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(CANCER RESEARCH 51.712-717, January 15. 1991] Inhibition of Cell Growth by Lovastatin Is Independent of ras Function Jeffrey E. DeClue, William C. Vass, Alex G. Papageorge, Douglas R. Lowy, and Berthe M. Willumsen Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892 ¡J.E. D., W. C. V., A. G. P., D. R. L.J, and University Microbiology Institute, Osterfarimagsgade 2A, D-1353 Copenhagen, Denmark ¡B.M. W.] ABSTRACT We have investigated the inhibition of cell growth by lovastatin (pre viously known as mevinolin), an antagonist of hydroxymethylglutaryl coenzyme A reducÃ-ase which blocks the processing and membrane local ization of ras proteins via inhibition of polyisoprenylation. A series of NIH 3T3 cells transformed by oncogenes with activities that are depend ent or independent of isoprenylated ras were studied, including cells transformed by myristylated ras protein that is isoprenylation independ ent. Treatment with lovastatin at concentrations ranging from 5 to IS MM for up to 96 h resulted in a time- and dose-dependent inhibition of cell growth in all lines tested. The inhibition ranged from 25 to 50% when cells were treated with 5 nM lovastatin for 48 h, to 72-90% for cells treated with 15 MMlovastatin for 96 h. Cells transformed by c-ras, \-ras, \-src, \-raf, and the myristylated ras genes displayed similar sensitivities; the parental NIH 3T3 line was the most resistant of the lines tested. Metabolic labeling of control and lovastatin-treated cells with [15S|methionine or tritiated lipids revealed that 15 MM lovastatin blocked the processing of both endogenous ras and \-rti.\ proteins yet had no effect on the lipidation of myristylated ras proteins. Addition of 300 MM mevalonic acid overcame the inhibition induced by 15 MM lovastatin. Thus the inhibition of cell growth in vitro by lovastatin did not show specificity for cells the transformation of which is dependent upon iso prenylated ras protein. It is therefore likely that the inhibition of other pathways affected by lovastatin, such as cholesterol biosynthesis or the processing of other cellular proteins, are responsible for the growth inhibition by lovastatin. INTRODUCTION The ras group of genes, which are a highly conserved sub family of the small GTPases, encode closely related M, 21,000 protein products (p21s) (1, 2). In addition to serving an impor tant function in the control of normal eukaryotic growth and proliferation, mutated versions of ras proteins have been found in a wide variety of human and animal tumor systems (3). Studies in which neutralizing antibodies to ras proteins have been microinjected into NIH 3T3 cells have shown that mitogenesis of nontransformed cells, as well as lines transformed with the v-irc, \-fes, and \-fms oncogenes, is dependent on functional endogenous ras protein (4). Human cells derived from normal tissues also require raÃ-for DNA synthesis follow ing mitotic stimulation, and a few tumor cell lines without activated ras are still inhibited to the same degree by the antibody as are tumor lines with activated ras genes (5). Thus some human tumor cells can be described as ras dependent, although their genetic lesion(s) lie(s) outside of the ras genes. A critical requirement for ras function is the localization of raÃ-proteins to the cell plasma membrane from their initial synthesis in the cytoplasm (6). Membrane association occurs following posttranslational modifications that take place at the COOH terminus and include the covalent addition of lipid (7). Genetic studies indicated that the last four amino acids, espe cially an invariant cysteine, were required for posttranslational changes and membrane localization (8). Although it was origi- nally believed that palmitic acid represented the obligatory lipid, it has recently been shown that palmitylation is not an obliga tory step for some raÃ-proteins. Rather, the acylation of ras proteins involves the addition of a polyisoprenoid derivative of M VA,' which is an intermediate in the sterol-biosynthetic path way (9-11). The polyisoprenoid is probably a farnesyl residue (11). Following translation of p21 raÃ-,the farnesyl moiety is apparently added to the invariant cysteine residue, which is located four amino acids from the COOH-terminus; the three amino acids downstream of this cysteine are removed; and the carboxyl group of the farnesylated cysteine is methylated (12). At this point, cysteine residue(s) NH2-terminal to the farnesy lated cysteine, if present, may be palmitylated. This series of processing steps results in an increasingly hydrophobic COOHterminal membrane anchor for the raÃ-proteins. The demonstration that proper processing of both normal and transforming raÃ-proteins is dependent on the availability of mevalonic acid-derived products has led to speculation that drugs such as compactin and lovastatin, which block the pro duction of M VA by inhibiting the rate-limiting enzyme HMGCoA reducÃ-ase(13), may prove to be useful as chemotherapeutic agents against rai-dependent tumors (9, 14). However, since HMG-CoA reducÃ-aselies far upstream of the protein farnesylation step, blocking this enzyme may also interfere with other critical cellular processes (15). Furthermore, protein isopreny lation appears to be a common modification; in addition to raÃ-, a large number of other proteins are labeled by MVA-derived compounds (16-18). Thus the question of specificity arises as one of critical importance in assessing the potential efficacy of lovastatin in treating rai-dependent tumors. Using a series of cell lines transformed by defined oncogenes, the current study has sought to determine if lovastatin may have greater activity against rai-dependent cell lines than against rai-independent lines. Our in vitro studies demonstrate that under the culture conditions used here, the inhibition of cell growth by lovastatin was not selective for polyisoprenylated rai-dependent cell lines, which implies that the growth effects of lovastatin in these lines are not mediated principally through its inhibition of raÃ-protein function. MATERIALS AND METHODS Construction of Gene Encoding a Myristylated raÃ-Protein. A doublestranded oligonucleotide encoding the first 15 amino acids of the v-src protein and the first four amino acids of ras was synthesized (sense strand: S'-GGCCACCATGGGGAGTAGCAAGAGCAAGCCTAAGGACCCCAGCCAGCGCCGGATGACAGAATACA-3'). For the annealed double stranded oligonucleotide, the 5' end encodes a Sad I site overhang, and the 3' end a HinDUl site overhang; the HindlU occurs naturally at amino acid 4 of v-rai". The main portion of the ras gene was derived from a Hind\\\/Xho\ fragment from the plasmid pBW1225. This fragment encodes amino acids 4-189 of the \-ras" protein including a cysteine-to-serine mutation at amino acid 186 which blocks the COOH-terminal posttranslational processing of raÃ-protein. These two fragments were inserted into a derivative of the expression Received 8/6/90; accepted 11/1/90. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1The abbreviations used are: MVA, mevalonic acid; HMG-CoA. hydroxy methylglutaryl coenzyme A; D-MEM. Dulbecco's modified Eagle's medium. 712 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. LOVASTATIN-INHIBITED CELL GROWTH AND raj FUNCTION vector pGV16 (19), pBWl 160 (20), by ligation to vector linearized by double digestion with Sacll and Xhol, which each cleave pBW1160 once. The resulting plasmid is designated pJDClOo. Derivation and Culture of Cell Lines. Morphological transformation was induced by calcium phosphate transfection of the NIH 3T3 (clone 7) line with the following plasmids: pBW'1423 for \-ras": pBW1631 for c-rtw" (21); pJDCl (22) for v-src, pJDClOo for the myristylated ras; and MSV3611 proviral DNA for v-ra/(23). With the exception of the v-ra/plasmid, each of the above plasmids contains neo as a linked selectable marker. All cell lines except the v-ra/line were generated by selection with G418 (600 MM)for 10-14 days, isolation of individual transformed colonies with cloning cylinders, and expansion into cell lines. The v-ra/line was generated by cloning an individual focus of transformed cells and expansion into a cell line. Cells were cultured in D-MEM supplemented with 10% fetal bovine serum (GIBCO) and 600 MMG418 where required. Growth Inhibition Assays. On day 0, cells were trypsinized and plated at 104/well in 8 triplicate sets in 24-well plates (Costar). On day 3, cells effects of lovastatin on these transformants should distinguish those of lovastatin in specifically blocking farnesylation of ras protein from its other effects in ras-transformed cells. Cells were also transformed with v-src and \-raf, which are ras dependent and ras independent, respectively (4). Fig. 1 represents photomicrographs of the cell lines. The parental NIH 3T3 line exhibits a flat, nonrefractile morphology, with fully contact-inhibited growth. In contrast, the v-ra/-transformed cells have an elongated, refractile appearance and tend to pile on top of each other, as do the v-src-transformed cells, although they have a rounded, refractile morphology. The cras, v-ras, and myristylated ras (myr-ras) transformed cells display a morphology somewhat in between the \-raf- and the v-src-transformed cells. Some of the cells are elongated, while others are rounded (especially for v-ras), and all the ras-trans formed lines lack contact inhibition and pile up extensively. Thus all of the oncogenes produce alterations in cell morphol were fed with fresh medium containing either 0, 5, 10, or 15 MM ogy and a loss of monolayer-restricted growth in NIH 3T3 cells. lovastatin. After 48 h (day 5), one triplicate set of each concentration In addition, the growth rates of the oncogene-transformed cells was trypsinized and counted in a Coulter Counter. The other triplicate sets were fed fresh medium with or without lovastatin at this time. exceed that of the parental line (data not shown). Two days later (96 h total treatment) the other triplicate sets were Growth Inhibition by Lovastatin. To assess their relative sen trypsinized and counted. For experiments in which MVA was included, sitivity to lovastatin, the cell lines were treated with 5, 10, and 300 MMMVA was added together with 15 MMlovastatin at both day 3 15 AIMlovastatin for up to 96 h. This concentration range has and day 5. been shown previously to inhibit the growth of cultured cell Preparation of Lovastatin. Lovastatin was generously provided by lines, including NIH 3T3. As noted by others, a dramatic, doseAlfred Alberts of Merck, Sharp & Dohme Co. To convert the inactive lactone form of lovastatin to the active form, the drug was dissolved in dependent rounding of the cells was observed after 24 h lovas ethanol, heated at 50°Cin 0.1 N NaOH, neutralized with HC1, and tatin treatment (28). This morphological effect, which was stored unfiltered as a 4 mg/ml stock at -20°C. especially pronounced at 15 /¿M lovastatin, was similar in the untransformed parental line and in the transformed lines (not Metabolic Labeling and Immunoprecipitation of ras Proteins. Cells were plated at a density of 2 x 106/75-cm2 flask (Costar). Labeling with shown). [35S]methionine was carried out in 2 ml of methioninc-free D-MEM Following 48 h treatment with lovastatin, cell growth was (Flow Laboratories) supplemented with 10% regular D-MEM, 2% fetal inhibited by 25-45% for 5 ¿¿M treatment and by 40-65% for 15 bovine serum, and 200 MCi/ml 15S-Trans-label (ICN). Labeling with ¿IMtreatment (Fig. 2A). All lines tested displayed a dose[3H]myristate or |'H|palmitate (NEN-Dupont) was carried out in reg dependent inhibition over the concentration range tested. Fur ular medium with 10% fetal bovine serum for 6 h with 0.5 and 2.0 thermore, the degrees of inhibition of the ras-transformed cell mCi/ml, respectively. For the labeling experiments, lovastatin treat lines, including two independently derived myr-ras transformment (15 MM)was for 24 h prior to cell lysis, including the labeling ants, were virtually identical and within 10% of the inhibition period. seen for the v-src- and the v-ra/-transformed lines. Only the Cells were lysed as described (24). For analysis of "S-labeled cells, lysates containing equal numbers of acid-precipitable counts were ana parental NIH 3T3 line differed significantly in its response to lyzed. The entire lysate from one flask was analyzed for the 'H-labeled lovastatin; the inhibition of this line was less than for any of cells. Lysates were immunoprecipitated with the ras-specific monoclo the transformed lines (Fig. 2A). Ninety-six h of treatment of nal antibody Y13-259 (25). Following a 2-h antibody incubation. 60 M' the cell lines with the same concentration range of lovastatin of a 10% suspension of protein A-Sepharose coated with rabbit-anti rat also resulted in a similar pattern of inhibition that was dose IgG were added for 45 min. The immunoprecipitates were washed 4 dependent for each line. At this time point, the inhibition of times with wash buffer as described (24), boiled in sodium dodecyl the v-src, v-ras, and myr-ras lines was almost identical (Fig. sulfate sample buffer, and loaded on 15% polyacrylamide gels. 2B), while the c-ras and v-ra/ lines were somewhat less inhib ited, and again, the NIH 3T3 line was the least inhibited of the RESULTS lines tested. Furthermore, the inhibition of every line, at each concentration, was greater at 96 h of treatment than at 48 h, Morphological Properties of Transformed Cell Lines. To com although the extent of inhibition and the variation among the pare the effect of lovastatin on cell lines that were transformed lines were greater at 96 h than after 48 h of treatment (Fig. by oncogenes that are ras dependent or ras independent, NIH 3T3 cells, which require normal (c-ras) function for passage IB). At 5 MM the inhibition ranged from 40 to 80%, while at 15 ¿IM the range was from 70 to 90%. Thus growth inhibition through the cell cycle, were transformed by a variety of onco genes and expanded as cell lines. For lines that would be in these studies was both time and concentration dependent. dependent upon exogenous ras, cells were transformed by muLovastatin Blocks Processing of ras Proteins. To confirm that ialiona IK activated v-ras or by overexpression of c-ras. We also lovastatin was inhibiting the processing of the authentic (nonmyristylated) endogenous and exogenous ras proteins, cells took advantage of the ability of ras protein to be transforming when its membrane association is driven by myristylation at its were labeled with ['5S]methionine, ['H]myristate, or ['HjpalmiNH2 terminus in the absence of COOH-terminal posttranslatate, in the presence or absence of lovastatin, and lysates of cell tional changes by inducing transformation with a nonfarnesyextracts were immunoprecipitated with the anti-ras monoclonal antibody Y13-259 (Fig. 3). In the absence of lovastatin, the ras lated, myristylated transforming ras protein the COOH termi nus of which had been mutated to prevent posttranslational proteins detected under these conditions represent fully proc processing (26, 27). Since the transforming activity of the essed forms, which migrate approximately 2-3 kDa faster than myristylated ras protein is independent of farnesylation, the an unprocessed primary ras translation product. Normal NIH 713 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. LOVASTATIN-INHIBITED CELL GROWTH AND ras FUNCTION ' Fig. I. Phase-contrast photomicrographs of the cell lines used in this study. A. normal NIH 3T3: B. v-ro/-transformcd line; C v-.wr-transformed line; I), c-ra.stransformed line; E. v-ra.v-transformed line; F. myr-rai-transformed line 4-3; 6", myr-ro.i-transformed line 4-4. Cells were plated at 8 x lO'/oO-mm dish 2 days prior to photomicroscopy. Original magnification. X-85. 3T3 cells labeled with [15S]methionine exhibited a low level of endogenous ras proteins at M, 21,000-22,000 (Fig. 3A. Lane /). As expected, the \-ras protein migrated as a doublet (29); the upper band represents the viral protein that is phosphorylated at Thr-59 and the lower band represents the nonphosphorylated form (Fig. 3A, Lane 2). The two myr-ra.v lines, since they also contain Thr-59, express a doublet of M, 26,000 and 28,000; their slower migration rate results from the additional 15 amino acids at their NH2 terminus and their lack of COOH terminal processing (Fig. 3A, Lanes 3 and 4). In the presence of 15 ßMlovastatin. the endogenous ras doublet was replaced by a single band (M, 22,000) (Fig. 3.-1,Lanes 5 and 6). while both the phosphorylated and nonphosphorylated v-ras proteins migrated approximately 2 kDa slower, indicating a lack of farnesylation, proteolytic cleavage, and carboxymethylation. In contrast, no effect on the migration of the myr-ras protein was seen (compare Lanes 4 and 6). We conclude that lovastatin has blocked the posttranslational modifications of newly synthe sized c-ras and \-ras proteins but has not altered the myr-ras proteins. When the cells were labeled with ['Hlmyrislate (Fig. 3B), as expected no label was incorporated into the endogenous ras or the \-rax proteins (Fig. 35, Lanes I and 2). The myr-ras proteins did, however, incorporate label in the presence or absence of lovastatin, indicating that they were myristylated, and no change in the migration of m\r-ras protein was seen in the lovastatin-treated cells (Fig. 3fi. Lanes 3-5). The slight reduc tion in label in Lane 5 compared to Lane 4 probably reflects the smaller number of cells in the flasks treated with lovastatin. Labeling of cells in the presence or absence of lovastatin showed that lovastatin blocked the processing of newly synthe sized endogenous ras and \-ras proteins, but no effect was noted on the processing of the myr-ras proteins. The cycling of palmitatc on and off previously processed ras proteins contin ued in the presence of lovastatin, as shown by metabolic labeling with ['H]palmitate (data not shown). Mevalonic Acid Overcomes the Effects of Lovastatin. Since lovastatin blocks the enzyme HMG-CoA reducÃ-ase,which con verts HMG to MVA, MVA can overcome effects of lovastatin that arc due to its inhibition of HMG-CoA reducÃ-ase. Rcpre714 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. LOVASTATIN-INHIBITED CELL GROWTH AND ras FUNCTION A Lov: - - 29 — ras B 123456 Lov: — — — — + 29 — 18.4 — 14.3 — 12345 Fig. 3. Expression of ras proteins in the presence or absence of lovastatin as detected by radiolabeling and immunoprecipitation. In A, cells were labeled for 20 h with |"S|methionine, with or without 24 h lovastatin treatment (15 MM)(top abscissa). Lane I, NIH 3T3; Lanes 2 and 5, v-rai-transformed line; Lane 3, myrras-transformed line 4-3; lanes 4 and 6, myr-rai-transformed line 4-4. In B, cells were labeled for 6 h with ['Hjmyristate with or without lovastatin treatment. Arrow, migration of the myr-raa proteins. Lane I, NIH 3T3; Lane 2, v-rastransformed line; Lane 3, myr-ras-transformed line 4-3; Lanes 4 and 5. myr-raitransformed line 4-4. Immunoprecipitation was carried out as described in "Materials and Methods." Left ordinate, migration of molecular weight standards (x IO3). Lovastatin Fig. 2. Inhibition of cell growth by 48- or 96-h lovastatin treatment. In A, cells were treated for 48 h with lovastatin at the concentrations indicated, and the number of cells obtained were presented as the percentage of untreated controls. In B, cells were treated for 96 h. this pathway, may specifically inhibit the growth of cells which are dependent on ras function for their growth. Our resulls demonstrated, in accordance with earlier reports (9-11), thai under ihe in vitro cullure condilions used here, lovaslalin blocked Ihe processing of both endogenous ras and \-ras pro teins. As expected, no effecl was seen by lovaslalin on the processing of a control transforming ras protein that became membrane associated via myristylation, which is a form of acylation lhal does noi depend on the sterol pathway. Despite these biochemical differences, lovastatin induced similar, dosedependent, biological changes (morphological changes and growth inhibition) in iransformed lines, regardless of whelher the transforming ras protein was prenylated. A similar degree of growih inhibition was also observed for lines Iransformed by \-src and \-raf, allhough transformation by v-raf has been shown to be independent of endogenous ras. Since it has been shown that transforming ras genes can provide the essential function of endogenous ras (30), and the myristylated ras gene should also provide this function, it is unlikely that the lack of specificity for growth inhibition seen here is due to the inhibi- sentative lines were therefore treated with lovastatin in the presence of MVA to determine the extent to which the effects of lovastatin on the cell lines resulted from HMG-CoA reduc Ã-aseinhibition (Fig. 4). When 300 /UMMVA was added together with 15 ßM lovastatin, cell growth was restored to within 15% of untreated controls, at both 48 and 96 h. In addition, no cell rounding was observed for cells treated simultaneously with MVA and lovastatin (data not shown). We conclude that the major effects of lovastatin on the cell lines depend upon its inhibition of HMG-CoA reducÃ-ase. DISCUSSION Based on the recent observation that proper processing of ras proteins depends on products derived from the sterol-biosynthetic pathway, we have here investigated the possibility lhat the compound lovastalin, which blocks ihe rate-limiling step in 715 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. LOVASTATIN-INHIBITED CELL GROWTH AND raj FUNCTION Effect of MVAon Growth Inhibition by Lovastatin by this compound may be overly optimistic, although it remains possible that specificity might be seen under different in vitro culture conditions or in tumorigenicity studies carried out in animals. The lack of specificity by lovastatin demonstrated here should not restrict further attempts to pharmacologically block the membrane localization of ras protein. However, it may be more appropriate to focus on targets downstream from HMG-CoA reducÃ-asewhich are more directly involved in the posttranslational processing of ras, such as the farnesylation step itself (35). Even such inhibitors of downstream targets may also affect macromolecules in addition to ras. As antagonists of farnesy lation, carboxymethylation, or related events are identified, their degree of specificity for ras transformation may be readily evaluated by comparison with their ability to inhibit myr-ras transformed cells. 50.0% 40.0% 30.0% 20.0% - v-ras tMVA myr v-ras 3 myr v-ras 3 +MVA 10.0% - myr v-ras 4 myf v-ras 4 +MVA Day Fig. 4. Effect of M VA on growth inhibition induced by lovastatin. The indicated cell lines were plated on day 0 and treated at day 3 with IS fiM lovastatin alone or together with 300 ^M MVA. Cell growth was expressed as in Fig. 2. tion of endogenous ras. The biological effects of lovastatin on the ras-transformed lines could be overcome by simultaneous treatment with MVA, which strongly implies that lovastatin was acting primarily via its inhibition of HMG-CoA reducÃ-ase (13). The results indicate that the lines transformed by HMGCoA reductase-dependenl ras protein were not more sensitive to the growth-inhibitory effects of lovastatin than were lines transformed by an oncogene the activity of which was HMGCoA reducÃ-aseindependent. We therefore conclude that, under the culture conditions tested here, the inhibitory growth effects of lovastatin on these lines are not mediated primarily via the effects of the drug on ras processing. Growth of NIH 3T3 cells transformed by a myristylated c-ras gene (27) is also inhibited when the cells are treated with compacting which also blocks HMG-CoA reducÃ-ase.This result further supports the conclu sion that ras-independent activities mediate Ihe growth inhibi tion observed when Ihis slep is blocked. Although we failed to detect any specificity of lovastatin toward cells the transformation of which depended upon farnesylated ras protein, rapidly growing, transformed lines were more sensitive to the growth-inhibitory effects of lovastatin than was the parental NIH 3T3 line, which exhibits a longer doubling time. Thus lovastatin may inhibit tumor cells by nonspecifically blocking critical cellular processes which are especially important during rapid cell proliferation. The finding that growth inhibition by lovastatin is not specific for ras-transformed cells is perhaps not surprising. In studies where cells have been treated with compactin (which also blocks HMG-CoA reducÃ-ase), then labeled with [3H]MVA, over 40 proteins have been shown, by 2-dimensional electrophoresis, to incorporate the label (16). These proteins include the nuclear lam ins A and B (18, 31, 32), which may play an essential role in the progression through the cell cycle, as well as several M, 20,000-25,000 proteins, which, like ras protein, may regulate critical functions involved in cellular growth control. Further more, MVA-derived products also include cholesterol, a regu lator of membrane fluidity, as well as dolichols, and the ubiquinones (14, 33, 34). In view of the wide variety of cellular processes that may be disrupted by lovastatin treatment, the hope that ras-transformed cells could be specifically inhibited 2 A. Cox and J. Buss, personal communication. REFERENCES 1. Marshall. C. J. The H4S oncogenes. J. Cell. Sci. (Suppl.), 10:, 157-169, 1988. 2. Barbacid. M. ras genes. Annu. Rev. 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Inhibition of Cell Growth by Lovastatin Is Independent of ras Function Jeffrey E. DeClue, William C. Vass, Alex G. Papageorge, et al. Cancer Res 1991;51:712-717. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/51/2/712 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.