Angiosarcoma is a rare and highly aggressive tumor of endothelial origin. The molecular mechanisms driving angiosarcoma growth have not been fully elucidated, although autocrine stimulation by vascular endothelial growth factor (VEGF) secretion may play a role in the pathogenesis of this tumor. We identified a patient with a very rare form of angiosarcoma arising from the retroperitoneum. Immunohistochemical analysis demonstrated widespread up-regulation of the hypoxic response pathway as a mechanism of enhanced VEGF expression. Disordered regulation of the hypoxic response pathway can result in the expression of factors such as VEGF and erythropoietin, which may promote autocrine tumor growth in angiosarcoma

Acs, Geza

Rathmell, W Kimryn

Simon, M Celeste

Vaughn, David J

Carolina Digital Repository

Abstract. Angiosarcoma is a rare and highly aggressivetumor of endothelial origin. The molecular mechanismsdriving angiosarcoma growth have not been fully elucidated,although autocrine stimulation by vascular endothelialgrowth factor (VEGF) secretion may play a role in thepathogenesis of this tumor. We identified a patient with a veryrare form of angiosarcoma arising from the retroperitoneum.Immunohistochemical analysis demonstrated widespread up-regulation of the hypoxic response pathway as a mechanismof enhanced VEGF expression. Disordered regulation of thehypoxic response pathway can result in the expression offactors such as VEGF and erythropoietin, which maypromote autocrine tumor growth in angiosarcoma.Angiosarcoma is a rare sarcoma of purely endothelialorigin, which has seldom been reported to arise in theretroperitoneum or kidney (1-4). These tumors arecomposed of poorly-differentiated endothelial cells, whichform diffuse tubular networks, that are highly vascular andoften hemorrhagic. Retroperitoneal angiosarcoma tends tobe aggressive and rapidly fatal if not amenable to primarysurgical management. Angiosarcomas, as endothelial cells,express the receptors for vascular endothelial growth factor(VEGF), flt-1 (VEGF-R1) and KDR/flk-1 (VEGF-R2).Previous studies have demonstrated that these tumors canalso produce VEGF, which provides a potential mechanismof autocrine-mediated growth stimulation (5,6). VEGF isprimarily expressed and secreted from cells of epithelialorigin, but the mechanism promoting VEGF expression inthese tumors has not been identified.A potential mechanism for the enhanced expression ofVEGF is the activation of the hypoxic response pathway. Thecellular hypoxic response includes the transcriptionalactivation of genes involved in angiogenesis, erythropoiesisand anaerobic metabolism. This response is mediated by thetranscription factors HIF1· (hypoxia inducible factor) andHIF2·, which activate the hypoxic response by promoting thetranscription of a large number of genes involved inangiogenesis (VEGF, angiopoietin 1 and angiopoietin 2),oxygen delivery (erythropoietin) and glycolysis (glut-1, lactatedehydrogenase, phosphofructokinase, carbonic anhydrases)(7-9). Under normal oxygen tension, HIF (this termdesignates either HIF1· or HIF2·) is rapidly targeted forproteasomal degradation through an interaction with the vonHippel-Lindau (VHL) tumor suppressor (10-12). Whenoxygen levels become limiting, this interaction is disruptedand HIF accumulates in local regions of hypoxia. HIF canalso be found at high levels in tumors independent of hypoxicstimulation (13). Clear cell renal cell carcinomas associatedwith VHL mutations demonstrate HIF expression, as well asexpression of HIF target genes, including VEGF (14). HIFregulation can also be mediated by transcriptional activationvia loss of the TSC2 tumor suppressor gene, which causesHIF accumulation as well as VEGF accumulation as a resultof loss of inhibition of mTOR (15). Additionally, HIFexpression may be modulated by the PTEN/AKT pathway(16). Although many potential mechanisms of HIF activationhave been postulated, tumors which express HIF and VEGFhave been demonstrated to correlate with both tumorvascularity and growth rate (17).We report an analysis of one patient presenting with aprimary retroperitoneal angiosarcoma, identifying constitutiveHIF overexpression in this tumor as a mechanism for aberrant167Correspondence to: W. Kimryn Rathmell, MD, PhD, Division ofHematology/Oncology, Lineberger Comprehensive Cancer Center,Campus Box 7295, Room 12-038, University of North Carolina atChapel Hill, Chapel Hill, NC 27599, U.S.A. Tel: (919) 962-2148,Fax: (919) 843-3160, e-mail: rathmell@med.unc.eduKey Words: VEGF, vascular endothelial growth factor,endothelium, angiosarcoma, retroperitoneal mass, HIF1, HIF2,hypoxia inducible factor, VHL, von Hippel-Lindau.ANTICANCER RESEARCH 24: 167-170 (2004)HIF Transcription Factor Expression and Induction of HypoxicResponse Genes in a Retroperitoneal AngiosarcomaW. KIMRYN RATHMELL1, GEZA ACS2, M. CELESTE SIMON3 and DAVID J. VAUGHN41Division of Hematology/Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill; 2Department of Pathology, University of Pennsylvania;3Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, University of Pennsylvania;4Division of Hematology/Oncology, Abramson Cancer Center of the University of Pennsylvania, U.S.A.0250-7005/2004 $2.00+.40VEGF expression. Furthermore, HIF expression promotestranscription of other growth factors including erythropoietin,suggesting multiple modes of autocrine-stimulated tumorgrowth.Materials and MethodsImmunohistochemistry. The tumor tissue was fixed immediatelyfollowing resection and paraffin embedded according to routineclinical protocols. Immunohistochemical stains were performed asdescribed previously (18). Primary antibodies included HIF1·(Novus), HIF2· (Novus), VEGF (NeoMarkers), VEGFR2 (Dako),Flk1 (Novus), erythropoietin (Genzyme) and carbonic anhydrase IX(Santa Cruz). Secondary antibody detection was performed withappropriate biotin-conjugated antibodies (Vector Laboratories).Detection was enhanced with ABC enhancement kit (Vectashield)and detected with DAB reagent (Vector Laboratories). Appropriatepositive and negative controls were performed (data not shown).ResultsA 49-year-old male with a history of cadaveric renal transplantfor glomerulonephritis-related renal failure was found to havea mass in the lower pole of the native left kidney withoutassociated lymphadenopathy. A radical nephrectomy wasperformed, which revealed a primary retroperitonealangiosarcoma with invasion and replacement of the native leftkidney. We performed immunohistochemistry on this tumorspecimen and found high levels of expression of both VEGFand the VEGF receptor, flk-1 (Figure 1, A and B). Tumor sections within this angiosarcoma demonstratedhigh level expression of other genes transcriptionallyactivated by the hypoxic response pathway, includingcarbonic anhydrase IX and erythropoietin (Figure 1, C andD), not normally expressed in endothelial cells. Endothelialcells in general and cells from this angiosarcoma, however,do express the erythropoietin receptor (data not shown),suggesting another autocrine mechanism supporting theproliferation of this tumor. Finally, we observed that bothHIF1· and HIF2· were expressed at high levels throughoutthe tumor, but limited to the tumor cells and sparing thesurrounding stroma, an uncommon expression pattern fornormal hypoxic regulation (Figure 1, E and F).  DiscussionEndothelial cell growth is an important mechanism forprocesses of both tissue development and tumorigenesis.While endothelial cells in general share many commonfeatures, subsets of endothelial cells, in particularendothelial cells of the glomerulus, express a uniquefootprint of endothelial specific growth promoting genes(19). Understanding mechanisms of disordered endothelialcell growth, especially in unique locations such as thekidney, is an important first step to unraveling the activitiesof angiogenesis and vasculogenesis which play an importantpart in the development of highly vascular tumors in thekidney or other organs.ANTICANCER RESEARCH 24: 167-170 (2004)168Figure 1. Expression of hypoxic response targets in angiosarcoma. Immunohistochemistry detected diffuse strong staining with antibodies targeting (A)VEGF, (B) VEGF-R2 (flk-1), (C) carbonic anhydrase, (D) erythropoietin, (E) HIF1·, (F) HIF2·. 20X resolution.Taken together, our observations suggest that, in thisangiosarcoma, the hypoxic response pathway was aberrantlyactivated as demonstrated by widespread high levelexpression of both HIF1· and HIF2·. This mechanism forthe promotion of angiosarcoma is also supported by recentstudies of mouse models of tumorigenesis. In a mosaicconditional knockout of VHL, the animals developedangiosarcomas of the liver in addition to hemangiomas andangiectasis in multiple tissues (20). Additionally, in theTSC2+/- mouse, angiosarcomas developed on the tail, pawsand mouth (21). In this angiosarcoma, two autocrinefeedback mechanisms (both the VEGF and erythropoietinsignaling pathways), which could support tumor growth,were activated in coordination with the HIF transcriptionalresponse pathway. While this correlation requires a morethorough evaluation, it provides a unique potential pathwayfor tumorigenesis which may provide targets for drugtherapy in this difficult to treat population.AcknowledgementsWKR is supported by a grant from the National Cancer Institute(CA098410), USA.References1 Mordkin RM, Dahut WL and Lynch JH: Renal angiosarcoma:a rare primary genitourinary malignancy. Southern Medical J90: 1159-60, 1997.2 Hiratsuka Y, Nishimura H, Kajiwara I et al: Renalangiosarcoma: a case report. International J Urology 4: 90-3,1997.3 Cason JD, Waisman J and Plaine L: Angiosarcoma of kidney.Urology 30: 281-3, 1987.4 Martinez-Pineiro L, Lopez-Ferrer P, Picazo M et al: Primaryrenal angiosarcoma. Case report and review of the literature.Scand J Urol Nephrol 29: 103-108, 1995.5 Amo Y, Masuzawa M, Hamada Y et al: Expression of vascularendothelial growth factor in a human hemangiosarcoma cellline (ISA-HAS). Arch Dermatol Res 293: 296-301, 2001.6 Zietz C, Rossle M, Haas C et al: MDM-2 oncoprotienoverexpression, p53 gene mutation, and VEGF up-regulation inangiosarcomas. Am J Pathol 153: 1425-1433, 1998.7 Pugh CW and Ratcliffe PJ: The von Hippel-Lindau tumorsuppressor, hypoxia-inducible factor-1 (HIF-1) degradation, andcancer pathogenesis. Seminars Cancer Biol 13: 83-9, 2003.8 Pugh CW, Maxwell PH, Harris AL et al: Hypoxia-inducibleexpression of tumor-associated carbonic anhydrases. Oncogene19: 6297-305, 2000.9 Iliopoulos O, Levy AP, Jiang C et al: Negative regulation ofhypoxia-inducible genes by the von Hippel-Lindau protein. ProcNat Acad Sci USA 93: 10595-9, 1996.10 Ohh M, Yang H, Klco JM et al: HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications forO2 sensing.[comment]. Human Molec Genetics 10: 1019-27, 2001.11 Masson N, Mole DR, Jaakkola P et al: The tumour suppressorprotein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.[comment]. J Biological Chem 275:25733-41, 2000.12 Tanimoto K, Makino Y, Pereira T et al: Mechanism ofregulation of the hypoxia-inducible factor-1 alpha by the vonHippel-Lindau tumor suppressor protein. EMBO J 19: 4298-309, 2000.13 Simons JW and Zhong H: Overexpression of hypoxia-induciblefactor 1alpha in common human cancers and their metastases.Biochem Biophys Res Commun 259: 523-6, 1999.14 Maher ER and Wiesener MS: Constitutive activation ofhypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1alpha in clear cell renal carcinomas. AdvCancer Res 82: 85-105, 2001.15 Liu MY, Poellinger L and Walker CL: Up-regulation ofhypoxia-inducible factor 2alpha in renal cell carcinomaassociated with loss of Tsc-2 tumor suppressor gene. CancerRes 63: 2675-80, 2003.16 Zhong H, Chiles K, Feldser D et al: Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growthfactor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathwayin human prostate cancer cells: implications for tumorangiogenesis and therapeutics. Cancer Res 60: 1541-5, 2000.17 Tsuzuki Y, Fukumura D, Oosthuyse B et al: Vascularendothelial growth factor (VEGF) modulation by targetinghypoxia-inducible factor-1a--hypoxia response element--VEGFcascade differentially regulates vascular response and growthrate in tumors. Cancer Res 60: 6248-6252, 2000.18 Mack FA, Rathmell WK, Arsham AM et al: Loss of pVHL issufficient to cause HIF dysregulation in primary cells but doesnot promote tumor growth. Cancer Cell 3: 75-88, 2003.19 Tsai JC ZJ, Minami T, Voland C, Zhao S, Yi X, Lassalle P,Oettgen P and Aird WC: Cloning and characterization of thehuman lung endothelial-cell-specific molecule-1 promoter. JVasc Res 39: 148-159, 2002.20 Ma W, Tessarollo L, Hong S et al: Hepatic vascular tumors,angiectasis in multiple organs, and impaired spermatogenesis inmice with conditional inactivation of the VHL gene. CancerRes 63: 5320-5328, 2003.21 Onda H, Crino PB, Zhang H et al: Tsc2 null murineneuroepithelial cells are a model for human tuber giant cells,and show activation of an mTOR pathway. Molec CellNeurosciences 21: 561-74, 2002.Received November 10, 2003Accepted December 16, 2003Kimryn Rathmell et al: HIF Activity and VEGF Expression in Angiosarcoma169

HIF Transcription Factor Expression and Induction of Hypoxic Response Genes in a Retroperitoneal Angiosarcoma

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HIF Transcription Factor Expression and Induction of Hypoxic Response Genes in a Retroperitoneal Angiosarcoma

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