The American Journal of Pathology, Vol. 178, No. 2, February 2011 Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. DOI: 10.1016/j.ajpath.2010.10.033 Tumorigenesis and Neoplastic Progression Deregulation of E2-EPF Ubiquitin Carrier Protein in Papillary Renal Cell Carcinoma Frederik C. Roos,*† Andrew J. Evans,*‡ way impinging on the positive feedback mechanism Walburgis Brenner,† Bill Wondergem,§ of HIF1-mediated regulation of E2-EPF in PRCC. (Am Jeffery Klomp,¶ Pardeep Heir,* Olga Roche,* J Pathol 2011, 178:853–860; DOI: 10.1016/j.ajpath.2010.10.033) Christian Thomas,†ʈ Heiko Schimmel,** Kyle A. Furge,¶ Bin T. Teh,§ Joachim W. Thüroff,† Renal cell carcinoma (RCC) accounts for 3% of all Christian Hampel,† and Michael Ohh* adult malignancies and its incidence continues to rise From the Department of Laboratory Medicine and Pathobiology,* at an approximate rate of 2.5% per year.1 RCC is a University of Toronto, Toronto, Ontario, Canada; the Department heterogeneous disease consisting of various subtypes † of Urology, Johannes Gutenberg University, Mainz, Germany; with diverse genetic and morphological features that ‡ the Department of Pathology, University Health Network, can be segregated into clear cell (conventional), pap- Princess Margaret Hospital, Toronto, Ontario, Canada; the illary (chromophil), chromophobe, collecting duct, and § ʈ Laboratories of Cancer Genetics, and Computational Biology, unclassified subtypes.2 Papillary RCC (PRCC) is the Van Andel Research Institute, Grand Rapids, Michigan; the second most common subtype comprising 10% to 15% Institute of Pathology,¶ Johannes Gutenberg University, Mainz, of kidney cancer with an estimated annual incidence of The ءء,Germany; and the Department of Urologic Sciences 3500 to 5000 cases in the United States.3 Although Vancouver Prostate Centre, University of British Columbia, PRCCs are genetically heterogeneous with complex Vancouver, British Columbia, Canada numerical chromosomal anomalies, PRCC can be mor- phologically classified into two subtypes: Type 1, which is twice as common as type 2, is characterized Molecular pathways associated with pathogenesis of by the presence of small cuboidal cells covering thin sporadic papillary renal cell carcinoma (PRCC), the papillae with a single line of small uniform nuclei and second most common form of kidney cancer, are basophilic cytoplasm, whereas type 2 is characterized poorly understood. We analyzed primary tumor spec- by the presence of large pseudostratified tumor cells imens from 35 PRCC patients treated by nephrectomy with eosinophilic cytoplasm.4 In general, type 2 pa- via gene expression analysis and tissue microarrays tients have poorer prognosis than patients with type 1 constructed from an additional 57 paraffin-embedded PRCC.5 However, the molecular basis underlying dif- PRCC samples via immunohistochemistry. Gene ferential prognosis of PRCC is largely unknown. products were validated and further studied by West- The vast majority of RCC, including PRCC, is spo- ern blot analyses using primary PRCC tumor samples radic or nonhereditary, whereas approximately 4% of and established renal cell carcinoma cell lines, and all renal cancers are attributed to familial RCC syn- potential associations with pathologic variables and 6 survival in 27 patients with follow-up information dromes. Hereditary PRCC is an autosomal dominant were determined. We show that the expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible fac- Supported by funds from the Kidney Foundation of Canada and the tor (HIF), von Hippel-Lindau protein, for proteasome- Canadian Cancer Society (18460). F.C.R. is a recipient of the German dependent degradation, is markedly elevated in the Research Foundation (DFG, Ro 3750/1-1). M.O. is a Canada Research majority of PRCC tumors exhibiting increased HIF1␣ Chair in Molecular Oncology. expression, and is associated with poor prognosis. In Accepted for publication October 13, 2010. addition, we identified multiple hypoxia-responsive Supplemental material for this article can be found on http://ajp. elements within the E2-EPF promoter, and for the amjpathol.org or at doi:10.1016/j.ajpath.2010.10.033. first time we demonstrated that E2-EPF is a hypoxia- Address reprint requests to Michael Ohh, Ph.D., Department of Labo- inducible gene directly regulated via HIF1. These find- ratory Medicine and Pathobiology, 1 King’s College Circle, University of ings reveal deregulation of the oxygen-sensing path- Toronto, Toronto, M5S 1A8 Canada. E-mail: [email protected]. 853 854 Roos et al AJP February 2011, Vol. 178, No. 2 disease characterized by multi-focal bilateral type 1 Materials and Methods PRCC and is caused by “gain-of-function” mutations on MET, which encodes the hepatocyte growth factor Patients and Specimens receptor, c-Met. Moreover, MET mutations have also Tissue samples were obtained under sterile conditions been associated with the vast majority of sporadic type from 91 patients with primary papillary RCC from two 1 PRCC.7 Hereditary leiomyomatosis renal cell cancer participating centers (Van Andel Research Institute, is a rare cancer syndrome characterized by the devel- Grands Rapids, MI; and Department of Urology of the opment of cutaneous and uterine leiomyomas and low- 6,7 University Medical Center of the Johannes Gutenberg penetrant type 2 PRCC. Hereditary leiomyomatosis University, Mainz, Germany). The diagnosis of PRCC was renal cell cancer is caused by mutations of fumarate based on H&E sections and classified as PRCC, accord- hydratase, which encodes a Krebs cycle enzyme that ing to the criteria previously described.4 Subtyping of 8 catalyzes the conversion of fumarate to malate. PRCC and re-evaluation of the diagnosis were performed Biallelic inactivation of von Hippel-Lindau (VHL) tu- from two pathologists in 65 tissue samples coming from mor suppressor gene is associated with sporadic Van Andel Research Institute, and in 26 samples col- clear-cell RCC and hereditary VHL disease-associated lected from the University Medical Center of the Jo- clear-cell renal cell carcinoma (CCRCC).9 VHL is the hannes Gutenberg University, by respective institute pa- substrate-conferring component of an E3 ubiquitin li- thologists. After re-evaluation, all patients with non-PRCC gase that targets ␣ subunit of hypoxia-inducible factor diagnosis (Van Andel Research Institute, n ϭ 8 and Uni- (HIF), the master transcriptional regulator of the ubiq- versity Medical Center of the Johannes Gutenberg Uni- uitous oxygen-sensing pathway, for ubiquitin-mediated versity, n ϭ 1) were excluded from further analysis. Clin- destruction.10 In the presence of oxygen, HIF␣ is hy- icopathologic features and information on oncologic droxylated on conserved prolines within the oxygen- follow-up were available from 27 PRCC patients present- dependent degradation domain by a family of prolyl- ing with PRCC (Table 1). The tumor size was based on hydroxylases, which enables direct binding to VHL.11 the pathologic tumor size, according to the maximum Under hypoxia, HIF␣ remains unmodified, escapes tumor diameter from the surgical specimens. The tumor VHL recognition, and subsequent ubiquitin-mediated staging was revised according to the 2003 TNM classifi- destruction, and partners with HIF␤ [also known as aryl cation system, and grading was based on the Fuhrman hydrocarbon receptor nuclear translocator (ARNT)] to classification system. Institutional review board approval form an active heterodimeric transcription factor that was obtained from the two participating institutions and engages hypoxia-responsive elements (HREs) in the informed consent was obtained from all participants. promoters of numerous hypoxia-inducible genes to trigger various adaptive responses to reduced oxygen Antibodies tension.12,13 Notably, several tyrosine kinase inhibitors (TKIs) are currently being used with promising results Monoclonal anti-HA antibody (12CA5) was obtained from to treat patients with metastatic CCRCC since the loss Roche Molecular Biochemicals (Basel, Switzerland). of VHL leads to the stabilization of HIF and transacti- Polyclonal rabbit anti-VHL antibody was obtained from Cell Signaling (Pickering, Ontario, Canada). Anti-CAIX vation of HIF-induced receptor tyrosine kinases and anti-HIF-2␣ antibodies were obtained from Novus (RTKs), such as vascular endothelial growth factor re- Biologicals Inc. (Littleton, CO). Anti-HIF1␣ antibody was ceptor and platelet-derived growth factor receptor.14 obtained from BD Biosciences (Mississauga, Ontario, Whereas the tumorigenesis of sporadic CCRCC is Canada). Anti-Vinculin was obtained from Sigma-Aldrich mainly associated with the loss of VHL function, the (Oakville, Ontario, Canada). Anti-GLUT-1 was obtained molecular pathways associated with the pathogenesis from Abcam (Cambridge, MA). Anti-E2EPF was obtained of sporadic PRCC are poorly understood. We show that from Abgent (San Diego, CA). the oxygen-sensing pathway in a significant fraction of sporadic PRCC is compromised, due at least in part to the overexpression of E2-EPF (also known as UBE2S) Tissue Microarrays ubiquitin carrier protein. E2-EPF has been shown pre- Tissue microarrays, consisting of representative 0.6 to viously to target VHL for 26S proteasome-mediated 1.0 mm cores from 37 PRCC and 20 PRCC of various 15 degradation. In addition, we identified multiple HREs Fuhrman grades (grades 1 to 4) and stages (pT1 to