Presented by Professor Agnes Kane from Brown University, USA.

1. MECHANISMS OF ASBESTOS-RELATED DISEASES:
IMPLICATIONS FOR EARLY DIAGNOSIS AND THERAPY
Agnes B. Kane, MD, PhD
Department of Pathology and Laboratory Medicine
Brown University
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1. Asbestos-Related Cancers
1. Classical and Emerging Hallmarks and Enabling Characteristics of Cancer
1. Tumor-Promoting Inflammation
1. Genomic Instability and Mutation
1. Avoiding Immune Destruction – Implications for Therapy
Biomarkers for
Early Diagnosis

2. 2
History of Asbestos-Related Diseases
Becklake, Am. Rev. Resp. Dis. 114:187-227, 1976
IOM (2006); IARC (2009)
ASBESTOS-RELATED CANCERS

3. CLASSICAL HALLMARKS OF CANCER ENABLING CHARACTERISTICS
Hanahan and Weinberg, Cell (2011)
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Differential Gene Expression in Malignant Pleural Mesothelioma
Røe et al. PLOS ONE (2009)
Hanahan and Weinberg, Cell (2000)
Sustained proliferation Resistance to apoptosis Inflammatory cytokines

4. INFLAMMATION IN THE TUMOR MICROENVIRONMENT
“Tumors behave like wounds that fail to heal.”
Harold F. Dvorak, 2007
Liguori et al. Cancers (2011)
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Healing Wound Tumor

5. ASBESTOS FIBERS CAUSE PERSISTENT
INFLAMMATION IN THE LUNGS AND PLEURA:
NLRP3 INFLAMMASOME ACTIVATION
Mossman et al. Am J Pathol (2013)
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Incomplete or Frustrated
Phagocytosis of Asbestos
Fibers by Macrophages

6. 6
ASBESTOS FIBERS AND CHRONIC INFLAMMATION
Murine 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)

7. PATHWAYS LINKING CHRONIC INFLAMMATION AND CANCER
Colotta et al. (2009), Multhoff et al. (2012), Kundu and Surh, (2012) 7

8. 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)
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TAMs contribute to cancer hallmarks:
sustained tumor growth
angiogenesis
invasion and metastasis

9. 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)
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MDSC: myeloid-derived suppressor cell
Overall, the tumor microenvironment is immunosuppressive –
an emerging hallmark of cancer

10. 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
glycoprotein
soluble serum protein biomarker
limited 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

11. TUMOR-ASSOCIATED INFLAMMATION AS A THERAPEUTIC TARGET
Coussens et al. Science (2013)
1. Deplete or reprogram tumor-associated macrophages
Miselis et al. Mol Cancer Ther (2008); Rogers and Holen (2011)
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Antiviral, antibacterial
M1 polarization Tumor resistance
Immunosuppressive
M2 polarization Tumor promotion
Macrophage Depletion Reduces Murine Mesothelioma Tumor Burden
Liposome-encapsulated clodronateLiposome-encapsulated PBS Tumor Burden

12. 2. Inhibit or sequester inflammatory mediators
arachidonic acid metabolites (COX-2 inhibitors)
inhibit inflammasome – IL1-RA (Anakinra), IL-18BP
cytokine blockade – IL-6 antibody, TNF-α blockers, CCL2 antagonists
inhibit signaling pathways – NF-ΚB, STAT3, JAK2 inhibitors
TUMOR-ASSOCIATED INFLAMMATION AS A THERAPEUTIC TARGET
Coussens et al. Science (2013)
3. Harness host cytotoxic T Cells
Cornelissen et al. Clin Dev Immunol (2012)
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13. 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)
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RONS – reactive oxygen and nitrogen species

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DNA DAMAGE AND GENOMIC INSTABILITY
McKinnon, Nature Rev Neuro (2009)
DNA strand breaks, oxidized bases dysregulated DNA repair mutations
defective cell cycle checkpoints, dysregulated homologous recombination
chromosomal instability

15. ASBESTOS FIBERS AND DNA DAMAGE
Nagai and Toyokuni, Arch Biochem Biophys (2010)
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multipolar mitoses
multinucleated
cells
Human lung epithelial cells
exposed to asbestos in vitro:
induction of aneuploidy

16. 16
GLOBAL SEQUENCING OF MESOTHELIOMA GENOME REVEALS
ANEUPLOIDY AND COMPLEX CHROMOSOMAL REARRANGEMENTS
Bueno et al. PLOS One (2010)
LOH/homozygous deletion 9p21.3 p16/CDKN2A
Copy number alterations 9q33.1 DBC1 deletion
Allelic imbalance 19p13 KEAP1 loss
LOH 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

17. OXIDATIVE DNA DAMAGE AND EPIGENETIC CHANGES
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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 MESOTHELIOMA
Christensen 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

19. 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 engulfment
multinucleation
multicellular clusters
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20. GENOMIC INSTABILITY AND CANCER PROGRESSION
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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 cytokines
NF-ΚB is also induced by ROS and DNA damage
increased expression of microRNAs
inhibits expression of DNA repair proteins

21. MicroRNA (miRNA) Expression Profiles in Asbestos-Related Lung Cancer
Nymark et al. Genes, Chromosomes & Cancer (2011)
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Downregulated Target Genes
GADD45A DNA repair
TXNRD1 oxidative stress
KDM4B 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 plasma
2. Proteomics for identification of new serum protein biomarkers
3. 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