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

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 pathwaysInactivation of cell cycle inhibitorsInactivation of apoptotic mechanismsTelomere maintenanceActivation of migration/invasion pathwaysActivation of angiogenic mechanisms

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.

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, 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.

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 oncogenesisRegulator of tumor growthDeterminant of metastasis

oncogenesis

Functional interactions between tumor cells and stroma

Mueller & Fusenig (2004) Nature Cancer Reviews

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

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

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

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

Stromal determinants of brain tumorigenesis

brain tumor incidence

0

5

10

15

20

25

Age in years

• 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

60-70%(81.8%, 4 yo)

15-20%(13.6%, 7 yo)

1-2%(2.3%, 13 yo)

1-2%(2.3%, 12 yo)

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

Developmental regulation of CXCL12 expression in human brain

Multiple sources of CXCl12 are present in OPG

CXCL12 neurofilament

CXCL12 CD68 pCXCR4

CXCL12 CXCL12

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

Neurofibromin loss alters CXCR4-mediated cAMP responses

Mutational modulation of stromal response pathways: neurofibromin and CXCR4

R4R4

L12L12

GRKs

R4R4

L12L12

PP

arrestin

AC

ATP cAMPGi

NF

RAS

growthgrowth

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 stromaLox-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 WarringtonB. Mark WoernerLihua YangErin GribbenMahil RaoShyam Rao

David GutmannArie Perry