Counterbalancing angiogenic regulatory factors control the rate of cancer progression and survival in a stage-specific manner Liang Xiea,1, Michael B. Duncana,1,2, Jessica Pahlerb, Hikaru Sugimotoa, Margot Martinoa, Julie Livelya,3, Thomas Mundela, Mary Soubasakosa, Kristofer Rubinc,d, Takaaki Takedae, Masahiro Inouee, Jack Lawlerf, Richard O. Hynesd, Douglas Hanahanb,4,5, and Raghu Kalluria,g,h,5 aDivision of Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; bDiabetes and Comprehensive Cancer Centers, University of California, San Francisco, CA 94143; cDepartment of Medical Biochemistry and Microbiology, Uppsala University, 75105 Uppsala, Sweden; eDepartment of Biochemistry, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan; fDivision of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; dHoward Hughes Medical Institute, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139; gDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215; and hHarvard–MIT Division of Health Sciences and Technology, Boston, MA 02215 Contributed by Douglas Hanahan, April 15, 2011 (sent for review November 12, 2010) Whereas the roles of proangiogenic factors in carcinogenesis are thelial cells (13). Thrombospondin-1 (TSP1) offers another ex- well established, those of endogenous angiogenesis inhibitors ample of how glycoproteins within the tumor microenvironment (EAIs) remain to be fully elaborated. We investigated the roles can serve as important functional regulators of angiogenesis (14). of three EAIs during de novo tumorigenesis to further test the In this study, we present genetic and pharmacological data, angiogenic balance hypothesis, which suggests that blood vessel which demonstrate that endogenous protein fragments are in- development in the tumor microenvironment can be governed by volved in regulating the angiogenic switch and controlling de a net loss of negative regulators of angiogenesis in addition to the novo tumor growth and survival associated with pancreatic well-established principle of up-regulated angiogenesis inducers. neuroendocrine tumorigenesis (PNET) in RIP-Tag2 (RT2) In a mouse model of pancreatic neuroendocrine cancer, adminis- mice. Genetic testing in a second cancer model, involving de- − − tration of endostatin, thrombospondin-1, and tumstatin peptides, letion of tumstatin and TSP1 in p53 / mice, generalizes the as well as deletion of their genes, reveal neoplastic stage-specific significance of these endogenous angiogenesis inhibitors in con- effects on angiogenesis, tumor progression, and survival, corre- trolling cancer progression and increasing overall survival. lating with endothelial expression of their receptors. Deletion of tumstatin and thrombospondin-1 in mice lacking the p53 tumor Results suppressor gene leads to increased incidence and reduced latency We documented the importance of VEGF-A gene expression for of angiogenic lymphomas associated with diminished overall the initial angiogenic switch, as illustrated in Fig. S1, assessing survival. The results demonstrate that EAIs are part of a balance hyperproliferative islets in RT2 mice in which the VEGF-A gene mechanism regulating tumor angiogenesis, serving as intrinsic had been specifically deleted within the oncogene-expressing microenvironmental barriers to tumorigenesis. islet β cells of the pancreas (RT2/VEGFRIPKO). When hyper- proliferative islet lesions (hyperplasias and dysplasias) were integrins | cell biology identified in the pancreas by BrdU labeling, both nonangiogenic islets as well as angiogenic islets were detected in VEGF wild- ngiogenesis, the formation of new blood vessels, is crucial type mice (RT2/VEGFWT), whereas the RT2/VEGFRIPKO mice Aduring transient physiological and pathological processes in had only hyperproliferative lesions without evident angiogenesis. MEDICAL SCIENCES the adult. An increasing body of evidence supports the hypoth- Hypoxia was prevalent in RT2/VEGFRIPKO islets (Fig. S1F). esis that angiogenesis is switched on and orchestrated by balance Although these results illustrate the importance of VEGF as an in the local abundance of endogenous proangiogenic and anti- inducing signal for triggering the initial angiogenic switch, the angiogenic factors (1, 2). Proangiogenic signaling is well un- data do not implicate a balance mechanism. A question remained derstood, in terms of intracellular networks and mechanistic as to whether there were significant negative regulatory signals effects (3, 4). By contrast, understanding of the roles, regulation, and effects of the counterbalancing endogenous antiangiogenic signals in regulating the angiogenic switch has lagged, due to Author contributions: R.O.H., D.H., and R.K. designed research; L.X., M.B.D., J.P., H.S., complexities in their molecular structures and receptors (5–7). M.M., J. Lively, T.M., M.S., K.R., T.T., and M.I. performed research; M.I., J. Lawler, R.O.H., Several endogenous antiangiogenic factors are proteolytic frag- D.H., and R.K. contributed new reagents/analytic tools; L.X., M.B.D., J.P., H.S., T.M., M.S., K.R., T.T., M.I., D.H., and R.K. analyzed data; and L.X., M.B.D., R.O.H., D.H., and R.K. wrote ments of structural proteins, including constituents of the vas- the paper. cular basement membrane (8). Most have several receptors or The authors declare no conflict of interest. coreceptors, in several cases including heterodimeric integrins Freely available online through the PNAS open access option. involved in anchoring endothelial cells to the vascular basement 1 membrane (9, 10). L.X. and M.B.D. contributed equally to this work. 2 Among the growing list of endogenous angiogenesis inhibitors, Present address: Section of Gastroenterology/Hepatology, Department of Medicine, Medical College of Georgia, Augusta, GA 30912. several of the best characterized are peptide fragments of natu- 3Present address: Department of Biology, Sewanee, The University of the South, Sewanee, rally occurring extracellular matrix (ECM) and basement mem- TN 37383. brane proteins (1). Endostatin, derived from the noncollagenous 4 α Present addresses: Swiss Institute for Experimental Cancer Research (ISREC) and Swiss (NC1) domain of the 1 chain of type XVIII collagen, attenuates Federal Institute of Technology Lausanne (EPFL), CH-1015 Lausanne, Switzerland. angiogenesis, tumor growth, and metastasis in experimental ani- 5To whom correspondence may be addressed. E-mail: [email protected] or mal models (11, 12). Another such fragment, tumstatin, consisting dh@epfl.ch. α of the NC1 domain of the 3 chain of type IV collagen, is anti- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. angiogenic and selectively proapoptotic to proliferating endo- 1073/pnas.1105041108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1105041108 PNAS | June 14, 2011 | vol. 108 | no. 24 | 9939–9944 Downloaded by guest on October 1, 2021 counterbalancing the proangiogenic signals exemplified by VEGF, slightly increased in the angiogenic islets, albeit without statisti- defining a balance mechanism for the initial angiogenic switch. cal significance (Fig. S2 A and D). β3 integrin was barely de- tectable in normal islets or in the premalignant stages of cancer Endostatin, Tumstatin, and TSP1 Restrict Tumor Angiogenesis and progression and then appeared robustly on most blood vessels of Growth in RT2 Mice. We used functionally active peptides derived nascent solid tumors. However, as tumors became larger, ex- from tumstatin and endostatin and a recombinant protein that pression levels decreased somewhat (Fig. S2 B and D). Coloc- includes the second type 1 repeat (TSR) of thrombospondin-1 alization of CD36 and CD31 by immunostaining revealed CD36 (TSP1), to probe the capabilities of the three endogenous in- expression in endothelial cells of normal islets and all stages of hibitors to suppress angiogenesis and tumor growth via phar- tumorigenesis (Fig. S2C). macological elevation of their levels in the circulatory system The mRNA levels of αV, β3, α5, and β1 integrin subunits along (15–17). To confirm the relative efficacy of these reagents, we with CD36 were then analyzed by quantitative real-time PCR of evaluated a 10-μM dose of each reagent (based on previous RNA prepared from physically isolated normal pancreatic islets, studies and chosen to ensure maximal activity while retaining hyperplastic/dysplastic islets, angiogenic islets, and tumors. The solubility) in cell-culture–based endothelial viability assays (15– expression of the integrin subunits increased in the tumors, 17). As shown in Fig. 1A,10μM of each peptide or recombinant compared with normal islets, by 50% to over 100%, in relation to protein reagent significantly inhibited endothelial cell viability baseline CD31 coexpression in the normal islets (Fig. S3). CD36 with similar efficacy. showed a modest decrease in gene expression in the tumor set- The endostatin, tumstatin, and TSR inhibitors were adminis- ting relative to CD31 expression. tered to RT2 mice in two trials to assess their efficacy during different stages of PNET tumorigenesis (18). A prevention trial Deficiency of Endogenous Angiogenesis Inhibitors Accelerates Islet from 5.5 to 10 wk of age was designed to assess the effect of the Carcinogenesis.