A Small Molecule Inhibitor of Isoprenylcysteine Carboxymethyltransferase Induces Autophagic Cell Death in PC3 Prostate Cancer Cells
A number of proteins involved in cell growth control, including members of the Ras family of GTPases, are modified at their C terminus by a three-step posttranslational process termed prenylation. The enzyme isoprenylcysteine carboxylmethyl-transferase (Icmt) catalyzes the last step in this process, and genetic and pharmacological suppression of Icmt activity significantly impacts on cell growth and oncogenesis. Screening of a diverse chemical library led to the identification of a specific small molecule inhibitor of Icmt, cysmethynil, that inhibited growth factor signaling and tumorigenesis in an in vitro cancer cell model (Winter-Vann, A. M., Baron, R. A., Wong, W., dela Cruz, J., York, J. D., Gooden, D. M., Bergo, M. O., Young, S. G., Toone, E. J., and Casey, P. J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4336–4341). To further evaluate the mechanisms through which this Icmt inhibitor impacts on cancer cells, we developed both in vitro and in vivo models utilizing PC3 prostate cancer cells. Treatment of these cells with cysmethynil resulted in both an accumulation of cells in the G1 phase and cell death. Treatment of mice harboring PC3 cell-derived xenograft tumors with cysmethynil resulted in markedly reduced tumor size. Analysis of cell death pathways unexpectedly showed minimal impact of cysmethynil treatment on apoptosis; rather, drug treatment significantly enhanced autophagy and autophagic cell death. Cysmethynil-treated cells displayed reduced mammalian target of rapamycin (mTOR) signaling, providing a potential mechanism for the excessive autophagy as well as G1 cell cycle arrest observed. These results identify a novel mechanism for the antitumor activity of Icmt inhibition. Further, the dual effects of cell death and cell cycle arrest by cysmethynil treatment strengthen the rationale for targeting Icmt in cancer chemotherapy. A number of proteins involved in cell growth control, including members of the Ras family of GTPases, are modified at their C terminus by a three-step posttranslational process termed prenylation. The enzyme isoprenylcysteine carboxylmethyl-transferase (Icmt) catalyzes the last step in this process, and genetic and pharmacological suppression of Icmt activity significantly impacts on cell growth and oncogenesis. Screening of a diverse chemical library led to the identification of a specific small molecule inhibitor of Icmt, cysmethynil, that inhibited growth factor signaling and tumorigenesis in an in vitro cancer cell model (Winter-Vann, A. M., Baron, R. A., Wong, W., dela Cruz, J., York, J. D., Gooden, D. M., Bergo, M. O., Young, S. G., Toone, E. J., and Casey, P. J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4336–4341). To further evaluate the mechanisms through which this Icmt inhibitor impacts on cancer cells, we developed both in vitro and in vivo models utilizing PC3 prostate cancer cells. Treatment of these cells with cysmethynil resulted in both an accumulation of cells in the G1 phase and cell death. Treatment of mice harboring PC3 cell-derived xenograft tumors with cysmethynil resulted in markedly reduced tumor size. Analysis of cell death pathways unexpectedly showed minimal impact of cysmethynil treatment on apoptosis; rather, drug treatment significantly enhanced autophagy and autophagic cell death. Cysmethynil-treated cells displayed reduced mammalian target of rapamycin (mTOR) signaling, providing a potential mechanism for the excessive autophagy as well as G1 cell cycle arrest observed. These results identify a novel mechanism for the antitumor activity of Icmt inhibition. Further, the dual effects of cell death and cell cycle arrest by cysmethynil treatment strengthen the rationale for targeting Icmt in cancer chemotherapy. Posttranslational processing of so-called CaaX proteins has received much attention in the past two decades due to the important roles these proteins play in biological regulations and diseases (1Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1754) Google Scholar, 2Gelb M.H. Brunsveld L. Hrycyna C.A. Michaelis S. Tamanoi F. Van Voorhis W.C. Waldmann H. Nat. Chem. Biol. 2006; 2: 518-528Crossref PubMed Scopus (163) Google Scholar). This processing is initiated by isoprenoid modification of the cysteine residue of the C-terminal CAAX motif of the protein, subsequent proteolytic removal of the three C-terminal amino acids, i.e. the –AAX residues, and the methylation of the newly exposed carboxyl group of the prenylated cysteine residue. The overall process, termed protein prenylation, has been shown to be important for the localization, stability, and ultimate functions of a broad array of CaaX proteins (3Magee T. Seabra M.C. Curr. Opin. Cell Biol. 2005; 17: 190-196Crossref PubMed Scopus (98) Google Scholar). Most members of the Ras superfamily of GTPases are CaaX proteins, and Ras proteins themselves, which are farnesylated, have been extensively studied due to the high prevalence of dysregulated Ras signaling in human cancers (4Malumbres M. Barbacid M. Nat. Rev. Cancer. 2003; 3: 459-465Crossref PubMed Scopus (1476) Google Scholar). Inhibitors of protein farnesyltransferase (FTase) 2The abbreviations used are: FTase, farnesyltransferase; Icmt, isoprenylcysteine carboxylmethyltransferase; LC3, microtubule-associated protein 1 light chain 3; LC3-II, activated form of LC3; J3, cysmethynil analog 1-octyl-m-tolyl-1H-indole; atg5, autophagy related 5 homolog; 4EBP1, eukaryotic initiation factor 4E-binding protein 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; 3-MA, 3-methyladenine; siRNA, small interfering RNA; DMSO, dimethyl sulfoxide. have been under development as anticancer agents for over a decade, but their efficacy, especially in solid tumors, has been disappointing (5Bishop W.R. Kirschmeier P. Baum C. Cancer Biol. Ther. 2003; 2: S96-S104Crossref PubMed Scopus (28) Google Scholar, 6Sebti S.M. Cancer Cell. 2005; 7: 297-300Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The realization that some CaaX proteins, including forms of Ras in which mutations are prevalent in human tumors, are subject to alternative prenylation by protein geranylgeranyltransferase I when FTase is inhibited (7Whyte D.B. Kirschmeier P. Hockenberry T.N. Nunez-Oliva I. James L. Catino J.J. Bishop W.R. Pai J.K. J. Biol. Chem. 1997; 272: 14459-14464Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar) spurred efforts to target the postprenylation processing steps of proteolysis and methylation since each of these steps is catalyzed by a single enzyme that acts on both farnesylated and geranylgeranylated proteins (8Ashby M.N. Curr. Opin. Lipidol. 1998; 9: 99-102Crossref PubMed Scopus (74) Google Scholar, 9Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (288) Google Scholar). In particular, targeting of CaaX protein methylation via inhibition of the enzyme responsible, isoprenylcysteine carboxylmethyltransferase (Icmt), through both genetic and pharmacological approaches, has been shown to dramatically impair oncogenesis in several tumor cell models (10Bergo M.O. Gavino B.J. Hong C. Beigneux A.P. McMahon M. Casey P.J. Young S.G. J. Clin. Investig. 2004; 113: 539-550Crossref PubMed Scopus (145) Google Scholar, 11Winter-Vann A.M. Baron R.A. Wong W. dela Cruz J. York J.D. Gooden D.M. Bergo M.O. Young S.G. Toone E.J. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4336-4341Crossref PubMed Scopus (156) Google Scholar). The mechanism(s) through which inhibition of Icmt impacts on cell proliferation and oncogenesis are still far from clear. Interference with cell cycle progression, however, is a cornerstone of many chemotherapeutic agents, and both FTase inhibition and geranylgeranyltransferase I inhibition have been demonstrated to arrest many types of tumor cells at G1 phase of the cell cycle; FTase inhibitors also trigger a G2/M arrest in certain cell types (5Bishop W.R. Kirschmeier P. Baum C. Cancer Biol. Ther. 2003; 2: S96-S104Crossref PubMed Scopus (28) Google Scholar, 6Sebti S.M. Cancer Cell. 2005; 7: 297-300Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 12Cox A. Der C. Curr. Opin. Pharmacol. 2002; 2: 388-393Crossref PubMed Scopus (127) Google Scholar). Another important property of cancer chemotherapeutic agents is the ability to induce cell death. The process of apoptosis in particular has been widely studied in this regard, and many current anticancer agents, including CaaX prenylation inhibitors, enhance apoptosis in cells (6Sebti S.M. Cancer Cell. 2005; 7: 297-300Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Evan G.I. Vousden K.H. Nature. 2001; 411: 342-348Crossref PubMed Scopus (2813) Google Scholar). Quite recently, autophagic cell death has stepped into the spotlight as a type of programmed cell death with the potential to be enhanced by cancer therapeutics (14Kondo Y. Kanzawa T. Sawaya R. Kondo S. Nat. Rev. Cancer. 2005; 5: 726-734Crossref PubMed Scopus (1511) Google Scholar, 15Hippert M.M. O'Toole P.S. Thorburn A. Cancer Res. 2006; 66: 9349-9351Crossref PubMed Scopus (360) Google Scholar). As with many biological regulatory processes, autophagy seems to be a double-edged sword in terms of an impact on cell functions. Most cell types display a baseline level of autophagy to clear damaged organelles and unwanted proteins, but dysregulation of the autophagy process can be detrimental to cell survival. Consequently, manipulation of autophagy is now considered to present therapeutic opportunities in several disease states, including cancer (16Huang J. Klionsky D.J. Cell Cycle. 2007; 6: 1837-1849Crossref PubMed Scopus (289) Google Scholar). We recently reported the identification of a specific small molecule inhibitor of Icmt, cysmethynil, and demonstrated a mechanism-based impact on tumorigenesis in an in vitro cancer cell model (11Winter-Vann A.M. Baron R.A. Wong W. dela Cruz J. York J.D. Gooden D.M. Bergo M.O. Young S.G. Toone E.J. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4336-4341Crossref PubMed Scopus (156) Google Scholar). We now report that treatment of PC3 prostate cancer cells with cysmethynil impairs cell cycle progression and, unexpectedly, activates an autophagic process in the cells and promotes autophagy-dependent cell death. Further, treatment of mice harboring xenograft tumors with cysmethynil results in dramatic impairment of tumor growth. These results identify a novel mechanism for the antitumor effects of Icmt inhibition and strengthen the support for targeting Icmt in cancer chemotherapy. Materials—Cysmethynil and biotin S-farnesylcysteine were synthesized by the Duke Small Molecule Synthesis Facility via established methods (11Winter-Vann A.M. Baron R.A. Wong W. dela Cruz J. York J.D. Gooden D.M. Bergo M.O. Young S.G. Toone E.J. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4336-4341Crossref PubMed Scopus (156) Google Scholar, 17Baron R.A. Casey P.J. BMC Biochem. 2004; 5: 19Crossref PubMed Scopus (34) Google Scholar). Cysmethynil analog 1-octyl-m-tolyl-1H-indole (J3) was synthesized via standard chemical procedures and characterized to confirm identity and purity (see the supplemental text and scheme). Stock solutions were prepared at 10 mm in DMSO and stored at –20 °C. Antibodies recognizing cyclins D1 and B1, p27, poly(ADP-ribose) polymerase-2, caspase 3, eukaryotic initiation factor 4E-binding protein 1 (4EBP1), and GAPDH were all from Cell Signaling. The LC3 antibody was from Abgent. Cell Culture and Proliferation Assays—The PC3 human prostate cell line was obtained from American Type Culture Collection (Rockville, MD). Cells were maintained at 37 °C with 5% CO2 in DMEM (Sigma) supplemented with 10% fetal bovine serum (Hyclone), 50 units/ml penicillin (Invitrogen), and 50 μg/ml streptomycin (Invitrogen). For proliferation assays, cells were seeded at 15–20% confluency in DMEM containing 5% fetal bovine serum in 96-well plates for 24 h prior to treatment with specific agents (e.g. cysmethynil) or vehicle at the concentrations and length of time indicated in the legends for Figs. 1, 2. The relative number of the live cells was determined using the CellTiter® 96 AQueous One Solution cell proliferation assay (Promega). Each condition was performed in quadruplicate, and data presented that obtained from at least three separate experiments. For proliferation studies performed with 3-methyladenine (3-MA) in addition to cysmethynil, cells were seeded as above and incubated with 0.5 mm 3-MA or vehicle at 37 °C overnight. Cells were then treated with the concentrations of cysmethynil as indicated in the legend for Fig. 1 in the continued presence of 0.5 mm of 3-MA for the indicated durations.FIGURE 2Cysmethynil treatment impacts on tumor growth in vivo. A, mice bearing PC3 cell-derived xenograft tumors were treated with vehicle (▪), 0.1 mg/g of cysmethynil or mg/g of cysmethynil h as under Each treatment group of and the data shown the and standard of each The has been with of were performed on from both in the data shown the and standard for the vehicle and mg/g of cysmethynil treatment at at cells were seeded in in DMEM containing 5% fetal bovine serum and incubated for 24 the cells were exposed to cysmethynil, the or vehicle at the and for the time indicated in the Cells were by at for 5 the cells were then in in containing μg/ml and 0.1 A from for 1 h at was by using an of to the indicated were with and with using a standard Cell Res. PubMed Scopus Google Scholar). with antibody to LC3 was performed at °C with antibody Analysis was performed using an with the and for (Sigma) was performed to a T. Kondo Y. H. Kondo S. I. Cancer Res. 2003; Google Scholar). cells were with and then with 1 for at 37 °C. Analysis was performed via using and a In the of the of cells were treated with for prior to the addition of targeting were to a the were using the by were with of and as well as time the as determined by of protein Cell proliferation on both the cells and the cells was then using the assay Cysmethynil Treatment of cells were in DMEM and 10% fetal serum and then with a standard using Cells were then with to in the The cell was then into the of For cysmethynil was prepared in DMSO, and (Sigma) the vehicle was with the In a mice were with vehicle control, and mg/g of The control, the 0.1 and the mice showed of the The mg/g and resulted in and in the 24 h all on the cysmethynil was at 0.1 and mg/g in two with the at The were for their and as well as tumors were with the standard tumor at the of the was and of tumor activity was determined by the in vitro assay R.A. Casey P.J. BMC Biochem. 2004; 5: 19Crossref PubMed Scopus (34) Google Scholar, R.A. J. Casey P.J. 2007; PubMed Scopus Google Scholar). the assay was using Icmt in cells, biotin S-farnesylcysteine as the and was by of of caspase and poly(ADP-ribose) through and of the of with as PubMed Scopus Google Scholar). To cells to the indicated treatment were and and protein was determined by protein assay were by and subsequent procedures were performed using an enhanced the Cysmethynil Cancer a of the mechanism-based of cysmethynil treatment on cells, we a analog of the that activity the of data on the chemical from which cysmethynil was Baron R. Casey P.J. Chem. 2007; 17: PubMed Scopus Google Scholar) that the of the be important in this analog the was termed and for in vitro activity This shown in Fig. that the analog was of Icmt activity chemical to The impact of by cysmethynil and the on the prostate cell line PC3 was using a cell Cysmethynil treatment resulted in a and in the number of PC3 cells, the analog at the was These with a using a cancer cell line in which the activity of cysmethynil was markedly by of Icmt in the cells (11Winter-Vann A.M. Baron R.A. Wong W. dela Cruz J. York J.D. Gooden D.M. Bergo M.O. Young S.G. Toone E.J. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4336-4341Crossref PubMed Scopus (156) Google that the impact of cysmethynil treatment on cells is due to inhibition of from this is that at cysmethynil to at both and activity are Cysmethynil in in a of the ability of cysmethynil to tumor growth in we a in mice and that an of mg/g was well We then established a xenograft model of PC3 cells in cells were in the tumors to in the of the tumor cells, mice were to and cysmethynil treatment bearing established PC3 tumors were of vehicle 0.1 or mg/g of cysmethynil h for The of the were by the tumor of the As shown in Fig. both of cysmethynil significantly the growth of PC3 tumors when with the vehicle was by as by of the treated mice when with the group These data that pharmacological inhibition of Icmt in vivo significantly impacts on tumor growth and further the of Icmt as an anticancer drug Cysmethynil Treatment Cell in PC3 in the is that inhibition of CaaX protein processing by of the protein can impact on cell cycle This with the that of cysmethynil have activity on PC3 cells to cell cycle in cells treated with this Icmt of PC3 cells treated h with cysmethynil showed a significantly of cells in G1 of with G1 arrest showed including reduced D1 and showed of These data are all with the G1 arrest in the Cysmethynil Treatment Cell the ability of cysmethynil treatment to trigger a G1 arrest in PC3 cells for the of this the in cell and in vitro that an in cell death was also by Icmt inhibition. of to this potential of cysmethynil treatment was on pathways since as inhibition of CaaX protein processing been to apoptosis in many cells types (6Sebti S.M. Cancer Cell. 2005; 7: 297-300Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). impact of cysmethynil treatment at the of cysmethynil that the number of cells was on as caspase and poly(ADP-ribose) in the treated PC3 cells was in PC3 cells cysmethynil treatment at the that resulted in cell death a of apoptosis to cell The to apoptosis to the cell death in the PC3 cells cysmethynil treatment to the drug a form of cell death. we a process that the of through an as this process has recently as important in cell growth and (14Kondo Y. Kanzawa T. Sawaya R. Kondo S. Nat. Rev. Cancer. 2005; 5: 726-734Crossref PubMed Scopus (1511) Google Scholar). cysmethynil treatment of PC3 cells resulted in a dramatic of the protein which is for the of autophagic Y. T. A. T. T. E. Y. T. J. PubMed Scopus Google Scholar). that the LC3 protein in cells both in and also into with the that Y. T. A. T. T. E. Y. T. J. PubMed Scopus Google Scholar). cell types of at baseline (14Kondo Y. Kanzawa T. Sawaya R. Kondo S. Nat. Rev. Cancer. 2005; 5: 726-734Crossref PubMed Scopus (1511) Google we the in the cells by of the of cells level of LC3 this showed that cysmethynil treatment significantly of Fig. The process of autophagy with the and then to through the of with M.M. O'Toole P.S. Thorburn A. Cancer Res. 2006; 66: 9349-9351Crossref PubMed Scopus (360) Google Scholar). of the live cells was also to in and PC3 cells. As shown in Fig. cysmethynil treatment markedly the of in the cells, providing further that the autophagic process was activated by drug treatment and that the is the of the inhibition of an inhibitor of the of to by the of to A. Y. T. Y. R. Y. Cell 1998; PubMed Scopus Google Scholar). a of the autophagy The treatment of the PC3 cells with markedly reduced the of that the by the treatment of cysmethynil the process with the with in the cells, a of that the of autophagy prior to were by this of PC3 Cells from Cell by Cysmethynil the of autophagy in PC3 cells to the cell death by treatment with the we 3-MA, a inhibitor of autophagy that acts through inhibition of type U. H. J. Biochem. 1997; PubMed Scopus Google cell death. PC3 cells were treated with vehicle cysmethynil, 3-MA or 3-MA cysmethynil, and of the cells was h As in Fig. 3-MA treatment impact on cell the of cells was markedly 3-MA was present the of the showed that the cells treated with both 3-MA and cysmethynil much of the autophagy when with the cells treated with cysmethynil that the autophagic process by cysmethynil is to the by type We also a to impair autophagy to further impact on cell death. of atg5, a of the autophagy (14Kondo Y. Kanzawa T. Sawaya R. Kondo S. Nat. Rev. Cancer. 2005; 5: 726-734Crossref PubMed Scopus (1511) Google markedly reduced cell death by cysmethynil treatment This has a for autophagy-dependent cell death in the of of also resulted in the of in cells a impact of the on the autophagic process in the cells. the with both 3-MA treatment and in the PC3 cells that cysmethynil autophagy but that the autophagy-dependent cell death significantly to the of cysmethynil in cancer cell death. Cysmethynil Treatment on in PC3 data presented above that cysmethynil treatment both G1 cell cycle arrest and a of these two is that can be by types of CaaX proteins are to be important in signaling, Ras GTPases and the Ras activates to the of which in activates by a of with a impact on cell cycle progression and impact on autophagy P.J. 2006; PubMed Scopus Google Scholar). of Ras methylation has been shown to impair Ras activity (11Winter-Vann A.M. Baron R.A. Wong W. dela Cruz J. York J.D. Gooden D.M. Bergo M.O. Young S.G. Toone E.J. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4336-4341Crossref PubMed Scopus (156) Google also be by Icmt and this further impact cysmethynil treatment of PC3 cells resulted in a of of as be from inhibition of In of the protein was markedly reduced in cells, and 4EBP1, showed the in with inhibition of the in PC3 cells were markedly reduced h of cysmethynil providing that the and activity of were on these we a model inhibition of Icmt to a of Ras and activity and inhibition of and signaling, to the effects on cell cycle progression and autophagy as a of of cancer cell death has attention recently, especially in of cancer cells. For the inhibitor rapamycin has been reported to induce cell death through autophagy in cell that are to many that induce apoptosis H. Kondo Y. Kanzawa T. H. Kondo S. Cancer Res. 2005; 65: PubMed Scopus Google Scholar). In this as a showed that treatment of cell autophagic cell death T. T. H. Kondo Y. Kondo S. Cell 2004; PubMed Scopus Google Scholar). the realization that autophagic cell death be an important of the of certain cancer is the the inhibition and autophagy has been well established (14Kondo Y. Kanzawa T. Sawaya R. Kondo S. Nat. Rev. Cancer. 2005; 5: 726-734Crossref PubMed Scopus (1511) Google Scholar). The signaling cell and growth and autophagy in many cells, and of the has been to oncogenesis in many cancers Cancer Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In many of the has been to of the and these to to many also the cancers to as the the cells on this for proliferation PubMed Scopus Google Scholar, C. R. P. J.J. H. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). this the PC3 prostate cancer cell used in the current studies is and signaling, which to the of this cell line to a of therapeutic The of autophagic cell death by cysmethynil that the inhibition of the CaaX protein processing in this group of cancers an therapeutic or signaling by cysmethynil of two mechanisms of of cell death and inhibition of cell cycle the of two and proteins play important roles in in particular, inhibition of of the G1 cell cycle regulatory protein D1 to G1 arrest in cells in which is inhibited C. R. P. J.J. H. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). The ability of cysmethynil to induce G1 arrest as well as autophagy as an anticancer The identification of cysmethynil as a specific and Icmt inhibitor a to the of Icmt in both and The current the rationale for targeting Icmt as an anticancer from a but also in vivo of cysmethynil through to mice bearing xenograft prostate data that an of autophagy by cysmethynil is a to the cell death that pharmacological inhibition of the specific CaaX this have been the Ras and GTPases are potential due to their to and We Wong and for and the Cancer for of the of this with