Stromal Influences on Tumor Formation and Growth

1. Stromal Influences on Tumor Formation and Growth Joshua B Rubin, M.D., Ph.D. Department of Pediatrics Division of Pediatric Hematology/Oncology Washington University School of Medicine

2. Outline • Historical perspectives on the mechanisms of oncogenesis • Hypothetical roles for stroma in oncogenesis • Experimental evidence for stromal action in oncogenesis and tumor growth • Stroma in brain tumorigenesis

3. Somatic Mutation Theory of Carcinogenesis • Cancer is derived from a single somatic cell that has acquired multiple 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

4. 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.

5. 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.

6. 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 mutagenized mammary fat pads but not when mutagenized epithelial cells were implanted into control fat pads.

7. How do you explain these findings?

8. 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. Cancer is a disease of tissue disorganization.

9. 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.

10. What constitutes tumor stroma • Vascular endothelial cells • Fibroblasts • Adipocytes • Inflammatory cells (mast cells, phagocytes, microglia) • Matrix

11. What kind of roles can we hypothesize for tumor stroma • Participant in oncogenesis • Regulator of tumor growth • Determinant of metastasis

12. Functional interactions between tumor cells and stroma Mueller & Fusenig (2004) Nature Cancer Reviews

13. 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 Three dimensional tissue organization:extracellular matrix Henry MD, Cohen MB, Campbell KP (2001) Human Pathol 32:791 Muschler J et al. (2002) Cancer Res. 62:7102

14. DAPI GFP CXCl12 The dimensional tissue organization:The Perivascular niche CXCR4 Properties of brain tumor initiating cells within the perivascular niche trophic support - Calabrese (2007) Cancer Cell Increased DNA repair, ABC transporter expression - Bao (2006) Nature

15. EC transplant % tumors 76 NMU 75 0 Veh 0 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

16. NPE NPE Tag-HPE Tag-HPE Fibroblasts and driving oncogenesis No tumor No tumor Normal fibroblasts No tumor Malignant progression CAFs Olumi AF et al (1999) Cancer Res. 59:5002

17. Stromal determinants of pediatric brain tumorigenesis

19. Glioma formation in NF1

20. 9 months Nf1 +/- Astro Nf1 +/- brain Hyperplasia Nf1 -/- Astro Nf1 +/- brain 98% OPGs Nf1 -/- Astro Nf1 +/+brain Hyperplasia Bajenaru et al. (2003) Cancer Research 63:8573-8577 Nf1flox/flox or Nf1flox/- crossed or not with GFAP-Cre transgenic mice Optic pathway glioma formation in NF1

21. Developmental regulation of CXCL12 expression in human brain Warrington et al. (2007) Cancer Research

22. CXCL12 neurofilament CXCL12 CD68 pCXCR4 CXCL12 CXCL12 Multiple sources of CXCL12 are present in OPG Warrington et al. (2007) Cancer Research

23. CXCL12 DDA CXCL12 + - + + - + FSK - + + - + + CXCL12 stimulates Nf1-/- but not Nf1+/+ astrocyte growth in a cAMP dependent manner Warrington et al. (2007) Cancer Research

24. Neurofibromin loss alters CXCR4-mediated cAMP responses Warrington et al. (2007) Cancer Research

25. L12 R4 AC cAMP ATP RAS growth L12 R4 P arrestin Mutational modulation of stromal response pathways: neurofibromin and CXCR4 NF Gi GRKs growth

26. Altering cAMP levels in the brain can alter the pattern of tumorigenesis in a mouse model of NF1

27. Conclusions Carcinogenesis is not always a cell autonomous event. Abnormal epithelial-stromal interactions can promote tumorigenesis. Stromal elements represent novel therapeutic targets

28. Thanks to Washington University Nicole Warrington B. Mark Woerner Lihua Yang Erin Gribben Mahil Rao Shyam Rao David Gutmann Arie Perry Erin Jackson David Piwnica-Worms