers. Here, we studied the effect of EP on the malignant phenotype of MM cells in tissue culture and on tumor growth in vivo using an orthotopic MM xenograft model. We found that EP impairs HMGB1 secretion by MM cells leading to reduced RAGE expression and NF-κB activation. As a consequence, EP impaired cell motility, cell proliferation, and anchorage-independent growth of MM cells. Moreover, EP reduced HMGB1 serum levels in mice and inhibited the growth of MM xenografts. Our results indicate that EP effectively hampers the malignant phenotype of MM, offering a novel potential therapeutic approach to patients afflicted with this dismal disease.

Transgenic Leucaena leucocephala expressing the Rhizobium gene pydA encoding a meta-cleavage dioxygenase shows reduced mimosine content

The use of the tree-legume Leucaena leucocephala (leucaena), which contains high levels of proteins in its foliage, is limited due to the presence of the toxic free amino acid mimosine. The goal of this research was to develop transgenic leucaena with reduced mimosine content. Two genes, pydA and pydB, encoding a meta-cleavage dioxygenase (EC 1.13.11.2) and a pyruvate hydrolase (EC 3.7.1.6), respectively, from the mimosine-degrading leucaena symbiont Rhizobium sp. strain TAL1145, were used to transform leucaena. These bacterial genes were sequence-optimized for expression in leucaena and cloned into the plant binary vector pCAMBIA3201 for Agrobacterium tumefaciens-mediated transformation. Using immature zygotic embryos as the start explant material, six pydA and three pydB transgenic lines were developed. The presence and expression of the bacterial genes in the transgenic lines were verified by PCR, reverse transcriptase PCR, and Southern analyses. HPLC analyses of the transgenic plants determined that the mimosine contents of the pydA-expressing lines were reduced up to 22.5% in comparison to the wild-type. No significant reduction in mimosine content was observed in the pydB-expressing lines. This is the first example of using a gene from a bacterial symbiont to reduce the toxicity of a tree-legume.

Immunization with hybrid recombinant Mycobacterium tuberculosis H37Rv proteins increases the TH1 cytokine response in mice following a pulmonary instillation of irradiated mycobacteria

The aim of this research was to identify subunit immunogens that can generate enhanced CD8 T cell and TH1 responses against Mycobacterium tuberculosis. A genomic comparison of the M. tuberculosis H(37)R(V) and M. bovis BCG identified 61 proteins that are unique to H(37)R(V). Further screening of these 61 proteins using in silico analyses mimicking proteasomal digestion, transporter-associated antigen processing and H-2 antigen presentation identified 13 proteins with high densities of predicted MHC class I epitopes. Two native proteins, Rv1986c and Rv3875, were selected on the basis of their secreted or transmembrane characteristics and relatively lower frequencies of predicted MHC class II epitopes. To further enhance the CD8 T cell and TH1 responses, a hybrid protein, H32, was constructed by combining the nucleotide sequences encoding the MHC class I antigen-rich segment of Rv1986c and the entire Rv3875 sequence. The two native proteins and the hybrid were used to immunize C57BL/6 and Balb/c mice, which was followed by pulmonary instillation with irradiated M. tuberculosis H(37)R(V). All three proteins elicited elevated IFN-gamma responses, with the hybrid showing significant increases over the native proteins in both mice. This strategy of immunogen selection might be used to improve the current subunit vaccines against M. tuberculosis as well as other intra-cellular pathogens.