Cell Signaling in Mesothelioma

1
Cell Signaling in Mesothelioma

• Brooke T. Mossman
• Professor of Pathology
• UVM College of Medicine

2
LECTURE OUTLINE

• Signaling, Proliferation and Multistage
  Carcinogenesis
• Signaling Pathways Induced by Asbestos Fibers are
  Constitutively Activated in Mesothelioma
• Therapeutic Strategies Acid Prepared Amorphous
  Silica Spheres (APMS)

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MULTISTAGE CARCINOGENESIS
Promotion
Initiation
Progression
OH.
O2.-
x
Additional genetic/ proliferative events
Expansion of initiated cells
DNA damage/mutations
Genetic changes
Altered gene expression
Invasion
Chronic cell proliferation
Metastasis
Genetic predisposition
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MULTIPLE MECHANISMS OF ASBESTOS-INDUCED CANCERS
INITIATION (Genetic Damage)
PROMOTION (Fibrosis)
-
Cell Replication
Cell Replication
PROGRESSION
PROGRESSION
Cell Replication
Cell Replication
? SV40 (Mesothelioma) ? Smoking (Lung Cancer)
MESOTHELIOMA
LUNG CANCER
5
CELL SIGNALING BY ASBESTOS FIBERS IS INITIATED AT
THE PLASMA MEMBRANE
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ASBESTOS SIGNALING, PROLIFERATION AND MIGRATION
BEGINS WITH ACTIVATION OF EXTRACELLULAR SIGNAL
REGULATED KINASES (ERKS)
Asbestos
ERK1/2, ERK5
fra-1
mRNA
sifra-1
x
Fra-1
jun
mRNA
AP-1
Cell Proliferation
cd44 c-met, etc.
sicd44
X
x
Migration, Invasion of Human Mesotheliomas
7
Asbestos Signaling
Asbestos Fibers
Oxygen radical formation
EGFR
Cytosol
P
P
P
Nucleus
8
Signaling Pathways Are Upregulated in
Mesotheliomas

• Mesothelial cell transformation requires
  increased AP-1 binding activity and ERK-dependent
  Fra-1 expression. Ramos-Nino ME, Timblin CR and
  Mossman BT. Cancer Res. 2002 Nov 162(21)6065-9.
  
• Human mesothelioma cells exhibit tumor
  cell-specific differences in phosphatidylinositol
  3-kinase/AKT activity that predict the efficacy
  of Onconase. Ramos-Nino ME, Vianale G,
  Sabo-Attwood T, Mutti L, Porta C, Heintz N and
  Mossman BT. Mol Cancer Ther 20054(5)835-842.
• Human and mouse mesotheliomas exhibit elevated
  AKT/PKB activity which can be targeted
  pharmacologically to inhibit tumor cell growth.
  Altomare DA, You H, Xiao G-H, Ramos-Nino ME,
  Skele KL, De Rienzo A, Jhanwar SC, Mossman BT,
  Kane AB and Testa JR. Oncogene 200524,
  6080-6089.

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Mesothelioma Treatment Regimens

• Surgery
• Radiation Therapy
• Palliative Procedures
• Chemotherapy
• Alimta (Pemetrexed)
• Onconase
• Doxorubicin (Adriamycin)
• Cisplatin
• Methotrexate
• Methotrexate and vincristine
• Cisplatin, vinblastine and mitomycin
• Cisplatin and doxorubicin
• Doxorubicin, cyclophosphamide (or ifosfamide) and
  cisplatin
• Even combination therapies (Alimta and
  Cisplatin) are ineffective, with a mean survival
  time of less than 1 year.

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Delivery to Target Cells

• Problems
• Ineffective delivery to target cells
• Systemic toxicity
• Novel method of delivery
• Acid-prepared mesoporous silica spheres (APMS)
• Patented by Christopher Landry, Ph.D., Department
  of Chemistry, UVM.

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Hypothesis

• Use of APMS as vehicles for intrapleural or
  intraperitoneal administration of therapeutics
  will increase the effectiveness of drug
  administration.

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Acid-Prepared Amorphous Silica Spheres (APMS)

• Can control diameter and pore size
• Easily chemically modified
• External and internal (pore) surfaces can be
  functionalized differently
• External surface is linked to a reactive group
• Pores left open for molecular exchange
• Advantage versus dense beads
• Surface area (and therefore the loading capacity)
  of a dense bead is much lower

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Basic Structure of APMS
Alexa 532
DNA-
Mg2
Mg2
DNA-
DNA-
40 70Å
Mg2
Mg2
DNA-
DNA-
Mg2
TEG
Antibody

Anti-mesothelioma
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Specific Questions

• How are APMS taken up by cells?
• Coatings binding and uptake
• How are they trafficked in cells
• Can the APMS deliver functional molecules to
  cells?
• Can this approach work in vivo?
• What is the fate of the APMS in vivo?
• Can APMS modify signaling pathways important in
  MM development and progression?

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Different Coatings on APMS Affect Cell Uptake
400x
Lipid Amine
Propylthiol Tetraethyleneglycol
(TEG)
Green (Cell nuclei, Sytox Green) Red (APMS, Alexa
568)
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APMS Are Not Toxic to Lung Epithelial Cells in
vitro
LDH Release (OD490) Compared to Cell-Free Media
APMS Concentration 2.5 x 107 per mL media
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Transfer and Expression of Plasmid Encoding Red
Fluorescent Protein (RFP) From APMS to Lung
Epithelial Cells in vitro (24 Hours)
APMS
RFP
Nucleus (Sytox Green)
Merge
(Green nuclei Sytox Green, Blue pseudocolor
APMS, Red RFP).
18
APMS Increase the Effectiveness of Doxorubicin
Killing of Human Mesothelioma Cells
A. Doxorubicin
B. APMS Loaded with Doxorubicin
LDH Release ()
LDH Release ()
LDH Release ()
Doxorubicin Concentration (nM)
APMS Doxorubicin Concentration (nM)
DOX concentrations in vitro range from 2µM to
15µM in published work.
APMS Concentration 2.5 x 107 per mL media
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Doxorubicin Delivered in APMS Increases Cell
Toxicity
APMS-DOX (80nM)
Control
DOX (80nM)
APMS-TEG
8 Hours
24 Hours
48 Hours
72 Hours
200x
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Doxorubicin Delivered in APMS Decreases Cell
Viability
Viability () Compared to Control
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Summary Question 2

• APMS can deliver both plasmids and anti-cancer
  drugs (doxorubicin) to cells.
• Delivered constructs and drugs are functional.
• Doxorubicin, when delivered by APMS, is more
  effective at killing cells than if administered
  alone.

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Specific Questions

• How are APMS taken up by cells?
• Coatings binding and uptake
• How are they trafficked in cells
• Can the APMS deliver functional molecules to
  cells?
• Can this approach work in vivo?
• What is the fate of the APMS in vivo?

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APMS in Ribs 72 Hours After Intrapleural
Injection
400x
400x
Location of APMS injection
APMS are red (Alexa 568), cell nuclei are blue
(TOTO3).
Injected 3.3 X 107 APMS in 100µL PBS per mouse
(C57Bl/6)
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APMS Enter Tissues 72 Hours After Intrapleural
Injection
Diaphragm
Lung
Nuclei
APMS
Nuclei
APMS
Merge
DIC
Merge
400x
200x
APMS are green (Pseudocolor) Nuclei are red
(Pseudocolor)
APMS are red (Alexa 568) Nuclei are blue (DAPI)
Injected 3.3 X 107 APMS in 100µL PBS per mouse
(C57Bl/6)
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APMS Injected Intrapleurally are Not Immunogenic
in the Lung as Indicated by Bronchoalveolar
Lavage Fluid Cell Populations
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APMS Injected Intrapleurally are Not Immunogenic
in the Lung as Indicated by Pleural Lavage Fluid
Cell Populations
3-Day PLF Cell Differential
7-Day PLF Cell Differential
Percent
Percent

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APMS Enter Lung Tissue When Administered Nasally
A. 6 hours post-instillation
B. 24 hours post-instillation
1,200X
APMS are red
Instilled mice with 3.3 x 107 APMS in 50µL PBS
28
APMS in CD45 Cells in the Lung 3-Days
Post-Intrapleural Injection
Nuclei
APMS
CD45 Cells
DIC/Merge
Nuclei Blue (DAPI) APMS Red (Alexa532) CD45
Green (Alexa488)
630x
29
Some APMS are Found in CD45 Cells of the Spleen
3-Days Post-Intrapleural Injection
Blue Cell nuclei Red APMS Yellow CD45
Spleen
1260x
30
Can APMS Be Found in Other Tissues? TEM and
Energy-Dispersive X-ray Spectroscopy (EDS)
APMS following OCl- treatment on 0.4uM Filter
(x15K)
APMS from OCl- digested lung on 0.4uM filter
(x20k)
X-ray spectra of silica APMS
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Summary Question 4

• APMS, when delivered intrapleurally in mice, are
  likely in CD45 cells, and possibly in
  epithelial-type cells.
• TEM / EDS is a technique that can be used to
  quantitate APMS in tissues.

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Conclusion

• APMS may be a viable approach for treatment of
  mesotheliomas, and following surgical debulking
  of tumors, and may be best suited as part of
  combination therapy.

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Future Directions

• Continue determination of fate of APMS in vitro
• Mechanisms of binding, uptake and intracellular
  trafficking
• Further characterize APMS for in vitro and in
  vivo transfection of constructs drugs (Dox
  LY29004)
• Determination of the fate of APMS in vivo
• Cell types, kinetics, other tissues
• Effect of APMS on signaling pathways
• ERK1/2, ERK5, AKT, NF-kB, AIF
• In vivo human mesothelioma xenograft model in
  nude mice

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ACKNOWLEDGMENTS
Mossman Laboratory Maria Ramos-Nino, Ph.D. Arti
Shukla, Ph.D. Tara Sabo-Attwood, Ph.D. Sylke
Buder-Hoffmann, Ph.D. Steven Blumen Christy
Barlow Jamie Levis Max MacPherson Trisha
Barrett Stacie Beuschel Joanna Gell
Collaborators Christopher Landry, Ph.D. Kai Cheng
Supported by grants from NIEHS (T32), NHLBI (PO1)
and NCI (KO1)