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MECHANISMS OF ASBESTOS-RELATED DISEASES: IMPLICATIONS FOR EARLY DIAGNOSIS AND THERAPY

Agnes B. Kane, MD, PhDDepartment of Pathology and Laboratory Medicine

Brown University

1. Asbestos-Related Cancers

2. Classical and Emerging Hallmarks and Enabling Characteristics of Cancer

3. Tumor-Promoting Inflammation

4. Genomic Instability and Mutation

5. Avoiding Immune Destruction – Implications for Therapy

Biomarkers for Early Diagnosis

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History of Asbestos-Related DiseasesBecklake, Am. Rev. Resp. Dis. 114:187-227, 1976

IOM (2006); IARC (2009)

ASBESTOS-RELATED CANCERS

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CLASSICAL HALLMARKS OF CANCER ENABLING CHARACTERISTICSHanahan and Weinberg, Cell (2011)

Differential Gene Expression in Malignant Pleural MesotheliomaRøe et al. PLOS ONE (2009)

Hanahan and Weinberg, Cell (2000)

Sustained proliferation Resistance to apoptosis Inflammatory cytokines

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INFLAMMATION IN THE TUMOR MICROENVIRONMENT“Tumors behave like wounds that fail to heal.”

Harold F. Dvorak, 2007

Liguori et al. Cancers (2011)

Healing Wound Tumor

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ASBESTOS FIBERS CAUSE PERSISTENT INFLAMMATION IN THE LUNGS AND PLEURA:

NLRP3 INFLAMMASOME ACTIVATION

Mossman et al. Am J Pathol (2013)

Incomplete or Frustrated Phagocytosis of Asbestos Fibers by Macrophages

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ASBESTOS FIBERS AND CHRONIC INFLAMMATIONMurine Models of Asbestosis

Fiber Penetration into Alveolar Walls Walls

J. Brain, Harvard School of Public Health

Signaling Networks Linking Inflammation and Fibrosis

Sabo-Attwood et al. Am J Pathol (2011)

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PATHWAYS LINKING CHRONIC INFLAMMATION AND CANCER

Colotta et al. (2009), Multhoff et al. (2012), Kundu and Surh, (2012)

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TUMOR ASSOCIATED MACROPHAGES (TAM):MASTER REGULATORS OF THE TUMOR

MICROENVIRONMENT

Human malignant mesothelioma

mesothelioma cells: cytokeratin + (pink)macrophages: CD68+ (brown)

Kim et al. Am J Respir Cell Mol Biol (2005)

Rogers and Holen, J Translational Medicine (2011)

TAMs contribute to cancer hallmarks: sustained tumor growth angiogenesis invasion and metastasis

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TUMOR MICROENVIRONMENT OF MURINE MESOTHELIOMA

Miselis et al. Cancer Microenvironment (2011)

Tumor-Associated Macrophages (F4/80)

MDSCs (CB11b & GR-1)

Regulatory T cells(CD4 & CD25)

Cytotoxic T cells(CD3 & CD8)

MDSC: myeloid-derived suppressor cell

Overall, the tumor microenvironment is immunosuppressive – an emerging hallmark of cancer

GENE EXPRESSION PROFILES OF MURINE AND HUMAN MESOTHELIOMAS

↑CCL2 (MCP-1) chemokine expression – inflammatory mediator

↑soluble mesothelin-related peptide (SMRP)

↓galectin-3 (LGALS3) expression

Mohr et al. Biochim Biophys Acta (2004)Miselis et al. Cancer Microenvironment (2011)

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Pleural Fluid Biomarkers for Early Diagnosis

SMRP –membrane-anchored glycoproteinsoluble serum protein biomarkerlimited sensitivity for diagnosis of mesothelioma

Gueugnon et al. Am J Pathol (2011)Blanquart et al. J. Thorac Oncology (2012)

Mesothelin expression in malignant pleural

mesothelioma

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TUMOR-ASSOCIATED INFLAMMATION AS A THERAPEUTIC TARGETCoussens et al. Science (2013)

1. Deplete or reprogram tumor-associated macrophagesMiselis et al. Mol Cancer Ther (2008); Rogers and Holen (2011)

Antiviral, antibacterialM1 polarization Tumor resistance

ImmunosuppressiveM2 polarization Tumor promotion

Macrophage Depletion Reduces Murine Mesothelioma Tumor BurdenLiposome-encapsulated clodronateLiposome-encapsulated PBS Tumor Burden

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2. Inhibit or sequester inflammatory mediatorsarachidonic acid metabolites (COX-2 inhibitors)inhibit inflammasome – IL1-RA (Anakinra), IL-18BPcytokine blockade – IL-6 antibody, TNF-α blockers, CCL2 antagonistsinhibit signaling pathways – NF-ΚB, STAT3, JAK2 inhibitors

TUMOR-ASSOCIATED INFLAMMATION AS A THERAPEUTIC TARGETCoussens et al. Science (2013)

3. Harness host cytotoxic T Cells Cornelissen et al. Clin Dev Immunol (2012)

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INFLAMMATION, DNA DAMAGE, AND GENOMIC INSTABILITY

Colotta et al. Carcinogenesis (2009), Grivennikov et al. Cell (2010), Aivaliotis et al. J Biomed Biotechnol (2012), Dizdaroglu, Cancer Letts (2012)

RONS – reactive oxygen and nitrogen species

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DNA DAMAGE AND GENOMIC INSTABILITYMcKinnon, Nature Rev Neuro (2009)

DNA strand breaks, oxidized bases dysregulated DNA repair mutations defective cell cycle checkpoints, dysregulated homologous recombination

chromosomal instability

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ASBESTOS FIBERS AND DNA DAMAGE

Nagai and Toyokuni, Arch Biochem Biophys (2010)

multipolar mitoses

multinucleated cells

Human lung epithelial cells exposed to asbestos in vitro: induction of aneuploidy

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GLOBAL SEQUENCING OF MESOTHELIOMA GENOME REVEALSANEUPLOIDY AND COMPLEX CHROMOSOMAL REARRANGEMENTS

Bueno et al. PLOS One (2010)

LOH/homozygous deletion 9p21.3 p16/CDKN2ACopy number alterations 9q33.1 DBC1 deletionAllelic imbalance 19p13 KEAP1 lossLOH 3p14 FHIT loss

MOLECULAR ALTERATIONS IN ASBESTOS-RELATED LUNG CANCER

Andujar et al. (2010)Nymark et al. (2009)Ruosaari et al. (2008)Pylkkänen et al. (2002)

Normal Lung DNA

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OXIDATIVE DNA DAMAGE AND EPIGENETIC CHANGES

O’Hagan et al. Cancer Cell (2011)

ROS DNA breaks oxidized guanine

recruitment of gene silencing complex to sites of oxidative damage: DNA damage proteins (γ-H2AX, SIRT1) DNA methylating enzymes (DNMT1, DNMT3B)

methylation of CpG promoter islands epigenetic gene silencing

“epigenetic therapy” – DNA demethylating agents

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METHYLATION PROFILES OF PLEURAL MESOTHELIOMAChristensen et al. Cancer Res (2009)

Methylation profiles discriminate between mesothelioma and nontumor pleura Methylation classes are associated with lung tissue asbestos body burden Pleural mesothelioma and lung adenocarcinomas have distinct methylation profiles

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PLEURAL FLUID CYTOLOGICAL MARKERS FOR MESOTHELIOMA Matsumoto et al. Cancer Cytopathol (2013)

Homozygous Deletion of 9p21 Gene Locus:

p16/CDKN2A tumor suppressor gene

FISH Cytology

arrows – 9p21 deletion in mesothelioma cells

arrowheads – normal lymphocytes (red signal)

cell-in-cell engulfmentmultinucleationmulticellular clusters

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GENOMIC INSTABILITY AND CANCER PROGRESSION

Kidane et al. Crit Rev Biochem Mol Biol (2014)

NF-ΚB activation at the crossroads of inflammation and DNA repair

miR – 21 IL10 expression (anti-inflammatory)

miR – 210 inhibits RAD52

miR – 155 inhibits mismatch repair

NF-ΚB is a master regulator of inflammation: increased expression of cytokinesNF-ΚB is also induced by ROS and DNA damage

increased expression of microRNAs

inhibits expression of DNA repair proteins

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MicroRNA (miRNA) Expression Profiles in Asbestos-Related Lung CancerNymark et al. Genes, Chromosomes & Cancer (2011)

Downregulated Target Genes

GADD45A DNA repairTXNRD1 oxidative stressKDM4B histone demethylation

Upregulated Target Genes

PTGS2 (COX2) inflammation

Differential expression of miRNAs in asbestos-related lung cancers

Increased expression of miRNAs downregulates target genes and inhibits protein expression

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SUMMARY

1. Asbestos fibers trigger chronic inflammation that enables tumor development

2. Asbestos fibers directly and indirectly generate ROS leading to genomic instability and mutation

3. The tumor microenvironment is immunosuppressive allowing evasion of host immune attack

4. Combination therapies that target chronic inflammation and harness the host immune response, in addition to cytotoxic agents, may be more effective against asbestos-related cancers

Research funding provided by the National Institute of Environmental Health Sciences

Additional information – Broaddus VC, Everitt JI, Black B, Kane AB: Non-neoplastic and neoplastic pleural endpoints following fiber exposure. J Toxicol Environ Health, Part B 14: 153-178 (2011)

Hanahan and Weinberg Cell (2011)

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FUTURE DIRECTIONS FOR EARLY DIAGNOSIS AND THERAPY

1. MicroRNA profiles in peripheral blood cells or plasma2. Proteomics for identification of new serum protein biomarkers3. Multifunctional diagnostic and therapeutic probes:

THERANOSTIC NANOPARTICLES Nanoparticle Drug Applications

gold doxorubicin diagnosis, PTT

iron oxide doxorubicin, targeting, docetaxel MRI, therapy

silica doxorubicin imaging, PTT

quantum doxorubicin, imaging, dots methotrexate therapyAhmed et al. Drug Discovery Today (2012)

PTT = laser photothermal therapy