Germline BARD1 variants predispose to mesothelioma by impairing DNA repair and calcium signaling.

We report that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare germline variants of the BRCA1 associated RING domain 1 (BARD1) gene that were predicted to be damaging by computational analyses. We conducted functional assays, essential for accurate interpretation of missense variants, in primary fibroblasts that we established in tissue culture from a patient carrying the heterozygous BARD1V523A mutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis. Investigating the underlying signaling pathways, we found that BARD1 forms a trimeric protein complex with p53 and SERCA2 that regulates calcium signaling and apoptosis. We validated these findings in BARD1-silenced primary human mesothelial cells exposed to asbestos. Our study elucidated mechanisms of BARD1 activity and revealed that heterozygous germline BARD1 mutations favor the development of mesothelioma and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to mesotheliomas in asbestos workers.

HMGB1 released by mesothelial cells drives the development of asbestos-induced mesothelioma.

Asbestos is the main cause of malignant mesothelioma. Previous studies have linked asbestos-induced mesothelioma to the release of HMGB1 from the nucleus to the cytoplasm, and from the cytoplasm to the extracellular space. In the cytoplasm, HMGB1 induces autophagy impairing asbestos-induced cell death. Extracellularly, HMGB1 stimulates the secretion of TNFα. Jointly, these two cytokines kick-start a chronic inflammatory process that over time promotes mesothelioma development. Whether the main source of extracellular HMGB1 were the mesothelial cells, the inflammatory cells, or both was unsolved. This information is critical to identify the targets and design preventive/therapeutic strategies to interfere with asbestos-induced mesothelioma. To address this issue, we developed the conditional mesothelial HMGB1-knockout (Hmgb1ΔpMeso) and the conditional myelomonocytic-lineage HMGB1-knockout (Hmgb1ΔMylc) mouse models. We establish here that HMGB1 is mainly produced and released by the mesothelial cells during the early phases of inflammation following asbestos exposure. The release of HMGB1 from mesothelial cells leads to atypical mesothelial hyperplasia, and in some animals, this evolves over the years into mesothelioma. We found that Hmgb1ΔpMeso, whose mesothelial cells cannot produce HMGB1, show a greatly reduced inflammatory response to asbestos, and their mesothelial cells express and secrete significantly reduced levels of TNFα. Moreover, the tissue microenvironment in areas of asbestos deposits displays an increased fraction of M1-polarized macrophages compared to M2 macrophages. Supporting the biological significance of these findings, Hmgb1ΔpMeso mice showed a delayed and reduced incidence of mesothelioma and an increased mesothelioma-specific survival. Altogether, our study provides a biological explanation for HMGB1 as a driver of asbestos-induced mesothelioma.

BAP1 is a novel regulator of HIF-1α.

BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1ß forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.

BAP1 forms a trimer with HMGB1 and HDAC1 that modulates gene × environment interaction with asbestos.

Carriers of heterozygous germline BAP1 mutations (BAP1+/-) are affected by the "BAP1 cancer syndrome." Although they can develop almost any cancer type, they are unusually susceptible to asbestos carcinogenesis and mesothelioma. Here we investigate why among all carcinogens, BAP1 mutations cooperate with asbestos. Asbestos carcinogenesis and mesothelioma have been linked to a chronic inflammatory process promoted by the extracellular release of the high-mobility group box 1 protein (HMGB1). We report that BAP1+/- cells secrete increased amounts of HMGB1, and that BAP1+/- carriers have detectable serum levels of acetylated HMGB1 that further increase when they develop mesothelioma. We linked these findings to our discovery that BAP1 forms a trimeric protein complex with HMGB1 and with histone deacetylase 1 (HDAC1) that modulates HMGB1 acetylation and its release. Reduced BAP1 levels caused increased ubiquitylation and degradation of HDAC1, leading to increased acetylation of HMGB1 and its active secretion that in turn promoted mesothelial cell transformation.

BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation.

BRCA1-associated protein 1 (BAP1) is a potent tumour suppressor gene that modulates environmental carcinogenesis. All carriers of inherited heterozygous germline BAP1-inactivating mutations (BAP1+/-) developed one and often several BAP1-/- malignancies in their lifetime, mostly malignant mesothelioma, uveal melanoma, and so on. Moreover, BAP1-acquired biallelic mutations are frequent in human cancers. BAP1 tumour suppressor activity has been attributed to its nuclear localization, where it helps to maintain genome integrity. The possible activity of BAP1 in the cytoplasm is unknown. Cells with reduced levels of BAP1 exhibit chromosomal abnormalities and decreased DNA repair by homologous recombination, indicating that BAP1 dosage is critical. Cells with extensive DNA damage should die and not grow into malignancies. Here we discover that BAP1 localizes at the endoplasmic reticulum. Here, it binds, deubiquitylates, and stabilizes type 3 inositol-1,4,5-trisphosphate receptor (IP3R3), modulating calcium (Ca2+) release from the endoplasmic reticulum into the cytosol and mitochondria, promoting apoptosis. Reduced levels of BAP1 in BAP1+/- carriers cause reduction both of IP3R3 levels and of Ca2+ flux, preventing BAP1+/- cells that accumulate DNA damage from executing apoptosis. A higher fraction of cells exposed to either ionizing or ultraviolet radiation, or to asbestos, survive genotoxic stress, resulting in a higher rate of cellular transformation. We propose that the high incidence of cancers in BAP1+/- carriers results from the combined reduced nuclear and cytoplasmic activities of BAP1. Our data provide a mechanistic rationale for the powerful ability of BAP1 to regulate gene-environment interaction in human carcinogenesis.

Asbestos induces mesothelial cell transformation via HMGB1-driven autophagy.

Asbestos causes malignant transformation of primary human mesothelial cells (HM), leading to mesothelioma. The mechanisms of asbestos carcinogenesis remain enigmatic, as exposure to asbestos induces HM death. However, some asbestos-exposed HM escape cell death, accumulate DNA damage, and may become transformed. We previously demonstrated that, upon asbestos exposure, HM and reactive macrophages releases the high mobility group box 1 (HMGB1) protein that becomes detectable in the tissues near asbestos deposits where HMGB1 triggers chronic inflammation. HMGB1 is also detectable in the sera of asbestos-exposed individuals and mice. Searching for additional biomarkers, we found higher levels of the autophagy marker ATG5 in sera from asbestos-exposed individuals compared to unexposed controls. As we investigated the mechanisms underlying this finding, we discovered that the release of HMGB1 upon asbestos exposure promoted autophagy, allowing a higher fraction of HM to survive asbestos exposure. HMGB1 silencing inhibited autophagy and increased asbestos-induced HM death, thereby decreasing asbestos-induced HM transformation. We demonstrate that autophagy was induced by the cytoplasmic and extracellular fractions of HMGB1 via the engagement of the RAGE receptor and Beclin 1 pathway, while nuclear HMGB1 did not participate in this process. We validated our findings in a novel unique mesothelial conditional HMGB1-knockout (HMGB1-cKO) mouse model. Compared to HMGB1 wild-type mice, mesothelial cells from HMGB1-cKO mice showed significantly reduced autophagy and increased cell death. Autophagy inhibitors chloroquine and desmethylclomipramine increased cell death and reduced asbestos-driven foci formation. In summary, HMGB1 released upon asbestos exposure induces autophagy, promoting HM survival and malignant transformation.

Heterozygous germline BLM mutations increase susceptibility to asbestos and mesothelioma.

Rare biallelic BLM gene mutations cause Bloom syndrome. Whether BLM heterozygous germline mutations (BLM+/-) cause human cancer remains unclear. We sequenced the germline DNA of 155 mesothelioma patients (33 familial and 122 sporadic). We found 2 deleterious germline BLM+/- mutations within 2 of 33 families with multiple cases of mesothelioma, one from Turkey (c.569_570del; p.R191Kfs*4) and one from the United States (c.968A>G; p.K323R). Some of the relatives who inherited these mutations developed mesothelioma, while none with nonmutated BLM were affected. Furthermore, among 122 patients with sporadic mesothelioma treated at the US National Cancer Institute, 5 carried pathogenic germline BLM+/- mutations. Therefore, 7 of 155 apparently unrelated mesothelioma patients carried BLM+/- mutations, significantly higher (P = 6.7E-10) than the expected frequency in a general, unrelated population from the gnomAD database, and 2 of 7 carried the same missense pathogenic mutation c.968A>G (P = 0.0017 given a 0.00039 allele frequency). Experiments in primary mesothelial cells from Blm+/- mice and in primary human mesothelial cells in which we silenced BLM revealed that reduced BLM levels promote genomic instability while protecting from cell death and promoted TNF-α release. Blm+/- mice injected intraperitoneally with asbestos had higher levels of proinflammatory M1 macrophages and of TNF-α, IL-1ß, IL-3, IL-10, and IL-12 in the peritoneal lavage, findings linked to asbestos carcinogenesis. Blm+/- mice exposed to asbestos had a significantly shorter survival and higher incidence of mesothelioma compared to controls. We propose that germline BLM+/- mutations increase the susceptibility to asbestos carcinogenesis, enhancing the risk of developing mesothelioma.

HMGB1 as a therapeutic target in disease.

High-mobility group box 1 (HMGB1) was initially recognized as a ubiquitous nuclear protein involved in maintaining the nucleosome integrity and facilitating gene transcription. HMGB1 has since been reevaluated to be a prototypical damage-associated molecular pattern (DAMP) protein, and together with its exogenous counterpart, pathogen-associated molecular pattern (PAMP), completes the body's alarmin system against disturbances in homeostasis. HMGB1 can be released into the extracellular matrix (ECM) by either granulocytes or necrotic cells to serve as a chemotaxis/cytokine during infection, endotoxemia, hypoxia, ischemia-reperfusion events, and cancer. Different isoforms of HMGB1 present with distinctive physiological functions in ECM-fully-reduced HMGB1 (all thiol) acts as the initial damage signal to recruit circulating myeloid cells, disulfide HMGB1 behaves as a cytokine to activate macrophages and neutrophils, and both signals are turned off when HMGB1 is terminally oxidized into the final sulfonate form. Targeting HMGB1 constitutes a favorable therapeutic strategy for inflammation and inflammatory diseases. Antagonists such as ethyl pyruvate inhibit HMGB1 by interfering with its cytoplasmic exportation, while others such as glycyrrhizin directly bind to HMGB1 and render it unavailable for its receptors. The fact that a mixture of different HMGB1 isoforms is present in the ECM poses a challenge in pinpointing the exact role of an individual antagonist. A more discriminative probe for HMGB1 may be necessary to advance our knowledge of HMGB1, HMGB1 antagonists, and inflammatory-related diseases.

High-density array-CGH with targeted NGS unmask multiple noncontiguous minute deletions on chromosome 3p21 in mesothelioma.

We used a custom-made comparative genomic hybridization array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1 (breast cancer 1)-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (<3 kb) in 46 of 251 genes: four were cancer-associated genes: SETD2 (SET domain-containing protein 2) (7 of 33), BAP1 (8 of 33), PBRM1 (polybromo 1) (3 of 33), and SMARCC1 (switch/sucrose nonfermentable- SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 1) (2 of 33). These four genes were further investigated by targeted next-generation sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9 of 33, 27%), BAP1 (16 of 33, 48%), PBRM1 (5 of 33, 15%), and SMARCC1 (2 of 33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous, but rather alternated with segments showing oscillating copy number changes along the 3p21 region. In summary, we found that in MM: (i) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; (ii) in addition to BAP1, mutations of SETD2, PBRM1, and SMARCC1 are frequent in MM; and (iii) our results suggest that high-density aCGH combined with tNGS provides a more precise estimate of the frequency and types of genes inactivated in human cancer than approaches based exclusively on NGS strategy.

Combined Genetic and Genealogic Studies Uncover a Large BAP1 Cancer Syndrome Kindred Tracing Back Nine Generations to a Common Ancestor from the 1700s.

We recently discovered an inherited cancer syndrome caused by BRCA1-Associated Protein 1 (BAP1) germline mutations, with high incidence of mesothelioma, uveal melanoma and other cancers and very high penetrance by age 55. To identify families with the BAP1 cancer syndrome, we screened patients with family histories of multiple mesotheliomas and melanomas and/or multiple cancers. We identified four families that shared an identical BAP1 mutation: they lived across the US and did not appear to be related. By combining family histories, molecular genetics, and genealogical approaches, we uncovered a BAP1 cancer syndrome kindred of ~80,000 descendants with a core of 106 individuals, whose members descend from a couple born in Germany in the early 1700s who immigrated to North America. Their descendants spread throughout the country with mutation carriers affected by multiple malignancies. Our data show that, once a proband is identified, extended analyses of these kindreds, using genomic and genealogical studies to identify the most recent common ancestor, allow investigators to uncover additional branches of the family that may carry BAP1 mutations. Using this knowledge, we have identified new branches of this family carrying BAP1 mutations. We have also implemented early-detection strategies that help identify cancers at early-stage, when they can be cured (melanomas) or are more susceptible to therapy (MM and other malignancies).

FTY720 inhibits mesothelioma growth in vitro and in a syngeneic mouse model.

BACKGROUND: Malignant mesothelioma (MM) is a very aggressive type of cancer, with a dismal prognosis and inherent resistance to chemotherapeutics. Development and evaluation of new therapeutic approaches is highly needed. Immunosuppressant FTY720, approved for multiple sclerosis treatment, has recently raised attention for its anti-tumor activity in a variety of cancers. However, its therapeutic potential in MM has not been evaluated yet. METHODS: Cell viability and anchorage-independent growth were evaluated in a panel of MM cell lines and human mesothelial cells (HM) upon FTY720 treatment to assess in vitro anti-tumor efficacy. The mechanism of action of FTY720 in MM was assessed by measuring the activity of phosphatase protein 2A (PP2A)-a major target of FTY720. The binding of the endogenous inhibitor SET to PP2A in presence of FTY720 was evaluated by immunoblotting and immunoprecipitation. Signaling and activation of programmed cell death were evaluated by immunoblotting and flow cytometry. A syngeneic mouse model was used to evaluate anti-tumor efficacy and toxicity profile of FTY720 in vivo. RESULTS: We show that FTY720 significantly suppressed MM cell viability and anchorage-independent growth without affecting normal HM cells. FTY720 inhibited the phosphatase activity of PP2A by displacement of SET protein, which appeared overexpressed in MM, as compared to HM cells. FTY720 promoted AKT dephosphorylation and Bcl-2 degradation, leading to induction of programmed cell death, as demonstrated by caspase-3 and PARP activation, as well as by cytochrome c and AIF intracellular translocation. Moreover, FTY720 administration in vivo effectively reduced tumor burden in mice without apparent toxicity. CONCLUSIONS: Our preclinical data indicate that FTY720 is a potentially promising therapeutic agent for MM treatment.

Increasing dietary selenium elevates reducing capacity and ERK activation associated with accelerated progression of select mesothelioma tumors.

To study the effect of the micronutrient selenium on malignant mesothelioma (MM) progression, we cultured four different MM cell lines in media containing increasing amounts of sodium selenite (30, 50, and 80 nmol/L). Increasing selenium levels increased density-dependent proliferation and mobility for CRH5 and EKKH5 but not AB12 and AK7. Comparing these cell lines revealed that extracellular regulated kinase (ERK) phosphorylation was sensitive to a selenium increase in CRH5 and EKKH5 but not AB12 and AK7 cells. Stable expression of a dominant-negative mutant ERK eliminated the effects of increasing selenium. Because ERK is redox sensitive, we compared the MM cell lines in terms of glutathione levels and the capacity to reduce exogenous hydrogen peroxide. Increasing selenium levels led to higher glutathione and reducing capacity in CRH5 and EKKH5 but not AB12 and AK7. The reducing agent N-acetylcysteine eliminated the effects of selenium on ERK activation, proliferation, and mobility. Mice fed diets containing increasing levels of selenium (0.08, 0.25, and 1.0 ppm) showed increased tumor progression for CRH5 but not AB12, MM cells, and in vivo N-acetylcysteine treatment eliminated these effects. These data suggest that the effects of dietary selenium on MM tumor progression depend on the arising cancer cells' redox metabolism, and the tumors able to convert increased selenium into a stronger reducing capacity actually benefit from increased selenium intake.

Numerous Iron-Rich Particles Lie on the Surface of Erionite Fibers from Rome (Oregon, USA) and Karlik (Cappadocia, Turkey).

Erionite samples from Rome, Oregon (USA) and Karlik, Cappadocia (Turkey) were analyzed by environmental scanning electron microscopy (E-SEM) coupled with energy-dispersive spectroscopy (EDS) to verify the chemical composition of this mineral phase, and the presence of iron in particular. By means of backscattered electron images, a large number of particles/grains were observed on the surface of the erionite fibers from both locations. The particles were found to be micrometric on samples from Rome and submicrometric on samples from Karlik, and always lighter than the hosting crystal in appearance. In different areas of the same fiber or bundle of fibers, several EDS spectra were recorded. Iron was detected only when a light particle was lying in the path of the electron beam. Iron was never identified in the EDS spectra acquired on the flat erionite surface. The results from E-SEM/EDS were confirmed by micro-Raman spectroscopy, showing bands ascribing to hematite­Fe2O3, goethite­FeO(OH), or jarosite­KFe3(3+)(SO4)2(OH)6 when the laser beam was addressed on the light particles observed on the fiber surface. The evidence that iron is on the surface of erionite fibers, rather than being part of the crystalline structure, may be relevant for the carcinogenic potential of these fibers.

Evaluation of clonal origin of malignant mesothelioma.

BACKGROUND: The hypothesis that most cancers are of monoclonal origin is often accepted as a fact in the scientific community. This dogma arose decades ago, primarily from the study of hematopoietic malignancies and sarcomas, which originate as monoclonal tumors. The possible clonal origin of malignant mesothelioma (MM) has not been investigated. Asbestos inhalation induces a chronic inflammatory response at sites of fiber deposition that may lead to malignant transformation after 30-50 years latency. As many mesothelial cells are simultaneously exposed to asbestos fibers and to asbestos-induced inflammation, it may be possible that more than one cell undergoes malignant transformation during the process that gives rise to MM, and result in a polyclonal malignancy. METHODS AND RESULTS: To investigate the clonality patterns of MM, we used the HUMARA (Human Androgen Receptor) assay to examine 16 biopsies from 14 women MM patients. Out of 16 samples, one was non-informative due to skewed Lyonization in its normal adjacent tissue. Fourteen out of the 15 informative samples revealed two electrophoretically distinct methylated HUMARA alleles, the Corrected Allele Ratio (CR) calculated on the allele peak areas indicating polyclonal origin MM. CONCLUSIONS: Our results show that MM originate as polyclonal tumors and suggest that the carcinogenic "field effect" of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.

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 ß-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.

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.

Fowlpox-based survivin vaccination for malignant mesothelioma therapy.

Survivin protein is an attractive candidate for cancer immunotherapy since it is abundantly expressed in most common human cancers and mostly absent in normal adult tissues. Malignant mesothelioma (MM) is a deadly cancer associated with asbestos or erionite exposure for which no successful therapies are currently available. In this study, we evaluated the therapeutic efficacy of a novel survivin-based vaccine by subcutaneous or intraperitoneum injection of BALB/c mice with murine fiber-induced MM tumor cells followed by vaccination with recombinant Fowlpox virus replicons encoding survivin. Vaccination generated significant immune responses in both models, leading to delayed tumor growth and improved animal survival. Flow cytometry and immunofluorescence analyses of tumors from vaccinated mice showed CD8(+) T-cell infiltration, and real-time PCR demonstrated increased mRNA and protein levels of immunostimulatory cytokines. Analyses of survivin peptide-pulsed spleen and lymph node cells from vaccinated mice using ELISPOT and intracellular cytokine staining confirmed antigen-specific, interferon-γ-producing CD8(+) T-cell responses. In addition pentamer-based flow cytometry showed that vaccination generated survivin-specific CD8(+) T cells. Importantly, vaccination did not affect fertility or induce autoimmune abnormalities in mice. Our results demonstrate that vaccination with recombinant Fowlpox expressing survivin improves T-cell responses against aggressive MM tumors and may form the basis for promising clinical applications.

BAP1 cancer syndrome: malignant mesothelioma, uveal and cutaneous melanoma, and MBAITs.

BACKGROUND: BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene located on chromosome 3p21. Germline BAP1 mutations have been recently associated with an increased risk of malignant mesothelioma, atypical melanocytic tumors and other neoplasms. To answer the question if different germline BAP1 mutations may predispose to a single syndrome with a wide phenotypic range or to distinct syndromes, we investigated the presence of melanocytic tumors in two unrelated families (L and W) with germline BAP1 mutations and increased risk of malignant mesothelioma. METHODS: Suspicious cutaneous lesions were clinically and pathologically characterized and compared to those present in other families carrying BAP1 mutations. We then conducted a meta-analysis of all the studies reporting BAP1-mutated families to survey cancer risk related to the germline BAP1 mutation (means were compared using t-test and proportions were compared with Pearson χ2 test or two-tailed Fisher's exact test). RESULTS: Melanocytic tumors: of the five members of the L family studied, four (80%) carried a germline BAP1 mutation (p.Gln684*) and also presented one or more atypical melanocytic tumors; of the seven members of W family studied, all carried a germline BAP1 mutation (p.Pro147fs*48) and four of them (57%) presented one or more atypical melanocytic tumors, that we propose to call "melanocytic BAP1-mutated atypical intradermal tumors" (MBAITs). Meta-analysis: 118 individuals from seven unrelated families were selected and divided into a BAP1-mutated cohort and a BAP1-non-mutated cohort. Malignant mesothelioma, uveal melanoma, cutaneous melanoma, and MBAITs prevalence was significantly higher in the BAP1-mutated cohort (p ≤ 0.001). CONCLUSIONS: Germline BAP1 mutations are associated with a novel cancer syndrome characterized by malignant mesothelioma, uveal melanoma, cutaneous melanoma and MBAITs, and possibly by other cancers. MBAITs provide physicians with a marker to identify individuals who may carry germline BAP1 mutations and thus are at high risk of developing associated cancers.

Asbestos-induced chronic inflammation in malignant pleural mesothelioma and related therapeutic approaches-a narrative review.

OBJECTIVE: The aim of this review is addressing the mechanisms of asbestos carcinogenesis, including chronic inflammation and autophagy-mediated cell survival, and propose potential innovative therapeutic targets to prevent mesothelioma development or improve drug efficacy by reducing inflammation and autophagy. BACKGROUND: Diffuse malignant pleural mesothelioma is an aggressive cancer predominantly related to chronic inflammation caused by asbestos exposure. Millions of individuals have been exposed to asbestos or to other carcinogenic mineral fibers occupationally or environmentally, resulting in an increased risk of developing mesothelioma. Overall patient survival rates are notably low (about 8-14 months from the time of diagnosis) and mesothelioma is resistant to existing therapies. Additionally, individuals carrying inactivating germline mutations in the BRCA-associated protein 1 (BAP1) gene and other genes are predisposed to developing cancers, prevalently mesothelioma. Their risk of developing mesothelioma further increases upon exposure to asbestos. Recent studies have revealed the mechanisms and the role of inflammation in asbestos carcinogenesis. Biomarkers for asbestos exposure and malignant mesothelioma have also been identified. These findings are leading to the development of novel therapeutic approaches to prevent or delay the growth of mesothelioma. METHODS: Review of full length manuscripts published in English from January 1980 to February 2021 gathered from PubMed.gov from the National Center of Biotechnology Information and the National Library of Medicine were used to inform this review. CONCLUSION: Key regulators of chronic inflammation mediate asbestos-driven mesothelial cell transformation and survival through autophagic pathways. Recent studies have elucidated some of the key mechanisms involved in asbestos-induced chronic inflammation, which are largely driven by extracellular high mobility group box 1 (HMGB1). Upon asbestos exposure, mesothelial cells release HMGB1 from the nucleus to the cytoplasm and extracellular space, where HMGB1 initiates an inflammatory response. HMGB1 translocation and release also activates autophagy and other pro-survival mechanisms, which promotes mesothelioma development. HMGB1 is currently being investigated as a biomarker to detect asbestos exposure and to detect mesothelioma development in its early stage when therapy is more effective. In parallel, several approaches inhibiting HMGB1 activities have been studied and have shown promising results. Moreover, additional cytokines, such as IL-1ß and TNF-α are being targeted to interfere with the inflammatory process that drives mesothelioma growth. Developing early detection methods and novel therapeutic strategies is crucial to prolong overall survival of patients with mesothelioma. Novel therapies targeting regulators of asbestos-induced inflammation to reduce mesothelioma growth may lead to clinical advancements to benefit patients with mesothelioma.

MicroRNA signature of malignant mesothelioma with potential diagnostic and prognostic implications.

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of target genes, and may behave as oncogenes or tumor suppressors. Human malignant mesothelioma is an asbestos-related cancer, with poor prognosis and low median survival. Here we report, for the first time, a cross-evaluation of miRNA expression in mesothelioma (MPP-89, REN) and human mesothelial cells (HMC-telomerase reverse transcriptase). Microarray profiling, confirmed by real-time quantitative RT-PCR, revealed a differential expression of miRNAs between mesothelioma and mesothelial cells. In addition, a computational analysis combining miRNA and gene expression profiles allowed the accurate prediction of genes potentially targeted by dysregulated miRNAs. Several predicted genes belong to terms of Gene Ontology (GO) that are associated with the development and progression of mesothelioma. This suggests that miRNAs may be key players in mesothelioma oncogenesis. We further investigated miRNA expression on a panel of 24 mesothelioma specimens, representative of the three histotypes (epithelioid, biphasic, and sarcomatoid), by quantitative RT-PCR. The expression of miR-17-5p, miR-21, miR-29a, miR-30c, miR-30e-5p, miR-106a, and miR-143 was significantly associated with the histopathological subtypes. Notably, the reduced expression of two miRNAs (miR-17-5p and miR-30c) correlated with better survival of patients with sarcomatoid subtype. Our preliminary analysis points at miRNAs as potential diagnostic and prognostic markers of mesothelioma, and suggests novel tools for the therapy of this malignancy.