Loss of Fibroblast HIF-1a Accelerates Tumorigenesis

Solid tumors consist of malignant cells and associated stromal components, including fibroblastic cells that contribute to tumor growth and progression. Although tumor fibrosis and aberrant vascularization contribute to the hypoxia often found in advanced tumors, the contribution of hypoxic signaling within tumor-associated fibroblasts to tumorigenesis remains unknown. In this study, we used a fibroblast-specific promoter to create mice in which key hypoxia regulatory genes, including VHL, HIF-1a, HIF-2a, and VEGF-A, were knocked out specifically in tumor stromal fibroblasts. We found that loss of HIF-1a and its target gene VEGF-A accelerated tumor growth in murine model of mammary cancer. HIF-1a and VEGF-A loss also led to a reduction in vascular density and myeloid cell infiltration, which correlated with improved tumor perfusion. Together, our findings indicate that the fibroblast HIF-1a response is a critical component of tumor vascularization. Cancer Res; 72(13); 3187–95. 2012 AACR. Introduction Tumor-associated stromal alterations influence the tumor microenvironment and significantly contribute to tumor growth and progression (1). Recently, it has also become clear that stroma coevolves with tumor cells (2, 3), and that stromal fibroblasts are a prominent cell type in that process (4, 5). Unlike fibroblasts residing in normal tissues, tumor-associated fibroblasts exhibit distinctive tumor-promoting features: they release growth and angiogenic factors, recruit inflammatory cells, and remodel extracellular matrix (6–11). Hypoxia, or low oxygen tension, is a significant characteristic of the tumor microenvironment. An inadequate blood supply to tumor tissue frequently results in the formation of hypoxic and necrotic regions. This in turn triggers the expression of hypoxia-inducible factors (HIFs; refs. 12 and 13). HIFs are heterodimeric helix-loop-helix transcription factors that are composed of 2 subunits: a constitutively expressed HIF-b subunit, and an oxygen-dependent HIF-a subunit. The HIF-a proteins are posttranslationally regulated by oxygenation (14–16), through a complex containing the pVHL tumor suppressor (17). Virtually all rapidly growing solid tumors contain multiple transient or chronic areas of severe hypoxia (oxygen tensions of less than 1%), and HIF-a levels are highly correlated with other prognostic markers of cancer, mortality, and metastasis (18–20). To determine how hypoxic signaling in tumor-associated fibroblasts affects tumorigenesis, a series of fibroblastspecific conditional knockout mice was created using an FSP-cre strain (6, 10, 21, 22). It was found that ablation of HIF-1a in stromal fibroblasts enhanced both tumor growth and perfusion while reducing vascular density. Fibroblastspecific deletion of VEGF-A had similar effects, indicating that hypoxic signaling and VEGF-A expression in stromal fibroblasts is an important aspect of solid tumor progression. Materials and Methods Cell culture Mammary epithelial carcinoma cells from female, FVB strain mice carrying the MMTV-PyMT transgene (23, 24) were isolated by digestion of advanced mammary tumors with collagenase and hyaluronidase. Cells were cultured in DMEM with 10% FBS and 1% penicillin–streptomycin in a humidified incubator with 5% carbon dioxide. To isolate tumor-associated fibroblasts, digested cell suspensions from late stage tumors were plated on a Petri (bacterial culture) dish; cells adherent within an hour were harvested for genomic DNA analysis. Transgenic mice Mice in FVB background harboring an oncogenic Polyoma virus middle T transgene under the control of the promoter Authors' Affiliations: Molecular Biology Section, Division of Biological Sciences, Department of Medicine, Division of Endocrinology andMetabolism, University of California, San Diego, La Jolla, California; Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom; Friedrich-Alexander-Universitat ErlangenNuremberg, Erlangen, Germany; Department of Cardiovascular Medicine, University of Tokyo, Graduate School of Medicine, Tokyo, Japan; Institut fur Physiologie, Universitat Duisburg-Essen, Essen, Germany; Department of Molecular Genetics, College of Biological Sciences, and Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Randall S. Johnson, Department of Physiology, Development & Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom. Phone: 44-0-1223-76593; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-0534 2012 American Association for Cancer Research. Cancer Research www.aacrjournals.org 3187 on January 6, 2018. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst May 3, 2012; DOI: 10.1158/0008-5472.CAN-12-0534