stromal influences on brain tumor formation and growth joshua b rubin, m.d., ph.d. department of...
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Stromal Influences on Brain Tumor Formation and Growth
Joshua B Rubin, M.D., Ph.D.Department of Pediatrics
Division of Pediatric Hematology/OncologyWashington University School of Medicine
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Outline
• Historical perspectives on the mechanisms of oncogenesis
• Hypothetical roles for stroma in oncogenesis
• Experimental evidence for stromal action in oncogenesis and tumor growth
• Targeting stroma in cancer therapy
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Somatic Mutation Model of Carcinogenesis
Cancer is derived from a single somatic cell that has acquired multiple DNA mutations.
This results in:
Activation of proliferation pathwaysInactivation of cell cycle inhibitorsInactivation of apoptotic mechanismsTelomere maintenanceActivation of migration/invasion pathwaysActivation of angiogenic mechanisms
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Support for the Somatic Mutation Theory1890: Hansemann notes mitotic abnormalities in cancer cells and postulates that some chromosomes might stimulate proliferation and others might block mitosis.
1914: Boveri observes that specific chromosomal abnormalities are associated with developmental anomalies in sea urchins and proposes that cancer might arise from somatic mutations.
1951: Armitage & Doll postulate the multistage theory of cancer including somatic mutations, genomic rearrangements and changes in tissue interactions.
1960: Nowell & Hungerford discover Philadelphia chromosome (9:22(BCR:ABL)). Soon afterward 8:14 and 8:22 were described (MYC:Ig).
1971: Knudson explains the epidemiology of retinoblastoma in the “two-hit hypothesis” and this work yields the term anti-oncogene or tumor suppressor.
1976: Varmus discovers a cellular homologue (Src) to the transforming protein of Rous Sarcoma Virus, thus identifying the first oncogene.
1983: Cavenee showed second hit involved a gross chromosomal mechanism.
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Observations that challenge the primacy of SMT
Stewart (1981) Injection of teratocarcinoma (TC) cells into mouse blastocyst generated normal tissues including germ cells.
DiBeradino (1982) Nuclear transplant from Lucke’s frog renal carcinoma cells into activated Ova produced normal tadpoles.
Martins-Green (1994) Integration of RSV into chicken genome only produced tumors in the setting of inflammation.
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and
Sternlicht (1999) Expression of stromalysin-1 in mammary gland produced epithelial tumors.
Olumi (1999) Xenograft of normal prostatic ECs and myofibroblasts (CAFs) led to intraepithelial neoplasia while co-injection of immortalized, non-transformed ECs and CAFs led to malignancy.
Maffini (2003) Mammary carcinomas in mouse arose after implantation of normal epithelial cells into irradiated mammary fat pads but not when mutagenized epithelial cells were implanted into control fat pads.
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What else could explain these findings
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Paget (1889) Tumor cells are like the seeds of plants, carried by the wind in all directions, but only able to live on congenial soil.
Boll (1890’s), Waddington (1935): Cancer results from abnormal inductive interactions between tissues.
Cancer is a disease of tissue disorganization.
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Theoretical support for the tissue organization hypothesis
Inherited cancer predisposition syndromes often result in cancers in a tissue and age restricted fashion.
During normal development organizing centers regulate growth and differentiation.
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What constitutes tumor stroma
• Vascular endothelial cells• Fibroblasts• Adipocytes• Inflammatory cells (mast cells,
phagocytes, microglia)• Matrix
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What kind of roles can we hypothesize for tumor stroma
Participant in oncogenesisRegulator of tumor growthDeterminant of metastasis
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oncogenesis
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Functional interactions between tumor cells and stroma
Mueller & Fusenig (2004) Nature Cancer Reviews
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Three dimensional tissue organization:Basement membrane effects
Normal breast epithelial cellsIn matrigel cultures
T4-2 breast carcinoma cellsIn matrigel
T4-2 breast carcinoma cellswith reconstituted alpha-dystroglycan
in matrigel
Henry MD, Cohen MB, Campbell KP (2001) Human Pathol 32:791Muschler J et al. (2002) Cancer Res. 62:7102
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The dimensional tissue organization:The Perivascular niche
DAPIGFPCXCl12
Properties of brain tumor initiating cells within the perivascular nichetrophic support - CalabreseIncreased DNA repair, ABC transporter expression - Bao
CXCR4
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Fibroblasts and driving oncogenesis
Normal fibroblasts
CAFs
NPE
Tag-HPE
NPE
Tag-HPE
No tumor
No tumor
No tumor
Malignant progression
Olumi AF et al (1999) Cancer Res. 59:5002
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Mutational activation of stromaMaffini et al.(2003) J Cell Sci 117:1495-1502
21 days old-remove epithelial cells from mammary glands52 days old-NMU or vehicle injection57 days old-NMU or vehicle treated EC transplant9 month experiment
NMU
Veh
EC transplant % tumors
76
75
0
0
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Stromal determinants of brain tumorigenesis
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brain tumor incidence
0
5
10
15
20
25
Age in years
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• NF1 loss is not sufficient for optic pathway glioma formation• NF1 loss results in hyperactivation of RAS and is associated with decreased generation of cAMP.• CXCR4 is Gi GPCR. CXCL12 binding results in activation of RAS and reduction in cAMP.
Could CXCL12 provide an anatomically localized growth signal
that promotes glioma formation in NF1?
Glioma formation in NF1
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60-70%(81.8%, 4 yo)
15-20%(13.6%, 7 yo)
1-2%(2.3%, 13 yo)
1-2%(2.3%, 12 yo)
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Optic pathway glioma formation in NF1
Nf1 +/- AstroNf1 +/- brain
Nf1 -/- AstroNf1 +/- brain
Nf1 -/- AstroNf1 +/+MC
9 months
Hyperplasia
100% OPGs
with microglial infiltrate.
Hyperplasia
Bajenaru et al. (2003) Cancer Research 63:8573-8577 Nf1flox/flox or Nf1flox/- crossed or not with GFAP-Cre transgenic mice
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Developmental regulation of CXCL12 expression in human brain
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Multiple sources of CXCl12 are present in OPG
CXCL12 neurofilament
CXCL12 CD68 pCXCR4
CXCL12 CXCL12
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CXCL12 stimulates Nf1-/- but not Nf1+/+ astrocyte growth in a cAMP dependent
manner
-40
-30
-20
-10
0
10
20Nf1+/+
Nf1-/-
CXCL12 + - + + - + FSK - + + - + +
CXCL12
Nf1+/+ Nf1-/-
-30
-20
-10
0
10
20
30Nf1+/+
Nf1-/-
DDA
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Neurofibromin loss alters CXCR4-mediated cAMP responses
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Mutational modulation of stromal response pathways: neurofibromin and CXCR4
R4R4
L12L12
GRKs
R4R4
L12L12
PP
arrestin
AC
ATP cAMPGi
NF
RAS
growthgrowth
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Induced Tumors
Warrington, et al. Cancer Res. 2010 Jul 15;70(14):5717-27.
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Suppression of cAMP is sufficient to promote gliomagenesis in a mouse model of NF1
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Targeting stromaLox-STOP-Lox L10a-GFP
Microglial transcriptome
Leukocyte transcriptome
Endothelial transcriptome
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Conclusions
Carcinogenesis is not always a cell autonomous event.
Abnormal epithelial-stromal interactions can promote tumorigenesis.
Stromal elements represent novel therapeutic targets
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Thanks to
Washington University
Nicole WarringtonB. Mark WoernerLihua YangErin GribbenMahil RaoShyam Rao
David GutmannArie Perry