Understanding the mechanisms of asbestos related diseases

University of Hawai i Cancer Center Understanding the mechanisms of asbestos related diseases Haining Yang, PhD Professor University of Hawai i Cancer Center

Marker of exposure: Bilateral pleural plaques Non-malignant diseases: i. Asbestosis (diffuse bilateral lung fibrosis, can lead to death) ii. Diffuse pleural thickening iii. Pleural effusion iv. Rounded atelectasis (folded lung) Malignant diseases i. Malignant Mesothelioma (MM) ii. Lung Cancer

Potential targets and strategies for mesothelioma therapy that have been proposed based on recent studies. Michele Carbone, and Haining Yang Clin Cancer Res 2012;18:598-604 2012 by American Association for Cancer Research

Normal human mesothelial cells (HM) HM Exposed to Crocidolite Asbestos

Asbestos is cytotoxic to HM in tissue culture. Haining Yang et al. PNAS 2006;103:10397-10402 2006 by National Academy of Sciences

TNF-α and TNF-α receptor TNF-R1 are induced in HM following asbestos exposure. Haining Yang et al. PNAS 2006;103:10397-10402 2006 by National Academy of Sciences

TNF-α significantly reduces asbestos cytotoxicity. Haining Yang et al. PNAS 2006;103:10397-10402 2006 by National Academy of Sciences

TNF-α induces the activation of NF-κB. Haining Yang et al. PNAS 2006;103:10397-10402 2006 by National Academy of Sciences

Inhibition of the NF-κB pathway by Bay11-7082 suppresses the anticytotoxicity effect induced by TNF-α. Haining Yang et al. PNAS 2006;103:10397-10402 2006 by National Academy of Sciences

RNA interference assays confirm that TNF-α inhibits asbestos induced cytotoxicity by means of a NF-κB dependent mechanism. Haining Yang et al. PNAS 2006;103:10397-10402 2006 by National Academy of Sciences

crocidolite+tnf- crocidolite+tnf- crocidolite asbestos 1 week 1 month

How does asbestos kill mesothelial cells? What kind of Cell death? Apoptosis? Necrosis?

Asbestos induced cell death -involves programmed necrosis New concept of necrosis -- It is a regulated event The process of necrosis normally involves-- 1) Activation of PARP (poly(adpribose)polymerase) 2) ATP depletion 3) Release of HMGB1(high mobility group box 1) 4) Induction of inflammation

Asbestos induces PARP activation in HM. (A) Increasing amounts of asbestos induced a parallel increase in PARP levels, without inducing PARP cleavage. Haining Yang et al. PNAS 2010;107:12611-12616 2010 by National Academy of Sciences

Asbestos-induced HM death is PARP dependent and causes ATP depletion. Haining Yang et al. PNAS 2010;107:12611-12616 2010 by National Academy of Sciences

HMGB1 http://stke.sciencemag.org/content/sigtrans/vol2002/issue151/images/large/1512002pe39f1.jpeg

HMGB1 is released from HM into the extracellular space after asbestos exposure. Haining Yang et al. PNAS 2010;107:12611-12616 2010 by National Academy of Sciences

Asbestos-induced cell death and the release of HMGB1 promote TNF-α secretion by macrophages. Haining Yang et al. PNAS 2010;107:12611-12616 2010 by National Academy of Sciences

Yang H et al, PNAS 2006, PNAS 2010, Clinical Cancer Res 2011

Asbestos Inhibition of HMGB1 decreases the foci formation in the in vitro MM model (HM/macrophages co-culture system)

Immunohistochemical analyses show strong HMGB1 and TNF-α staining around areas of asbestos deposits in two representative murine specimens. Haining Yang et al. PNAS 2010;107:12611-12616 2010 by National Academy of Sciences

HMGB1 levels in the serum of mice injected with different types of fibers 40 HMGB1 ELISA - Mice injected w/ fibers 35 * HMGB1 (ng/ml) 30 25 20 15 10 * * * * * 5 0 PBS Glass CHRY CRO ND OR TK TRE

HMGB1 serum levels are long-term persistent after exposure to crocidolite but not to chrysotile (Qi et al, Am J Pathol, 2013) HMGB1 levels in the serum of mice injected with with a total of 5 mg of crocidolite or chrysotile in a high-dose, short-term protocol with two weekly injections of 2.5 mg each

HMGB1 levels in the serum of mice injected with with a total of 5 mg of crocidolite or chrysotile in a low-dose, long-term protocol with 10 weekly injections of 0.5 mg each

Model for asbestos carcinogenesis: Yang H et al, PNAS 2006, 2010, Clinical Cancer Res 2011

Yang H et al, PNAS 2010

HMGB1 is highly expressed in mesothelioma tissue. Normal pleura MM 100X 400X

MM cells contain high levels of HMGB1 in both nucleus and cytoplasm MM cell lines secrete HMGB1 HMGB1 ELISA using cell culture medium

MM cells migration assay

Inhibition of HMGB1 reduces MM cells proliferation and mobility Wound-healing assay

Inhibition of HMGB1 reduces anchorage independent growth of MM cells

Anti-HMGB1 mab inhibits MM growth in mouse model

Jube S, M Carbone and H Yang. Cancer Res. 2012

Anti-inflammatory drug: Aspirin low toxicity, and proven efficacy in reducing the incidence of colorectal cancer. The Physician's Health Study In the study, 22,067 physicians were followed for 24 years. <aspirin (325 mg on alternate days) or placebo> 17 cases of MM reported. An intention to treat analysis comparing aspirin to placebo revealed a relative risk of 0.7 (0.3-1.9 95% confidence interval). The risk was 30% lower in the aspirin treatment group The result indicates a possible association between aspirin use and reduced MM incidence.

Effect of different anti-inflammatory drugs on MM motility Aspirin: acetylsalicylic acid (ASA)

Anti-inflammatory drug: Aspirin Aspirin reduces HMGB1 serum levels in mice exposed to asbestos Aspirin reduces HMGB1 serum levels in mice with MM

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Salicylates inhibit colony formation of MM cells

Salicylates inhibit colony formation of high HMGB1-secreting MM cells (A-B) but not of low HMGB1-secreting cells (C-D).

Effect of SA on HMGB1-induced migration is COX-2 independent

Aspirin inhibits MM tumor growth in mice

Conclusion: HMGB1 is a critical player in asbestos carcinogenesis and MM pathogenesis. Targeting HMGB1 can be a novel strategy for mesothelioma prevention and therapy Some of the aspirin anticancer activity may be mediated through inhibition of HMGB1. Our results are relevant to mesothelioma and to other inflammation-related cancers in which HMGB1 plays a role. Specific HMGB1 inhibitors, such as BoxA, may have stronger anti-cancer activity and less side effects than aspirin.

HMGB1 serum level is significantly higher in MM patients than in healthy controls.

- NCI R01 (Yang PI) Current Active Grants HMGB1 and Its Isoforms As Biomarkers For Mineral Fiber Exposure and MM Detection (Yang and Carbone, Co PIs) Identification and validation of novel germline DNA variants associated to increased risk of malignant mesothelioma - The EDRN Mesothelioma Biomarker Discovery Laboratory - V Foundation Translational Grant (Yang-Basic Science PI, Carbone-Clinical PI) Riviera United-4 A Cure Foundation grant