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 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 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 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. 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 mutagenized mammary fat pads but not when mutagenized epithelial cells were implanted into control fat pads. How do you 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. 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 Functional interactions between tumor cells and stroma Mueller & Fusenig (2004) Nature Cancer Reviews Three dimensional tissue organization: extracellular matrix 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 (2007) Cancer Cell Increased DNA repair, ABC transporter expression - Bao (2006) Nature 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 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 Stromal determinants of pediatric brain tumorigenesis Glioma formation 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 Nf1 -/- Astro Nf1 +/+brain Hyperplasia 98% OPGs Hyperplasia Developmental regulation of CXCL12 expression in human brain Warrington et al. (2007) Cancer Research Multiple sources of CXCL12 are present in OPG CXCL12 CXCL12 CXCL12 CD68 neurofilament pCXCR4 CXCL12 Warrington et al. (2007) Cancer Research CXCL12 stimulates Nf1-/- but not Nf1+/+ astrocyte growth in a cAMP dependent manner CXCL12 DDA CXCL12 FSK + - + + + + - + + + Warrington et al. (2007) Cancer Research Neurofibromin loss alters CXCR4-mediated cAMP responses Warrington et al. (2007) Cancer Research Mutational modulation of stromal response pathways: neurofibromin and CXCR4 L12 R4 NF AC Gi ATP GRKs cAMP RAS L12 growth R4 P arrestin Altering cAMP levels in the brain can alter the pattern of tumorigenesis in a mouse model of NF1 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 Erin Jackson David Piwnica-Worms