Transcriptional Switch of Dormant Tumors to Fast-Growing Angiogenic Phenotype Nava Almog, 1,2 Lili Ma, 1 Raktima Raychowdhury, 1 Christian Schwager, 3 Ralf Erber, 4 Sarah Short, 2 Lynn Hlatky, 1 Peter Vajkoczy, 4 Peter E. Huber, 1,3 Judah Folkman, 2 and Amir Abdollahi 1,2,3 1 Center of Cancer Systems Biology, Caritas St. Elizabeth’s Medical Center, Tufts University School of Medicine; 2 Vascular Biology Program, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts; 3 Department of Radiation Oncology, German Cancer Research Center (DKFZ) and University of Heidelberg Medical School, Heidelberg, Germany; and 4 Department of Neurosurgery, Charite´-Universitaetsmedizin Berlin, Berlin, Germany Abstract Tumor dormancy has important implications for early detection and treatment of cancer. Lack of experimental models and limited clinical accessibility constitute major obstacles to the molecular characterization of dormant tumors. We have developed models in which human tumors remain dormant for a prolonged period of time (>120 days) until they switch to rapid growth and become strongly angiogenic. These angiogenic tumors retain their ability to grow fast once injected in new mice. We hypothesized that dormant tumors undergo a stable genetic reprogramming during their switch to the fast-growing phenotype. Genome- wide transcriptional analysis was done to dissect the molecular mechanisms underlying the switch of dormant breast carci- noma, glioblastoma, osteosarcoma, and liposarcoma tumors. A consensus expression signature distinguishing all four dormant versus switched fast-growing tumors was generated. In alignment with our phenotypic observation, the angiogen- esis process was the most significantly affected functional gene category. The switch of dormant tumors was associated with down-regulation of angiogenesis inhibitor thrombospondin and decreased sensitivity of angiogenic tumors to angiostatin. The conversion of dormant tumors to exponentially growing tumors was also correlated with regulation and activation of pathways not hitherto linked to tumor dormancy process, such as endothelial cell–specific molecule-1, 5¶-ecto-nucleotidase, tissue inhibitor of metalloproteinase-3, epidermal growth factor receptor, insulin-like growth factor receptor, and phosphatidylinositol 3-kinase signaling. Further, novel dor- mancy-specific biomarkers such as H2BK and Eph receptor A5 (EphA5) were discovered. EphA5 plasma levels in mice and mRNA levels in tumor specimens of glioma patients correlated with diseases stage. These data will be instrumental in identifying novel early cancer biomarkers and could provide a rationale for development of dormancy-promoting tumor therapy strategies. [Cancer Res 2009;69(3):836–44] Introduction It is widely accepted that human tumors can arise in the absence of angiogenic activity and exist in a microscopic dormant state for months to years without neovascularization (1). The disease stage of cancer, therefore, seems to be a late event in tumor development. Dormant tumors are defined as microscopic (diameter of f1 mm) and asymptomatic cancerous lesions that remain occult for prolonged periods of time. They represent the earliest stages in tumor development and are highly prevalent in humans (2). Moreover, tumor dormancy is also attributed to the long latency periods frequently observed in cancer patients between the primary diagnosis and treatment and the potential clinical evidence of local recurrence or distant metastasis. Based on tumor type and stage, the dormancy period may range from years to even decades between the initial therapy and the occurrence of relapsed tumors or recurrent metastatic disease (3). Although the clinical implication of tumor dormancy in prevention and treatment of tumors has intrigued the medical community for years, there is a paucity of molecular markers and mechanistic understanding. A critical limitation confronting the field of tumor dormancy is the lack of suitable experimental models as well as consistent and abundant sources of dormant tumor cells. Although several mechanisms have been proposed to affect tumor dormancy (1, 4–10), it is still unclear what keeps these tumors in a microscopic size and prevents their expansion. We have previously established in vivo models of human breast cancer, glioblastoma, osteosarcoma, and dormant liposarcoma in immunocompromised [severe combined immunodeficient (SCID) mice; refs. 8, 11]. Using these models, we have shown that dormant microscopic tumors reside in mice for a long period (>90 days) until they switch to become fast-growing angiogenic tumors. In this work, we aimed to characterize the consensus molecular fingerprint of tumor dormancy using genome-wide expression profiling. We found angiogenesis-related genes to be the most significantly enriched functional category among the differentially regulated genes. The endogenous angiogenesis inhibitor thrombospondin, as well as angiostatin and endostatin binding proteins (angiomotin and tropomyosin, respectively), were found to be up-regulated in all dormant tumor cell lines examined. Of note, we found that angiostatin selectively binds and inhibits tumor migration in angiomotin-expressing dormant tumor cells. Furthermore, we found elevated RNA levels of key cancer pathways in angiogenic fast-growing tumors, including epithelial growth factor receptor (EGFR)-1, insulin-like growth factor type I receptor (IGF-IR), and phosphatidylinositol 3-kinase (PI3K). Importantly, Eph receptor A5 (EphA5) levels in plasma of mice bearing occult, dormant glioma was significantly higher than in control mice. It also correlated with disease stage in cancer patients. The consensus dormancy signature reported here may suggest molecular instructions to address the unmet medical need of Note: J. Folkman died on January 14, 2008. Requests for reprints: Amir Abdollahi, Center of Cancer Systems Biology, Caritas St. Elizabeth’s Medical Center, Tufts University School of Medicine, 736 Cambridge Street CBR1, Boston, MA 02135. Phone: 617-779-6569; Fax: 617-562-7142; E-mail: Amir.Abdollahi@Tufts.edu or Nava Almog, E-mail: Nava.Almog@Tufts.edu. I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-2590 Cancer Res 2009; 69: (3). February 1, 2009 836 www.aacrjournals.org Research Article Research. on February 29, 2016. © 2009 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 27, 2009; DOI: 10.1158/0008-5472.CAN-08-2590