Stromal Influences on Brain Tumor Formation
and Growth
Joshua B Rubin, M.D., Ph.D.
Department of Pediatrics
Division of Pediatric Hematology/Oncology
Washington University School of Medicine
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
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 pathways
 Inactivation of cell cycle inhibitors
 Inactivation of apoptotic mechanisms
 Telomere maintenance
 Activation of migration/invasion pathways
 Activation of angiogenic mechanisms
Support for the Somatic Mutation Theory
1890: 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.
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.
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, nontransformed 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.
What else could explain these
findings
 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.
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.
What constitutes tumor stroma
•
•
•
•
Vascular endothelial cells
Fibroblasts
Adipocytes
Inflammatory cells (mast cells,
phagocytes, microglia)
• Matrix
What kind of roles can we
hypothesize for tumor stroma
 Participant in oncogenesis
 Regulator of tumor growth
 Determinant of metastasis
Multi-stage oncogenesis
evading
apoptosis
telomere
maintenance
enhanced
proliferation
oncogenesis
invasion
metastasis
angiogenesis
Functional interactions between tumor
cells and stroma
Mueller & Fusenig (2004) Nature Cancer Reviews
Three dimensional tissue organization:
Basement membrane effects
Normal breast epithelial cells
In matrigel cultures
T4-2 breast carcinoma cells
In matrigel
T4-2 breast carcinoma cells
with reconstituted alpha-dystroglycan
in matrigel
Henry MD, Cohen MB, Campbell KP (2001) Human Pathol 32:791
Muschler J et al. (2002) Cancer Res. 62:7102
The dimensional tissue organization:
The Perivascular niche
DAPI
GFP
CXCl12
CXCR4
Properties of brain tumor initiating cells within the perivascular niche
trophic support - Calabrese
Increased DNA repair, ABC transporter expression - Bao
Fibroblasts and driving oncogenesis
No tumor
NPE
No tumor
Normal fibroblasts
Tag-HPE
No tumor
NPE
Malignant progression
CAFs
Tag-HPE
Olumi AF et al (1999) Cancer Res. 59:5002
Mutational activation of stroma
Maffini et al.(2003) J Cell Sci 117:1495-1502
21 days old-remove epithelial cells from mammary glands
52 days old-NMU or vehicle injection
57 days old-NMU or vehicle treated EC transplant
9 month experiment
EC transplant
% tumors
76
NMU
75
0
Veh
0
Stromal determinants of brain tumorigenesis
Glioma formation in NF1
• 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 Gi 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?
1-2%
(2.3%, 12 yo)
60-70%
(81.8%, 4 yo)
1-2%
(2.3%, 13 yo)
15-20%
(13.6%, 7 yo)
Tumor localization in NF1
Optic pathway glioma formation in NF1
Bajenaru et al. (2003) Cancer Research 63:8573-8577
Nf1flox/flox or Nf1flox/- crossed or not with GFAP-Cre transgenic mice
9 months
Nf1 +/- Astro
Nf1 +/- brain
Nf1 -/- Astro
Nf1 +/- brain
Hyperplasia
100% OPGs
with microglial infiltrate.
Nf1 -/- Astro
Nf1 +/+MC
Hyperplasia
Developmental regulation of CXCL12
expression in human brain
Multiple sources of CXCl12 are present in
OPG
CXCL12
CXCL12
CXCL12
CD68
neurofilament
pCXCR4
CXCL12
CXCL12 stimulates Nf1-/- but not Nf1+/+
astrocyte growth in a cAMP dependent
manner
CXCL12
DDA
CXCL12
FSK
+ - +
+
+
+
-
+
+
+
Neurofibromin loss alters CXCR4-mediated
cAMP responses
Mutational modulation of stromal response
pathways: neurofibromin and CXCR4
L12
R4
NF
AC
Gi
ATP
GRKs
cAMP
RAS
L12
growth
R4
P
arrestin
Induced Tumors
Warrington, et al. Cancer Res. 2010 Jul 15;70(14):5717-27.
Suppression of cAMP is sufficient to promote
gliomagenesis in a mouse model of NF1
Targeting stroma
Lox-STOP-Lox L10a-GFP
Microglial transcriptome
Leukocyte transcriptome
Endothelial transcriptome
Conclusions
 Carcinogenesis is not always a cell autonomous event.
 Abnormal epithelial-stromal interactions can promote
tumorigenesis.
 Stromal elements represent novel therapeutic targets
Thanks to
Washington University
Nicole Warrington
B. Mark Woerner
Lihua Yang
Erin Gribben
Mahil Rao
Shyam Rao
David Gutmann
Arie Perry