Depletion of Methionine Aminopeptidase 2 Does Not Alter Cell Response to Fumagillin Or Bengamides

[CANCER RESEARCH 64, 2984–2987, May 1, 2004] Advances in Brief

Depletion of Methionine Aminopeptidase 2 Does Not Alter Cell Response to Fumagillin or Bengamides

Sunkyu Kim, Kenneth LaMontagne, Michael Sabio, Sushil Sharma, Richard W. Versace, Naeem Yusuff, and Penny E. Phillips Novartis Pharmaceuticals, East Hanover, New Jersey

Abstract DMSO at 1000-fold final concentrations and added to cells in culture. DMSO was included at 0.1% final concentration in all vehicle control samples. Cell Inhibition of endothelial cell growth by fumagillin has been assumed to culture, production of recombinant human MetAp1 and 2, and MetAp enzyme be mediated by inhibition of the molecular target methionine aminopep- assays have been described previously (10). For Fig. 2, C and D, cell prolif- tidase 2 (MetAp2). New data show that depletion of MetAp2 by siRNA eration was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy- does not inhibit endothelial cell growth. Moreover, MetAp2-depleted en- phenyl)-2-(4-sulfonyl)-2H-tetrazolium assay. One-hundred percent growth re- dothelial cells remain responsive to inhibition by either fumagillin or a fers to cell number of vehicle-treated cells after 72 h, corrected for initial newly identified MetAp2 enzyme inhibitor. These data suggest that plating density. Cell growth was normalized to initial cell number as described MetAp2 function is not required for endothelial cell proliferation. (10). For Figs. 2B and 3A–C, cell growth was measured by bromodeoxyuridine (BrdUrd) incorporation. BrdUrd was added to cells in the final2hofthe Introduction indicated times of cell growth. BrdUrd incorporation into cells was measured The methionine aminopeptidases (MetAp) are a class of proteases using a cell proliferation ELISA kit (catalogue no. RPN250; Amersham that selectively remove methionine from the NH terminus of newly Bioscience) according to manufacturer’s specifications. One-hundred percent 2 growth refers to the incorporation of BrdUrd in cells treated with vehicle synthesized proteins (1). Two isoforms of MetAps have been identi- control. MetAp2 suppression was performed using an siRNA sequence de- fied in humans (2–4). The human MetAp2 isoform has been of signed as described previously (13). The targeting sequence was AAUGCCG- considerable interest as a target for cancer chemotherapy, after the GUGACACAACAUGA (Dharmacon Research). The control mismatch sequence discovery that the antiangiogenic natural compound fumagillin is a was AAUGCCGGCGCUACAACAUGA. Duplex RNA was introduced into ep- covalent inhibitor selective for MetAp2 (5–7). On the basis of the ithelial tumor cells using LipofectAMINE 2000 (Invitrogen) according to the antiangiogenic activity observed in vitro, a fumagillin analog TNP- manufacturer’s protocol. Duplex RNA was introduced into endothelial cells by 470 has progressed to clinical trials (8); however, the compound has electroporation. All data are the average Ϯ SE of triplicate determinations from a demonstrated dose limiting neurotoxicity (9). representative experiment repeated at least twice. A second family of natural products, the bengamides, has recently Human Endothelial Cell Response to Basic Fibroblast Growth Factor. been identified as inhibitors of both MetAp isoforms (10). In contrast Human umbilical vein endothelial cells and human aortic endothelial cells were transfected with siRNA as previously described (10), plated at 12,500 to fumagillin, bengamides and a synthetic analogue, LAF389, inhibit cells/well of a 24-well plate, and incubated overnight at 37°C, 5% CO2. Fresh growth of tumor epithelial cells as well as endothelial cells. In an media (EBM-2, Clonetics, ϩ 2% fetal bovine serum) containing fumagillin effort to test the hypothesis that inhibition of MetAp2 results in were added for 30 min. After this pretreatment, cells received either vehicle selective inhibition of endothelial cell growth, we prepared a series of control or 10 nM basic fibroblast growth factor and cultured for 72 h. Cells bengamide analogues and tested their activity in MetAp enzyme and were trypsinized, and cell number was determined by Coulter counter. cell proliferation assays. The current data demonstrate that MetAp2 can be selectively inhibited by a bengamide analogue but that this Results compound does not selectively inhibit endothelial cell proliferation. Furthermore, endothelial cell proliferation is unaffected by selective A number of analogues of the bengamide LAF389 were prepared depletion of MetAp2 through use of a targeting small interfering and screened for MetAp2 specificity. A methyl-substituted analogue (si)RNA. Finally, endothelial cells depleted of MetAp2 by siRNA of LAF389 (Fig. 1), despite a relatively modest structural modifica- treatment remain sensitive to growth inhibition by either fumagillin or tion, was found to be specific for the desired isoform. LBM648 ␮ the MetAp2-selective bengamide. Taken together, these data show inhibited MetAp2 with a 0.38 M IC50, equivalent to LAF389 but had ␮ that MetAp2 is not the target for the antiangiogenic effects of fum- no activity on MetAp1, even at 10 M (Table 1). agillin. The effect of LBM648 on cell proliferation was assayed on several epithelial cell lines and on human umbilical vein endothelial cells. Materials and Methods Compared with the nonselective MetAp inhibitor LAF389, LBM648 was 3–10-fold less potent on epithelial tumor lines (Table 2). The Cell and Enzyme Assays. A549 and H1299 non-small cell lung cancer cell increase in MetAp2 enzyme selectivity of LBM648 did not result in lines were purchased from American Type Culture Collection. Fumagillin was increased selectivity for endothelial growth inhibition, compared with purchased from Sigma. Human umbilical vein endothelial cells and human LAF389. Several additional MetAp2-selective bengamide analogues aortic endothelial cells were purchased from Clonetics and cultured as recom- mended by the vendor. LBM648 and LAF389 were synthesized as described were also as potent on epithelial cells as on endothelial cells (data not previously (11, 12). LBM648, LAF389, and fumagillin were dissolved in shown), thus challenging the hypothesis that small molecule inhibition of MetAp2 necessarily results in selective inhibition of endothelial cell growth. Received 1/7/04; revised 3/12/04; accepted 3/17/04. The costs of publication of this article were defrayed in part by the payment of page In an alternative approach to address the role of MetAp2 in cell charges. This article must therefore be hereby marked advertisement in accordance with proliferation, we prepared cells depleted in MetAp2 using siRNA. As 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Penny Phillips, Novartis Pharmaceuticals, One Health Plaza, has previously been reported (10), treatment of epithelial cells with East Hanover, NJ 07936. MetAp2 siRNA reduced MetAp2 protein within 24 h to a level at or 2984

superimposable in H1299 cells treated with the mismatch siRNA sequence versus cells treated with the MetAp2 siRNA sequence (Fig. 2C). A549 cells treated with MetAp2 siRNA also showed no change in response to LBM648 compared with cells treated with the mismatch sequence (Fig. 2D). Fig. 1. Structures of LAF389 and LBM648 A more stringent test of MetAp2’s role in cell growth was performed using primary endothelial cells. Endothelial cells have been

Table 1 IC50s for LAF389 and LBM648 on human methionine aminopeptidase suggested to have an absolute requirement for MetAp2 function for (MetAp)1 and MetAp2 growth based on the antiproliferative effects seen with fumagillin and MetAp1 and MetAp2 enzyme assays were performed using recombinant enzyme and a methionine-alanine-serine substrate as described previously (10). analogues (6, 7, 14). This would suggest that reduction of MetAp2 protein should inhibit endothelial cell growth. To test this hypothesis, IC , ␮M (average Ϯ SE) 50 MetAp2 protein levels were specifically reduced by treatment with MetAp1 MetAp2 siRNA in human umbilical vein and human aortic endothelial cells. In LAF389 0.70 Ϯ 0.01 0.37 Ϯ 0.01 both types of endothelial cells, proliferation in complete growth Ͼ Ϯ LBM648 10 0.38 0.04 medium was unaffected by reduction of MetAp2 protein to Ͻ5% of control levels (Fig. 3A). This experiment could not rule out the possibility that the remaining levels of MetAp2 protein in siRNA- below the detection level of Western blots (Fig. 2). In H1299 epithelial cells, reduction of MetAp2 by the targeting sequence was specific; MetAp2 protein levels were not reduced in cells treated with a Table 2 Antiproliferative IC50s for LAF389 and LBM648 sequence containing 3 mismatch bases, and actin levels were identical Cell proliferation was measured at 72 h by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy- in cells treated with the MetAp2 targeting sequence and the mismatch methoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium. Details of the procedures have been re- sequence. Reduction of MetAp2 protein by siRNA caused appearance ported previously (10). ␮ Ϯ of an unprocessed form of 14-3-3␥, showing that MetAp2 enzyme IC50, M (average SE) function was also substantially reduced in these cells. No difference in Human growth was observed in H1299 cells depleted of MetAp2, as measured umbilical vein by incorporation of BrdUrd (Fig. 2B), suggesting that in these cells endothelial MetAp2 activity is not limiting for cell growth. Surprisingly, H1299 A549 H1299 HCT116 cells cells depleted of MetAp2 by siRNA remained completely responsive LAF389 0.09 Ϯ 0.01 0.23 Ϯ 0.02 0.04 Ϯ 0.04 0.05 Ϯ 0.01 to growth inhibition by LBM648; growth inhibition curves were LBM648 0.74 Ϯ 0.20 0.99 Ϯ 0.76 0.14 Ϯ 0.15 0.14 Ϯ 0.05

Fig. 2. Methionine aminopeptidase 2 (MetAp2) small interfering (si)RNA effects on epithelial tumor cells. A, H1299 cells were treated with MetAp2 siRNA (2) or control siRNA (2C) and analyzed by Western blotting as described in “Materials and Methods.” Uncleaved 14-3-3␥ was recognized using a monoclonal antibody specific for the unprocessed, methionine containing form (10). Total 14-3-3␥ was recognized using a gamma isoform-specific antibody (Santa Cruz Biotechnology). B, effects of siRNA treatment for 48, 72, and 96 h on cell proliferation were measured by incorporation of bromodeoxyuridine (BrdUrd). Western blots were performed in parallel on cell lysates to confirm that MetAp2 down-regulation persisted for 96 h (data not shown). C and D, growth of siRNA-treated H1299 (C) and A549 (D) cells was measured after 72-h exposure to LBM648 using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium. Insets show MetAp2 and actin levels measured in cells after 72 h. 2985

Fig. 3. Methionine aminopeptidase 2 (MetAp2) small interfering (si)RNA effects on endothelial cells. A, human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) were treated with MetAp2 siRNA (2) or control siRNA (2C). Bromodeoxyuridine incorporation was measured after 48 h as described in “Materials and Methods.” B and C, growth of siRNA-treated HAECs was measured after 72-h exposure to LBM648 (B) or fumagillin (C). Insets show MetAp2 levels measured in cells after 72 h; actin levels (data not shown) were identical in control siRNA and MetAp2 siRNA-treated cells. D, proliferation of HUVECs after treatment with control siRNA (left) or MetAp2 siRNA (right). Cell number was measured by counting trypsinized cells in a Coulter counter after 72 h of growth in the presence of vehicle or basic fibroblast growth factor (bFGF) and in the presence of the indicated concentrations of fumagillin. Each sample was counted twice, and the average of the triplicate control wells (i.e., stimulated cells with DMSO only) determined the percentage of control values for the test wells and the negative control wells. The graphs were generated by SigmaPlot, and the significance (P) was determined by a t test (SigmaStat). Asterisks indicate P Ͻ 0.01.

treated cells were sufficient for endothelial cell growth. Human aortic active in endothelial cells but able to be inhibited by both fum- endothelial cells treated with MetAp2-specific siRNA were therefore agillin and bengamide analogues. Indeed, a recent publication has challenged with the MetAp2 enzyme inhibitors LBM648 (Fig. 3B) proposed such an enzyme based on sequence homology, although and fumagillin (Fig. 3C). The ability of both compounds to inhibit no enzymatic activity of the putative new MetAp isoform, was proliferation of human aortic endothelial cells was unaltered in cells shown (16). Regardless of the existence of a third MetAp isoform depleted of MetAp2 protein compared with either control, untreated or some other unrelated antiproliferative target, the current data are human aortic endothelial, or to human aortic endothelial cells treated sufficient to challenge the hypothesis that inhibition of MetAp2 by with the mismatch siRNA sequence. Human umbilical vein endothe- fumagillin or bengamide analogues is the mechanism by which lial cells depleted of MetAp2 also retained the ability to respond to these compounds exert their antiangiogenic effects. Given that fumagillin, whether growth was measured under serum-depleted con- clinical trials are in progress with fumagillin analogues (17), ditions, or in response to a basic fibroblast growth factor stimulus additional target-finding experiments are clearly called for to elu- (Fig. 3D). cidate the molecular targets of these compounds. Taken together, these data demonstrate that MetAp2 is not required for endothelial cell proliferation and that two different References classes of MetAp2 enzyme inhibitors remain effective inhibitors of 1. Lowther WT, Matthews BW. Structure and function of the methionine aminopepti- cell growth on cells depleted of their putative target. One question dases. Biochim Biophys Acta 2000;1477:157–67. left unanswered by these surprising results is the nature of the 2. Li X, Chang Y-H. Molecular cloning of a human complementary DNA encoding an initiation factor 2-associated protein (p67). Biochim Biophys Acta 1995;1260:333–6. target(s) for fumagillin and LBM648 and whether this target is 3. Li X, Chang Y-H. Evidence that the human homologue of a Rat initiation factor-2 common to both inhibitors. The activity of the compounds in associated protein (p67) is a methionine aminopeptidase. Biochem Biophys Res MetAp2-depleted endothelial cells is particularly puzzling because Comm 1996;227:152–9. 4. Nagase T, Miyajima N, Tanaka A, et al. Prediction of the coding sequences of both classes of inhibitors have been cocrystalized with human unidentified human genes. III. The coding sequences of 40 new genes (KIAA0081- MetAp2 (10, 15). One possibility is that another member of the KIAA0120) deduced by analysis of cDNA clones from human cell line KG-1. DNA Res 1995;2:37–43. MetAp family exists in humans; the current data could be resolved 5. Ingber D, Fujita T, Kishimoto S, et al. Synthetic analogues of fumagillin that inhibit by showing the presence of a third MetAp enzyme expressed and angiogenesis and suppress tumour growth. Nature (Lond.) 1990;348:555–7. 2986

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. [CANCER RESEARCH 64, 9230, December 15, 2004] Corrections p53 and BCNU Resistance in Astrocytes In the article on p53 and BCNU Resistance in Astrocytes in the June 15, 1996 issue of Cancer Research (1), the title was incorrect. The title should have read “Wild-Type p53 Renders Mouse Astrocytes Resistant to 1,3-Bis(2-chloroethyl)-1-nitrosourea Despite the Absence of a p53-dependent Cell Cycle Arrest.” 1. Nutt CL, Chambers AF, Cairncross JG. Wild-type p53 renders mouse astrocytes resistant to 1,3-Bis(2-chloroethyl)-1-nitrosourea despite the absence of a p53-dependent cell cycle arrest. Cancer Res 1996;56:2748–51.

AChE in Apoptosis In the article on AChE in Apoptosis in the April 15, 2004, issue of Cancer Research (1), there is an error on page 2652, in the section under “Materials and Methods” on “siRNA Transfection”. The AChE target sequence should have read 5Ј-AAGAGUGUCUGCUAC- CAAUAU-3Ј. 1. Park SE, Kim ND, Yoo YH. Acetylcholinesterase plays a pivotal role in apoptosome formation. Cancer Res 2004;64:2652–5.

Depletion of Methionine Aminopeptidase 2 In the article on Depletion of Methionine Aminopeptidase 2 in the May 1, 2004, issue of Cancer Research (1), there is an error on page 2984, in the section under “Materials and Methods” on “Cell and Enzyme Assays”. The text near the end of the section should have read the following: “The targeting sequence was AAUGCCGGUGA- CACAACAGUA (Dharmacon Research). The control mismatch sequence was AAUGCCGGCGCUACAACAGUA.” 1. Kim S, LaMontagne K, Sabio M, Sharma S, Versace RW, Yusuff N, Phillips PE. Depletion of methionine aminopeptidase 2 does not alter cell response to fumagillin or bengamides. Cancer Res 2004;64:2984–7.

NIS Gene Therapy of Hepatocarcinoma In the article on NIS Gene Therapy of Hepatocarcinoma in the November 1, 2004, issue of Cancer Research (1), a note should have been included indicating that J. Faivre and J. Clerc contributed equally to the study. 1. Faivre J, Clerc J, Ge´rolami R, Herve´ J, Longuet M, Liu B, Roux J, Moal F, Perricaudet M, Bre´chot C. Long-term radioiodine retention and regression of liver cancer after sodium iodide symporter gene transfer in Wistar rats. Cancer Res 2004;64:8045–51.

Novel Functions of BRAK In the article on Novel Functions of BRAK in the November 15, 2004, issue of Cancer Research (1), the following grant support information should have appeared: This work was supported in part by the University of Texas M.D. Anderson Cancer Center SPORE in Head and Neck Cancer NIH-NCI P50 CA097007 (G. Clayman and M. Frederick), NIH R01 DE013954 (G. Clayman), Cancer Center Support Grant NIH P30 CA016672, Alando J. Ballantyne Distinguished Chair in Head and Neck Surgery Award (G. Clayman), Michael A. O’Bannon Endowment for Cancer Research (G. Clayman), Betty Berry Cancer Research Fund (G. Clay- man), and NIH INRS Award T32 CA060374 (G. Clayman). 1. Shellenberger TD, Wang M, Gujrati M, Jayakumar A, Strieter RM, Burdick MD, Ioannides CG, Efferson CL, El-Naggar AK, Roberts D, Clayman GL, Frederick MJ. BRAK/CXCL14 is a potent inhibitor of angiogenesis and a chemotactic factor for immature dendritic cells. Cancer Res 2004;64:8262–70.

9230 Depletion of Methionine Aminopeptidase 2 Does Not Alter Cell Response to Fumagillin or Bengamides

Sunkyu Kim, Kenneth LaMontagne, Michael Sabio, et al.

Cancer Res 2004;64:2984-2987.

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