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• W2076227992 abstract "Activation of oncogenes or inactivation of tumor suppressors in urothelium is considered critical for development of urothelial cancer. Here we report cloning of the urothelium-specific promoter uroplakin-II (UPK II) and generation of transgenic mice in which expression of SV40 large T antigen is driven by UPK II promoter. Inactivation of tumor suppressor p53 and pRb in urothelium by SV40 T antigen resulted in urothelial carcinoma, resembling human high-grade carcinoma in situ. Specific deletion of p53 in urothelial cells using the newly generated UPK II-Cre mice results in normal bladders without any evidence of cancer. The high-grade carcinoma in situ in the UPK II-SV40 mice is associated with significant activation of angiogenic signals consisting of hypoxia-inducible factor-1α (HIF-1α) and VEGF and a down-regulation of thrombospondin-1. Interestingly, such pro-angiogenic activity was not associated with progression to invasive cancer. Analysis of bladder-associated microRNAs in carcinoma in situ lesions reveals a pro-angiogenic profile, with specific overexpression of miR-18a and miR-19a and down-regulation of miR-107. A group of microRNAs (miRs) identified as associated with invasive human urothelial cancer remained unchanged in this mouse model. Collectively, our results support the notion that activation of angiogenesis and loss of p53 are not sufficient for progression to invasive cancer. Our studies identify a new mouse model for bladder cancer that can be used to study factors that determine progression to an invasive phenotype of bladder cancer. Activation of oncogenes or inactivation of tumor suppressors in urothelium is considered critical for development of urothelial cancer. Here we report cloning of the urothelium-specific promoter uroplakin-II (UPK II) and generation of transgenic mice in which expression of SV40 large T antigen is driven by UPK II promoter. Inactivation of tumor suppressor p53 and pRb in urothelium by SV40 T antigen resulted in urothelial carcinoma, resembling human high-grade carcinoma in situ. Specific deletion of p53 in urothelial cells using the newly generated UPK II-Cre mice results in normal bladders without any evidence of cancer. The high-grade carcinoma in situ in the UPK II-SV40 mice is associated with significant activation of angiogenic signals consisting of hypoxia-inducible factor-1α (HIF-1α) and VEGF and a down-regulation of thrombospondin-1. Interestingly, such pro-angiogenic activity was not associated with progression to invasive cancer. Analysis of bladder-associated microRNAs in carcinoma in situ lesions reveals a pro-angiogenic profile, with specific overexpression of miR-18a and miR-19a and down-regulation of miR-107. A group of microRNAs (miRs) identified as associated with invasive human urothelial cancer remained unchanged in this mouse model. Collectively, our results support the notion that activation of angiogenesis and loss of p53 are not sufficient for progression to invasive cancer. Our studies identify a new mouse model for bladder cancer that can be used to study factors that determine progression to an invasive phenotype of bladder cancer. Urothelial carcinomas of the bladder are among the most frequent human cancers and account for more than 14,000 deaths annually in the United States (1Jemal A. Siegel R. Ward E. Hao Y. Xu J. Thun M.J. CA Cancer J. Clin. 2009; 59: 225-249Crossref PubMed Scopus (9937) Google Scholar). Most cases of urothelial cancer are superficial bladder tumors, but around 20% of them are invasive carcinomas that can generate metastasis at the initial presentation or later in the evolution of the disease. Two groups of superficial bladder tumors have been described (2Pasin E. Josephson D.Y. Mitra A.P. Cote R.J. Stein J.P. Rev. Urol. 2008; 10: 31-43PubMed Google Scholar). Low grade papillary neoplasms are the most frequent, with a low potential of transformation into invasive cancer. Conversely, carcinomas in situ (CIS) 3The abbreviations used are: CIScarcinoma in situmiRmicroRNATSP-1thrombospondin-1UPK IIuroplakin II. are biologically more aggressive and carry a high probability of turning into an invasive carcinoma. The molecular basis of urothelial bladder cancer is being progressively unveiled, and the generation of transgenic mouse models of bladder cancer obtained through the urothelial expression of oncogenes (SV40) (3Zhang Z.T. Pak J. Shapiro E. Sun T.T. Wu X.R. Cancer Res. 1999; 59: 3512-3517PubMed Google Scholar) or deletion of tumor suppressor genes remains a viable strategy for unraveling this mechanism. It is acknowledged that there are at least two different pathways driving the malignant transformation of urothelium, each of them corresponding to one of the two types of urothelial tumors. The pathway leading to CIS, which is speculated to involve a loss of function of p53 (4Cheng J. Huang H. Pak J. Shapiro E. Sun T.T. Cordon-Cardo C. Waldman F.M. Wu X.R. Cancer Res. 2003; 63: 179-185PubMed Google Scholar), is particularly important due to its role as precursor of invasive disease. However, p53 deletion does not seem to be sufficient for urothelial tumorigenesis unless there is either a concurrent deletion of pRb (5Mo L. Cheng J. Lee E.Y. Sun T.T. Wu X.R. Am. J. Physiol. Renal Physiol. 2005; 289: F562-f568Crossref PubMed Scopus (34) Google Scholar, 6He F. Mo L. Zheng X.Y. Hu C. Lepor H. Lee E.Y. Sun T.T. Wu X.R. Cancer Res. 2009; 69: 9413-9421Crossref PubMed Scopus (62) Google Scholar) or Pten (7Puzio-Kuter A.M. Castillo-Martin M. Kinkade C.W. Wang X. Shen T.H. Matos T. Shen M.M. Cordon-Cardo C. Abate-Shen C. Genes Dev. 2009; 23: 675-680Crossref PubMed Scopus (254) Google Scholar) or a concurrent H-ras activation (8Gao J. Huang H.Y. Pak J. Cheng J. Zhang Z.T. Shapiro E. Pellicer A. Sun T.T. Wu X.R. Oncogene. 2004; 23: 687-696Crossref PubMed Scopus (46) Google Scholar). carcinoma in situ microRNA thrombospondin-1 uroplakin II. Bladder urothelial carcinoma in situ poses a difficult conundrum in the clinical setting. Its high recurrence rate (90% of cases) and its frequent evolution to invasive disease make it a target for aggressive treatment regimens, such as bladder bacillus Calmette-Guerin therapy or cystectomy as in cases of extensive or persistent disease presentation (9Kaufman D.S. Shipley W.U. Feldman A.S. Lancet. 2009; 374: 239-249Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar). Angiogenesis is among the biological factors that have been studied as potential predictors of progression to invasive tumors in superficial bladder carcinoma and is considered by some groups as a useful biomarker (10Mitra A.P. Datar R.H. Cote R.J. J. Clin. Oncol. 2006; 24: 5552-5564Crossref PubMed Scopus (252) Google Scholar). In this regard, some clinical studies have suggested that elevated microvessel density, an indicator of angiogenic phenotype, is associated with progression of carcinoma in situ to invasive cancer, but some other clinical observations did not confirm these results (11Sağol O. Yörükoğlu K. Sis B. Tuna B. Ozer E. Güray M. Mungan U. Kirkali Z. Urology. 2001; 57: 895-899Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 12Stavropoulos N.E. Bouropoulos C. Ioachim I.E. Michael M. Hastazeris K. Tsimaris I. Kalogeras D. Liamis Z. Stefanaki S. Agnantis N.I. Int. Urol. Nephrol. 2004; 36: 163-167Crossref PubMed Scopus (22) Google Scholar). Similar conflicting results have been reported for VEGF levels in urine or plasma (13Crew J.P. O'Brien T. Bicknell R. Fuggle S. Cranston D. Harris A.L. J. Urol. 1999; 161: 799-804Crossref PubMed Scopus (123) Google Scholar) or for VEGF and HIF-1α expression in the tumor, which some authors suggest as important for progression to invasive cancer (14Izawa J.I. Slaton J.W. Kedar D. Karashima T. Perrotte P. Czerniak B. Grossman H.B. Dinney C.P. Oncol. Rep. 2001; 8: 9-15PubMed Google Scholar, 15Fauconnet S. Bernardini S. Lascombe I. Boiteux G. Clairotte A. Monnien F. Chabannes E. Bittard H. Oncol. Rep. 2009; 21: 1495-1504Crossref PubMed Scopus (35) Google Scholar, 16Ioachim E. Michael M. Salmas M. Michael M.M. Stavropoulos N.E. Malamou-Mitsi V. Urol. Int. 2006; 77: 255-263Crossref PubMed Scopus (37) Google Scholar). Most studies include both CIS and pT1a types of tumors, thus making their interpretation confounding. Consequently, the emergence of angiogenic phenotype and its relevance for progression toward invasive bladder carcinoma are still controversial and need experimental validation. The goal of our study was to determine the role of p53 and angiogenic phenotype in the initiation and progression of bladder cancer. SV40 T antigen expression is able to inactivate both p53 and pRb tumor suppression pathways, and its urothelial conditional expression driven by uroplakin II (UPK II) has been previously shown to generate urothelial bladder carcinoma in situ, with late muscle invasion and metastasis in a few mice (3Zhang Z.T. Pak J. Shapiro E. Sun T.T. Wu X.R. Cancer Res. 1999; 59: 3512-3517PubMed Google Scholar). We generated a new transgenic mouse, UPK II-SV40, and observed that the development of high-grade urothelial carcinoma in situ was associated with a significant activation of angiogenic signals. This pro-angiogenic phenotype was independent of invasion and associated with specific changes in the profile of microRNAs regulating angiogenesis, without any effect on urothelial expression of invasion-related microRNA (miR). Lastly, loss of p53 alone in the urothelium did not result in initiation of cancer or angiogenic activation, and mice were normal. Collectively, these results suggest that progression to invasive bladder cancer is not solely dependent on angiogenesis, and therefore, the notion that markers of angiogenesis or angiogenesis-related miRs may serve as useful markers to predict invasive bladder cancer needs to be carefully evaluated. For the specific urothelial expression of SV40 large T antigen, a construct containing the urothelium-specific promoter uroplakin II (3.6 kb) and the SV40 T antigen was designed. First, we cloned mouse UPK II promoter (accession number: EF467361) from bacterial artificial chromosome clone RP24-308H8 with a PCR-based approach. During the cloning process, we found that about 1500 bp of the UPK II promoter region, which was deposited in GenBankTM (accession number U14421), was oppositely inserted between the two SacI restriction enzyme sites (from −1262 to −2805 from exon 1) in UPK II promoter. SV40 early sequence DNA was obtained from the Rip-TAG vector (kindly provided by Dr. Douglas Hanahan). The chimeric gene containing (5′ to 3′) the UPK II promoter (3.6 kb), the SV40 T antigen sequence (3.0 kb), and a 250-bp poly(A) signal was excised from the vector with EcoRI and BamHI digestion and purified with the Illustra GFX PCR DNA and gel band purification kit (GE Healthcare). Additionally, we also generated a transgenic mouse that expresses the Cre recombinase enzyme in bladder uroepithelium by utilizing the same UPK II promoter. Microinjection of the purified constructs in the pronuclei of fertilized eggs from C57/BL mice was performed for generation of transgenic mice (UPK II-SV40 and UPK II-Cre) at the Beth Israel Deaconess Medical Center transgenic facility. For specific urothelial knock-out of p53, the UPK II-Cre mouse strain described before was interbred to p53 floxed mice (National Institutes of Health, NCI, Mouse Repository) in which LoxP sites have been inserted in introns 1 and 10 of the p53 gene. To confirm urothelium-restricted expression of UPK II, a reporter strain was generated by interbreeding the UPK II-Cre with Rosa-R26R-YFP floxed mice (17Srinivas S. Watanabe T. Lin C.S. William C.M. Tanabe Y. Jessell T.M. Costantini F. BMC Dev. Biol. 2001; 1: 4Crossref PubMed Scopus (2340) Google Scholar). Genotyping of transgenic mice was determined by PCR in genomic DNA isolated from mice tails. The term “controls” refers to littermates of mice with a negative UPK II-SV40, UPK II-Cre;p53 f/f, or UPK II-Cre;YFP genotype. Mice were sacrificed at different time points, and bladders were obtained, weighted, and processed for histological analysis and molecular biology techniques. Thirty minutes before sacrifice, pimonidazole (HypoxyprobeTM-1, HPI Inc.) was intraperitoneally administered at a dose of 60 mg/kg. Mice were maintained at the Beth Israel Deaconess Medical Center animal facility under standard conditions. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center. Paraformaldehyde-fixed, paraffin-embedded 5-μm sections were treated with 10 mm citrate buffer for antigen retrieval, and standard immunohistochemical techniques were applied. The following primary antibodies and dilutions were used: anti-SV40 T antigen (1:50; Santa Cruz Biotechnology), anti-VEGF Ab-1 (10 μg/ml; Thermo Fisher Scientific), anti-HIF-1α (1:50; Santa Cruz Biotechnology), anti-Glut-1 (1:50; Abcam), anti-p53 (1:50; Santa Cruz Biotechnology), and anti-CD34 (1:50; Abcam). Biotinylated secondary antibodies (1:200) and VECTASTAIN ABC kit were used according to the manufacturer's instructions (Vector Laboratories). For hypoxia staining, the HypoxyprobeTM-1 Kit (HPI, Inc.) was used according to the manufacturer's instructions (primary antibody: 1:50 dilution). For immunofluorescence, 5-μm frozen sections were fixed in 100% acetone at −20 °C for 10 min. Primary antibodies were anti-CD31 (1:50; BD Biosciences Pharmingen), anti-CD34 (1:50; Abcam), anti-VEGF Ab-1 (10 μg/ml; Thermo Fisher Scientific), and anti-NG2 (1:100; Santa Cruz Biotechnology). Sections were subsequently labeled with fluorescein- or rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories), and DAPI was used for nuclear counterstaining. For blood vessel evaluation, a sufficient number of sections to cover the whole area of bladder stroma and urothelium were analyzed at 200×, and the relative area of CD31 staining was quantified with the ImageJ software (National Institutes of Health, Bethesda, MD). For evaluation of pericyte coverage, the proportion of CD31 vessels with perivascular NG2 staining was calculated. Samples (purified protein from whole bladder) were denatured with SDS sample buffer. Primary antibodies were anti-VEGF (1:500; Oncogene), anti-SV40 (1:100; Santa Cruz Biotechnology), and anti-actin (1:1000; Sigma). After incubation with horseradish peroxide-conjugated secondary antibodies (Sigma), an enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology) was used. Total RNA was isolated from whole bladder tissues using TRIzol® according to the manufacturer's recommendations (Invitrogen). After DNase I treatment (Invitrogen), reverse transcription for mRNA was carried out using the High Capacity cDNA reverse transcription kit (Applied Biosystems). For microRNA studies, RNA reverse transcription was performed with the ABI TaqMan microRNA reverse transcription kit (Applied Biosystems) using specific stem-loop primers, as described previously (18Chen C. Ridzon D.A. Broomer A.J. Zhou Z. Lee D.H. Nguyen J.T. Barbisin M. Xu N.L. Mahuvakar V.R. Andersen M.R. Lao K.Q. Livak K.J. Guegler K.J. Nucleic Acids Res. 2005; 33: e179Crossref PubMed Scopus (4083) Google Scholar). Quantification of expression levels of VEGF, thrombospondin-1 (TSP-1), HIF-1α, HIF-1β, Dicer, Drosha, and microRNAs was determined by quantitative real time-PCR using SYBR Green (Applied Biosystems) and an Applied Biosystems 7300 detection system. GAPDH and RNU6B (for microRNA) were used for normalization, and all reactions were run in triplicate. Relative quantification of genes expression was analyzed with the comparative cycle threshold method (2−ΔΔCT). Sequences of miR, stem-loop primers, and primers for real time RT-PCR are shown in supplemental Table 1. Other primers for real time RT-PCR are shown in supplemental Table 2. Data are expressed as mean ± S.E. Statistical analysis was performed using SPSS version 15.0 (SPSS Inc.). Tests used were non-parametric Mann-Whitney (unpaired, two-tailed), and when appropriate, Student's t test. Statistical significance was established as p ≤ 0.05. SV40 T large antigen expression is able to inactivate both p53 and pRb, and its uroplakin II conditional expression has been previously shown to induce the development of bladder CIS (3Zhang Z.T. Pak J. Shapiro E. Sun T.T. Wu X.R. Cancer Res. 1999; 59: 3512-3517PubMed Google Scholar). We generated a transgenic mouse model in which the newly cloned UPK II promoter drives the expression of the SV40 T antigen (Fig. 1A). UPK II-SV40 (+) mice developed bladder urothelial carcinoma in situ with a 100% (39/39) penetrance starting from the first week of their life (Fig. 1, B–D and F–O). Although both males and females developed bladder carcinomas with similar disease progression kinetics, the bladder weight was higher in males, reflecting a higher tumor burden, as reported previously (19Johnson A.M. O'Connell M.J. Miyamoto H. Huang J. Yao J.L. Messing E.M. Reeder J.E. BMC Urol. 2008; 8: 7Crossref PubMed Scopus (54) Google Scholar) (Fig. 1E). Immunohistochemistry evaluation of paraffin-embedded bladder tissue revealed specific expression of SV40 in urothelial cells from UPK II-SV40 mice (Fig. 1, P–Q). Bladder transitional carcinomas developed with characteristics of human CIS or high-grade intraurothelial neoplasia (20Epstein J.I. Amin M.B. Reuter V.R. Mostofi F.K. Am. J. Surg. Pathol. 1998; 22: 1435-1448Crossref PubMed Scopus (1403) Google Scholar), exhibiting a flat growth, with large and pleomorphic nuclei and frequent mitoses (Fig. 1R). A papillary high-grade pattern appeared later but was infrequent. Although previous reports of similar models showed muscle invasion and occasional metastasis (3Zhang Z.T. Pak J. Shapiro E. Sun T.T. Wu X.R. Cancer Res. 1999; 59: 3512-3517PubMed Google Scholar, 4Cheng J. Huang H. Pak J. Shapiro E. Sun T.T. Cordon-Cardo C. Waldman F.M. Wu X.R. Cancer Res. 2003; 63: 179-185PubMed Google Scholar), this was an infrequent and late event; accordingly, in our UPK II-SV40 lines, we observed one incidence (1 out of 39) of lamina propria invasion but without muscle involvement or distant metastases (4–6 months). The development of urothelial carcinomas in renal pelvis and ureter followed a similar, but slower, progression (Fig. 1, S–V). The expression of VEGF, the main pro-angiogenic factor in cancer, has been considered a critical factor in SV40-induced tumor progression (21Catalano A. Romano M. Martinotti S. Procopio A. Oncogene. 2002; 21: 2896-2900Crossref PubMed Scopus (55) Google Scholar). To analyze the angiogenic pattern in our model of bladder carcinoma in situ, we performed immunofluorescence for CD31 and CD34. Staining for both markers was considerably increased in the bladder of UPK II-SV40 mice when compared with controls (Fig. 2, A–D). Tumor growth was associated with the development of a prominent vasculature, as shown by a statistically significant increase in the CD31 area (p = 0.017) (Fig. 2G). To evaluate the maturation status of carcinoma-associated bladder vessels, we double-labeled the bladder for CD31 and NG2, a pericyte marker (Fig. 2, E and F). Pericyte coverage of vessels was similar in both groups, thus suggesting a normal maturation and function of bladder tumor-associated vasculature in this stage (Fig. 2H). Differences in CD31 or NG2 staining (corrected by bladder area) were not observed between males and females. To provide further evidence of a pro-angiogenic phenotype and to explore the potential mechanism/s of vessel proliferation, Western blot of VEGF was performed using SV40 as a control for the presence of transformed urothelium. The analysis of Western blot revealed higher VEGF levels in UPK II-SV40 (+) mice (Fig. 3A). VEGF expression in SV40 urothelial tumors, as evaluated by immunofluorescence, was detected in stroma (Fig. 3, B–G) but was stronger in the urothelium, where it co-localized with UPK II (Fig. 3, H–M). Because hypoxia-related pathways have a role in the generation of a tumor-associated angiogenic phenotype, hypoxia was next assessed in bladder tissues. When compared with control, UPK II-SV40 mice exhibited a higher level of hypoxia as evaluated by Hypoxyprobe labeling (Fig. 4, A and B). HIF-1α participates in angiogenesis, and it is expressed in human CIS (16Ioachim E. Michael M. Salmas M. Michael M.M. Stavropoulos N.E. Malamou-Mitsi V. Urol. Int. 2006; 77: 255-263Crossref PubMed Scopus (37) Google Scholar, 22Jones A. Fujiyama C. Blanche C. Moore J.W. Fuggle S. Cranston D. Bicknell R. Harris A.L. Clin Cancer Res. 2001; 7: 1263-1272PubMed Google Scholar). We found an intense expression of HIF-1α both in the transformed urothelium and in the tumor stroma (Fig. 4, C and D), thus highlighting the participation of the hypoxia response pathway in VEGF production and neoplastic progression of bladder CIS. Staining for Glut-1, a glucose transporter whose up-regulation is usually dependent on HIF-1α (23Palit V. Phillips R.M. Puri R. Shah T. Bibby M.C. Oncol. Rep. 2005; 14: 909-913PubMed Google Scholar), was also increased in bladder tumoral epithelium when compared with normal urothelium and with tumor stroma (supplemental Fig. 1, A–D). Analysis of the expression of angiogenic growth factors and hypoxia markers, as evaluated by real time RT-PCR, showed an increase in VEGF and HIF-1β and down-regulation of thrombospondin-1 (TSP-1), a well known inhibitor of angiogenesis (19Johnson A.M. O'Connell M.J. Miyamoto H. Huang J. Yao J.L. Messing E.M. Reeder J.E. BMC Urol. 2008; 8: 7Crossref PubMed Scopus (54) Google Scholar) (Fig. 4E). We did not find any difference between females and males in the expression of VEGF, HIF-1α, or TSP-1 (supplemental Table 3). Dysregulation in the expression of miRs, a group of small non-coding molecules of RNA with an important role in post-transcriptional gene regulation, is a frequent phenomenon in cancer (24Iorio M.V. Croce C.M. J. Clin. Oncol. 2009; 27: 5848-5856Crossref PubMed Scopus (853) Google Scholar). Thus, we next sought to identify modifications in the profile of miRs related to the regulation of angiogenesis (25Wang S. Olson E.N. Curr. Opin. Genet. Dev. 2009; 19: 205-211Crossref PubMed Scopus (367) Google Scholar), such as the miR-17-92 cluster (26Dews M. Homayouni A. Yu D. Murphy D. Sevignani C. Wentzel E. Furth E.E. Lee W.M. Enders G.H. Mendell J.T. Thomas-Tikhonenko A. Nat. Genet. 2006; 38: 1060-1065Crossref PubMed Scopus (913) Google Scholar). Whole bladder from UPK II-SV40 mice revealed an increase in the expression of miR-18a and miR-19a, which are considered responsible for pro-angiogenic effects (27Doebele C. Bonauer A. Fischer A. Scholz A. Reiss Y. Urbich C. Hofmann W.K. Zeiher A.M. Dimmeler S. Blood. 2010; 115: 4944-4950Crossref PubMed Scopus (314) Google Scholar), whereas miR-17/20 and miR-92a were unchanged (Fig. 5A). These results, taken together with TSP-1 down-regulation (Fig. 4E), are consistent with a pro-angiogenic effect of miR-17-92 in this model. Additionally, miR-107, another angiogenesis-related miR whose expression is thought to be dependent on p53 status (28Yamakuchi M. Lotterman C.D. Bao C. Hruban R.H. Karim B. Mendell J.T. Huso D. Lowenstein C.J. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 6334-6339Crossref PubMed Scopus (357) Google Scholar), was significantly decreased in bladder carcinoma in situ when compared with normal bladder (Fig. 5B). A similar pattern of angiogenesis-related miR was found in both males and females, although there was a trend toward increased pro-angiogenic microRNAs, such as miR-18a, miR-19, and miR-20a in the females (supplemental Table 3). Because expression of Dicer and Drosha, two enzymes participating in miR processing, may be up-regulated in the setting of increased angiogenesis (29Kuehbacher A. Urbich C. Dimmeler S. Trends Pharmacol. Sci. 2008; 29: 12-15Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 30Kuehbacher A. Urbich C. Zeiher A.M. Dimmeler S. Circ. 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Oncol. 2010; 28: 39-48Crossref PubMed Scopus (82) Google Scholar), miR-205 (32Neely L.A. Rieger-Christ K.M. Neto B.S. Eroshkin A. Garver J. Patel S. Phung N.A. McLaughlin S. Libertino J.A. Whitney D. Summerhayes I.C. Urol. Oncol. 2010; 28: 39-48Crossref PubMed Scopus (82) Google Scholar, 33Wiklund E.D. Bramsen J.B. Hulf T. Dyrskjøt L. Ramanathan R. Hansen T.B. Villadsen S.B. Gao S. Ostenfeld M.S. Borre M. Peter M.E. Ørntoft T.F. Kjems J. Clark S.J. Int. J. Cancer. 2011; 128: 1327-1334Crossref PubMed Scopus (328) Google Scholar), miR-34a (34Catto J.W. Miah S. Owen H.C. Bryant H. Myers K. Dudziec E. Larré S. Milo M. Rehman I. Rosario D.J. Di Martino E. Knowles M.A. Meuth M. Harris A.L. Hamdy F.C. Cancer Res. 2009; 69: 8472-8481Crossref PubMed Scopus (283) Google Scholar), miR-222 (35Veerla S. Lindgren D. Kvist A. Frigyesi A. Staaf J. Persson H. Liedberg F. Chebil G. Gudjonsson S. Borg A. Månsson W. Rovira C. Höglund M. Int. J. Cancer. 2009; 124: 2236-2242Crossref PubMed Scopus (200) Google Scholar), miR-129 (36Dyrskjøt L. Ostenfeld M.S. Bramsen J.B. Silahtaroglu A.N. Lamy P. Ramanathan R. Fristrup N. Jensen J.L. Andersen C.L. Zieger K. Kauppinen S. Ulhøi B.P. Kjems J. Borre M. Orntoft T.F. Cancer Res. 2009; 69: 4851-4860Crossref PubMed Scopus (334) Google Scholar), miR-200c (37Olson P. Lu J. Zhang H. Shai A. Chun M.G. Wang Y. Libutti S.K. Nakakura E.K. Golub T.R. Hanahan D. Genes Dev. 2009; 23: 2152-2165Crossref PubMed Scopus (243) Google Scholar), and miR-145 (36Dyrskjøt L. Ostenfeld M.S. Bramsen J.B. Silahtaroglu A.N. Lamy P. Ramanathan R. Fristrup N. Jensen J.L. Andersen C.L. Zieger K. Kauppinen S. Ulhøi B.P. Kjems J. Borre M. Orntoft T.F. Cancer Res. 2009; 69: 4851-4860Crossref PubMed Scopus (334) Google Scholar, 38Ostenfeld M.S. Bramsen J.B. Lamy P. Villadsen S.B. Fristrup N. Sørensen K.D. Ulhøi B. Borre M. Kjems J. Dyrskjøt L. Orntoft T.F. Oncogene. 2010; 29: 1073-1084Crossref PubMed Scopus (128) Google Scholar, 39Ichimi T. Enokida H. Okuno Y. Kunimoto R. Chiyomaru T. Kawamoto K. Kawahara K. Toki K. Kawakami K. Nishiyama K. Tsujimoto G. Nakagawa M. Seki N. Int. J. Cancer. 2009; 125: 345-352Crossref PubMed Scopus (366) Google Scholar). As shown in Fig. 5D, their relative expression in bladder carcinoma in situ did not significantly change when compared with the normal bladder. Only miR-145 expression, related to urothelial transformation versus muscle invasion (38Ostenfeld M.S. Bramsen J.B. Lamy P. Villadsen S.B. Fristrup N. Sørensen K.D. Ulhøi B. Borre M. Kjems J. Dyrskjøt L. Orntoft T.F. Oncogene. 2010; 29: 1073-1084Crossref PubMed Scopus (128) Google Scholar, 39Ichimi T. Enokida H. Okuno Y. Kunimoto R. Chiyomaru T. Kawamoto K. Kawahara K. Toki K. Kawakami K. Nishiyama K. Tsujimoto G. Nakagawa M. Seki N. Int. J. Cancer. 2009; 125: 345-352Crossref PubMed Scopus (366) Google Scholar), was down-regulated in UPK II-SV40 CIS. miR-21:miR-205 ratio, which has been recently reported as a sensitive marker of bladder tumor invasion, was unaltered (32Neely L.A. Rieger-Christ K.M. Neto B.S. Eroshkin A. Garver J. Patel S. Phung N.A. McLaughlin S. Libertino J.A. Whitney D. Summerhayes I.C. Urol. Oncol. 2010; 28: 39-48Crossref PubMed Scopus (82) Google Scholar). p53 allelic loss or mutations usually coexist in SV40 T-induced carcinomas and high-grade human urothelial tumors and are associated with poor prognosis. Because the precise cellular origin of bladder carcinomas is still unclear, a mouse model in which p53 deletion involves all layers of the urothelium was generated. In the UPK II-Cre;p53 f/f mouse model (Fig. 6A), deletion of p53 in the urothelium was not associated with formation of bladder carcinomas or other morphological alterations (Fig. 6, B–E). Immunolabeling confirmed the lack of p53 expression in all cell layers of urothelium (Fig. 6, G–H). Further confirmation of urothelium-restricted expression of the Cre was obtained using reporter mice generated after breeding of UPK-II-Cre with Rosa-R26R-YFP floxed mice (supplemental Fig. 2A). When compared with control mice, YFP expression was specifically detected in the urothelial cells of bladder (supplemental Fig. 2, B and C) and ureter (supplemental Fig. 2, D and E) of UPK II-Cre; Rosa-Stop-YFP+/+ mice. Cre-mediated excision of the STOP cassette was also demonstrated by PCR for the recombinant YFP in the bladder DNA from UPK II-Cre; Rosa-Stop-YFP+/+ mice (supplemental Fig. 2F). To determine whether the pro-angiogenic profile observed in the UPK II-SV40 mice was related to p53 deletion, we examined the vasculature of bladders from UPK II-Cre;p53 f/f mice. As shown in Fig. 7, A and B, increased vascularization was not observed in UPK II-Cre;p53 f/f mice, when evaluated using immunofluorescence for CD31 (Fig. 7C). Pericyte coverage of blood vessels was also similar in both groups. We did not observe any difference in hypoxia response as evaluated using HIF-1α staining (Fig. 7, D and E) and VEGF staining (data not shown) F" @default.
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• W2076227992 title "Loss of p53 and Acquisition of Angiogenic MicroRNA Profile Are Insufficient to Facilitate Progression of Bladder Urothelial Carcinoma in Situ to Invasive Carcinoma" @default.
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