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Use of mitogenic cascade blockers for treatment of C-Raf induced lung adenoma in vivo: CI-1040 strongly reduces growth and improves lung structure

Use of mitogenic cascade blockers for treatment of C-Raf induced lung adenoma in vivo: CI-1040... Background: Signaling networks promoting cell growth and proliferation are frequently deregulated in cancer. Tumors often are highly dependent on such signaling pathways and may become hypersensitive to downregulation of key components within these signaling cascades. The classical mitogenic cascade transmits stimuli from growth factor receptors via Ras, Raf, MEK and ERK to the cell nucleus and provides attractive molecular targets for cancer treatment. For example, Ras and Raf kinase inhibitors are already in a number of ongoing phase II and phase III clinical trials. In this study the effect of the Raf kinase inhibitor BAY 43-9006 and of the MEK inhibitor CI-1040 (PD184352) on a Raf dependent lung tumor mouse model was analyzed in detail. Methods: We have generated a lung cancer mouse model by targeting constitutively active C-Raf kinase to the lung. These mice develop adenomas within 4 months of life. At this time-point they received daily intraperitoneal injections of either 100 mg/kg BAY 43-9006 or CI-1040 for additional 21 days. Thereafter, lungs were isolated and the following parameters were analyzed using histology and immunohistochemistry: overall lung structure, frequency of adenoma foci, proliferation rate, ERK activity, caspase-3 activation, and lung differentiation. Results: Both inhibitors were equally effective in vitro using a sensitive Raf/MEK/ERK ELISA. In vivo, the systemic administration of the MEK inhibitor CI-1040 reduced adenoma formation to a third and significantly restored lung structure. The proliferation rate of lung cells of mice treated with CL-1040 was decreased without any obvious effects on differentiation of pneumocytes. In contrast, the Raf inhibitor BAY 43-9006 did not influence adenoma formation in vivo. Conclusion: The MEK inhibitor CI-1040 may be used for the treatment of Ras and/or Raf- dependent human malignancies. Background tin for the treatment of advanced breast cancer and Oncogene-based therapeutics is a novel approach to Gleevec for chronic myelogenous leukemia have proven inhibit proteins, which are essential for the initiation and that cancer therapies targeting specific molecular altera- maintenance of malignancies [1]. Agents such as Hercep- tions in signaling pathways are successful [2]. The Ras- Page 1 of 6 (page number not for citation purposes) BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 MAP kinase pathway has a central role in regulating tumor 1040: daily intraperitoneal injections at a dose of 100 mg/ cell growth and survival, differentiation and angiogenesis kg from 4 months of age over a period of 21 days, n = 12 and has been targeted for therapeutic intervention in the each group). At this dose both drugs were well tolerated past [3,4]. C-Raf kinase and MEK are downstream effec- (data not shown). Serum concentration of BAY 43-9006 tors of the Ras signaling cascade. Both kinases are essential was determined by liquid-liquid extraction and consecu- for cellular homeostasis and induce both proliferation tive HPLC analysis [14]. Lungs were isolated and analyzed and survival by suppression of apoptosis [5]. Raf and Ras at the end of the treatment period. Animals were com- mutations found in human malignancies convey constitu- pared to non-treated or placebo-treated transgenic mice of tive activity to these signaling molecules thereby convert- the same litter. Placebo treatment did not cause any ing them into an oncogenic state [6]. changes in comparison to untreated mice (data not shown). In this study the Raf inhibitor BAY 43-9006 [7] and the MEK inhibitor CI-1040 [8] were tested as potential drugs Histology and immunohistochemistry in a transgenic mouse lung cancer model [9]. We have pre- Lungs were fixed under 25 cm water pressure with forma- viously established this mouse model by expressing lin. Histology was done on formalin-fixed, paraffin- mutated, constitutively active C-Raf kinase (C-Raf BxB) embedded lung specimen. 4 µm-cut sections were depar- under the control of the human surfactant protein C (SP- affinized, rehydrated in graded alcohols and hematoxylin C) promoter [10]. C-Raf BxB lacks the regulatory NH -ter- and eosin stained. Antigen retrieval was performed with minal sequences including the Ras interaction domain. citrate buffer, pH 6.0, and microwave treatment. Endog- Lung targeted expression of constitutively active C-Raf enous peroxidase activity was blocked by incubation with induced lung adenomas within 4 months of life [10]. 3% methanol. Unspecific binding was blocked by normal Although these adenomas are stable for more than one goat serum. The primary antibody for p-ERK (Cell signal- year, deficiencies in other genes such as Bcl-2 or p53 were ing 9101), PCNA (DAKO M 0879), for Ki-67 (DAKO M found to modulate adenoma growth or even switch the 7249) and Bmi-1 (Santa Cruz sc-10745) were detected by phenotype of tumor cells, respectively [11,12]. The effects a peroxidase labelled secondary antibody (DAKO P0450). of BAY 43-9006 and CI-1040 were assessed in this lung Corresponding secondary antibodies were used to detect adenoma model by daily intraperitoneal injections of activated caspase-3 primary antibody (Cell signaling these drugs at concentrations of 100 mg per kg body 9664) and pro-SP-C antibody (generous gift of Dr. Jeffrey weight over a period of three weeks to four months old C- A. Whitsett, Cincinnati Children's Hospital Medical Raf-BxB mice. Center, OH/USA). Staining was performed using diami- nobenzidine and hematoxylin counterstaining. Slides were analyzed in a blinded fashion. Random pictures Methods ELISA were taken of each specimen. Adenoma foci were counted Enzyme-linked immunosorbent assay for the MEK signal- per mm . Positive cells in adenoma foci were counted and ing cascade was done as previously described [13]. In expressed as percentage of all adenoma cells. brief, plates were coated with an anti ERK-antibody (Santa Cruz, sc-94). A kinase reaction was performed in the pres- Results ence of ERK, Raf, MEK and the respective inhibitor. Phos- BAY 43-9006 and CI-1040 are equally effective in vitro but differ in their in vivo properties phorylated ERK was detected by a sandwich technique using a secondary antibody (BioLabs, #9106L) and a per- To determine whether the inhibitors used in our study are oxidase-linked species-specific tertiary antibody (Amer- active we performed at first an in vitro assay. The activity sham Pharmacia Biotech, #NA 931). Similar results were of BAY 43-9006 and CI-1040 was measured using an obtained with constitutively active C-Raf BxB (data not ELISA method that detects phosphorylation of ERK that is shown). dependent on Raf and MEK activity [13]. Both substances were equally effective in vitro with complete inhibition of Animals ERK phosphorylation at concentrations of less than 1 µM Lung targeted expression of constitutively active C-Raf (Fig. 1a). The IC values for inhibition of C-Raf by BAY (SP-C C-Raf BxB), lacking the regulatory N-terminal 43-9006 and by CI-1040 in our assays were 16 nM and 12 sequences, which comprise the Ras interaction domain, nM, respectively. In addition, the effect of both inhibitors induced lung adenomas within 4 months of life [10]. The was also tested on cell lines. At a concentration of 0,01 µM adenomas were indistinguishable from those induced by the inhibitory effect of CI-1040 on ERK phosphorylation expression of wild type C-Raf (data not shown). Litters of was more than 90 %, whereas BAY 43-9006 at 4 µM lead the transgenic mice were randomly assigned to the control to an inhibition in the range between 60 – 80 % as deter- groups (no treatment, n = 11, respectively placebo treat- mined by Western blotting and immunohistochemistry ment, n = 12) and to the study group (BAY 43-9006 or CI- (data not shown). Page 2 of 6 (page number not for citation purposes) BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 BAY 43-9006 BAY 43-9006 P<0.05 A CI-1040 P<0.05 CI-1040 0 0 -3 -2 -1 0 1 2 10 10 10 10 10 10 BAY 43-9006 CI-1040 Placebo µM E F CI-1040 Placebo BAY 43-9006 MEK inh Figure 1 ibitor CI-1040 but not Raf inhibitor BAY 43-9006 reduces lung adenomas in vivo MEK inhibitor CI-1040 but not Raf inhibitor BAY 43-9006 reduces lung adenomas in vivo. (A) MEK activity was completely inhibited in vitro by BAY 43-9006 and CI-1040 as determined by ERK activation enzyme-linked immunosorbent assay. IC50 values for BAY 43-9006 and CI-1040 were 16 nM and 12 nM respectively. (B, C) The amount of P-ERK and the number of P-ERK positive cells were reduced in vivo by treatment with CI-1040 but not with BAY 43-9006. (D, E, F) Treat- ment with CI-1040 but not with BAY 43-9006 reduced adenomas and improved lung structure after 21d (hematoxylin and eosin staining; scale bars = 60 µm). Lung adenomas as well as the overall lung morphology was indistinguishable in placebo treated and untreated animals (data not shown). At next, the in vivo effects of a systemic administration of phosphorylated ERK was clearly reduced as determined by the inhibitors were analyzed using our Raf dependent immunohistochemistry (Fig. 1b) or Western blotting lung tumor mice. Comparable serum concentrations of (data not shown). The number of cells that were stained both inhibitors were reached after intraperitoneal injec- by the Phospho-ERK antibody was reduced almost three- tion as determined by liquid-liquid extraction of mouse fold (Fig. 1c). In contrast, phosphorylation of ERK in lung serum and consecutive HPLC analysis [14] and Sirrenberg adenomas in vivo was virtually not affected by systemic unpublished data). Lung sections of transgenic mice that treatment of mice with BAY 43-9006 (Fig. 1b,1c) or in have been treated for three weeks with either CI-1040 or controls (data not shown). Moreover, treatment with CI- BAY 43-9006 were examined for the amount of phospho- 1040 did not only reduce adenomas but also improve the rylated ERK. After treatment with CI-1040 the amount of overall lung structure with thin alveolar walls, whereas Page 3 of 6 (page number not for citation purposes) Percentage Inhibition P-ERKpositive Cells% BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 BAY 43-9006 again has no detectable effect (Fig. not be useful as anticancer drugs because inhibition of Raf 1d,1e,1f). is always counterbalanced by reactivation [16]. Finally, it was recently suggested that C-Raf has signaling properties The number of adenoma foci per mm was determined that are independent of its protein kinase activity [17]. and found to be reduced more than 75 % after CI-1040 Therefore, it has also to be considered that BAY 43-9006- treatment in comparison to BAY 43-9006 treated or pla- bound kinase-inactive C-Raf has still transforming activity cebo treated animals (Fig. 2a). This treatment results in a in pneumocyte type II cells. But this possibility appears reduced ratio of lung to body weight of lungs obtained unlikely in the light of earlier results demonstrating the from mice after CI-1040 treatment (7.5 ± 0.3 mg/g) in loss of transforming activity and concomitant gain of comparison to lungs isolated from mice after BAY 43- dominant-negative function of c-Raf genes with muta- 9006 treatment (10.4 ± 0.6 mg/g, P < 0.05). tions that inactivate the protein kinase activity of C-Raf [18-20]. Moreover, there is a direct correlation between CI-1040 reduces proliferation in lung adenomas but has no reversal of a transformed phenotype by BAY43-9006 and influence on apoptosis or lung specific differentiation Phospho-ERK suppression (Sirrenberg and Rapp, unpub- The adenoma foci were further analyzed for proliferation, lished data). apoptosis and differentiation to decipher the mechanism of action by which CI-1040 reduced adenomas. We Previous work on CI-1040 has already established that assessed proliferation by immunohistochemistry for pro- this drug has a cytostatic effect on subcutaneously liferating cellular antigen (PCNA), Ki-67 and Bmi-1. The implanted colon tumors of human and mouse origin har- percentage of PCNA positive cells was clearly reduced by boring RAS mutations [21]. In addition, in a mouse half after CI-1040 treatment (Fig. 2b). The percentage of model of metastatic melanoma that was dependent on a Ki-67 and Bmi-1 positive cells was reduced to a third after B-Raf mutation CI-1040 was preventing the formation of treatment with CI-1040 (Fig. 2c,2d). Apoptotic cells were new pulmonary metastases and caused rapid regression of detected by immunohistochemistry for activated caspase- already established pulmonary metastases [22]. Both pub- 3 and no difference was detected after BAY 43-9006 or CI- lications as well as our report indicate that CI-1040 has a 1040 treatment (Fig. 2e). Adenoma cells were analyzed broad spectrum of antitumor activity and will be useful for the expression of pro-SP-C as a marker of alveolar type for the treatment of cancers that are dependent on muta- II cell differentiation. The percentage of pro-SP-C positive tions or overexpression of members of the mitogenic cas- cells did not change after BAY 43-9006 or CI-1040 treat- cade upstream of ERK. ment (Fig. 2f). We also determined the effects of both inhibitors on expression of Bmi-1. The number of Bmi-1 positive cells Discussion We were able to show inhibition of ERK phosphorylation in lung sections was reduced more than twofold by CI- in vitro by both, the MEK inhibitor CI-1040 and the Raf 1040, whereas BAY 43-9006 had no effect. The observed inhibitor BAY 43-9600. In addition, both drugs inhibited effects of CI-1040 may be limited to proliferation as nei- ERK phosphorylation to a comparable level when tested ther apoptosis nor alveolar differentiation was directly on cell lines in tissue culture (data not shown). Although affected. Bmi-1 was originally cloned as c-Myc cooperat- both inhibitors reached comparable serum concentra- ing oncogene in murine lymphomas [23,24]. A role for tions after intraperitoneal administration (data not Bmi-1 in human tumorigenesis was recently suggested by shown), the MEK inhibitor was active in mice whereas the the discovery of Bmi-1 amplification in mantle cell lym- Raf inhibitor was not. Why BAY 43-9006 is not active in phomas [25] and of Bmi-1 overexpression in a variety of our Raf dependent lung tumor mouse model and whether tumors including non-small cell lung cancer [26]. Since Raf and MEK might fulfill different functional roles with CI-1040 might be more effective in inhibiting the prolif- respect to lung adenoma formation needs to be further eration of Bmi-1 positive adenoma cells, we want to addressed in detail in future. address in future whether Bmi-1 is a self-renewal factor of lung stem cells or lung adenoma stem cells. One possible explanation for the observed effects may relate to differences in the in vivo accessibility of these Conclusions inhibitors to sub-cellular sites where C-Raf is localized. These experiments demonstrate an essential role of MEK/ Alternatively, inhibition of Raf kinases might be more dif- ERK signaling for the maintenance of Raf induced trans- ficult to achieve due to multiple feedback mechanisms formation in vivo. Effects on the ERK pathway in vivo may [15], whereas the regulation of MEK is less complex and be predictive for effective tumor drug design and therapy inhibition of MEK is easier achieved in the living organ- of Raf driven tumors. CI-1040 is currently evaluated in ism. Along this line it was already discussed previously phase II trials and may be a promising anti-proliferative that inhibitors targeting the kinase activity of Raf might drug for Ras and/or Raf-dependent human malignancies. Page 4 of 6 (page number not for citation purposes) BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 7 P<0.05 BAY 43-9006 CI-1040 Placebo BAY 43-9006 CI-1040 Placebo MEK Figure 2 inhibitor CI-1040 reduces proliferation in lung adenomas but has no influence on apoptosis or lung specific differentiation MEK inhibitor CI-1040 reduces proliferation in lung adenomas but has no influence on apoptosis or lung spe- cific differentiation. (A) The number of adenoma foci per mm was reduced after treatment with CI-1040 but not with BAY 43-9006. (B) CI-1040 reduced the PCNA positive cells in lung adenomas by half but not BAY 43-9006. (C, D) Ki-67 and Bmi-1 positive cells were reduced to a third in lung adenomas after CI-1040 treatment. (E) Neither BAY 43-9006 nor CI-1040 acti- vated the caspase-3 mediated apoptotic pathway in lung adenomas. (F) BAY 43-9006 or CI-1040 treatment did not change cell differentiation in lung adenomas, which was assessed by expression of pro-SP-C, a marker for alveolar type II cells. Page 5 of 6 (page number not for citation purposes) PCNA positive Cells Ki-67 positive Cells Number of Adenoma Foci per mm Pro SP-C positive Cells Bmi-1 positive Cells Activ. Caspase 3 positive Cells ‰ BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 BAY 43-9006 in small samples of serum. J Chromatogr B Biomed Competing interests Appl in press. None declared. 15. Cohen PT, Browne GJ, Delibegovic M, Munro S: Assay of protein phosphatase 1 complexes. Methods Enzymol 2003, 366:135-144. 16. Hall-Jackson CA, Eyers PA, Cohen P, Goedert M, Boyle FT, Hewitt N, Authors' contributions Plant H, Hedge P: Paradoxical activation of Raf by a novel Raf BWK was primarily involved and responsible for work inhibitor. Chem Biol 1999, 6:559-568. 17. Huser M, Luckett J, Chiloeches A, Mercer K, Iwobi M, Giblett S, Sun related to histology and immunohistochemistry. RG car- XM, Brown J, Marais R, Pritchard C: MEK kinase activity is not ried out or supervised all work related to the ELISA and necessary for Raf-1 function. Embo J 2001, 20:1940-1951. the drug administration. URR conceived the study and 18. Kolch W, Heidecker G, Lloyd P, Rapp UR: Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature 1991, participated in its design and coordination. All authors 349:426-428. were involved in drafting the manuscript and all approved 19. Bruder JT, Heidecker G, Rapp UR: Serum-, TPA-, and Ras- the final manuscript. induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev 1992, 6:545-556. 20. Troppmair J, Bruder JT, Munoz H, Lloyd PA, Kyriakis J, Banerjee P, Acknowledgements Avruch J, Rapp UR: Mitogen-activated protein kinase/extracel- We thank Ralf Schreck for assistance in preparation of the manuscript, lular signal-regulated protein kinase activation by onco- genes, serum, and 12-O-tetradecanoylphorbol-13-acetate Tamara Potapenko for expert help with the mouse work and Ludmilla Wix- requires Raf and is necessary for transformation. J Biol Chem ler and Renate Metz for Phospho-ERK assays. Furthermore, we thank 1994, 269:7030-7035. Christian Sirrenberg (Merck KGaA, Darmstadt) for the collaboration and 21. Sebolt-Leopold JS, Dudley DT, Herrera R, Van Becelaere K, Wiland his support. Work was funded by the DFG (SFB487) and the Mildred Scheel A, Gowan RC, Tecle H, Barrett SD, Bridges A, Przybranowski S, Leopold WR, Saltiel AR: Blockade of the MAP kinase pathway Foundation. suppresses growth of colon tumors in vivo. Nat Med 1999, 5:810-816. References 22. Collisson EA, De A, Suzuki H, Gambhir SS, Kolodney MS: Treat- 1. Weinstein IB: Cancer. Addiction to oncogenes--the Achilles ment of metastatic melanoma with an orally available inhib- heal of cancer. Science 2002, 297:63-64. itor of the Ras-Raf-MAPK cascade. Cancer Res 2003, 2. Shawver LK, Slamon D, Ullrich A: Smart drugs: tyrosine kinase 63:5669-5673. inhibitors in cancer therapy. Cancer Cell 2002, 1:117-123. 23. Haupt Y, Alexander WS, Barri G, Klinken SP, Adams JM: Novel zinc 3. Herrera R, Sebolt-Leopold JS: Unraveling the complexities of finger gene implicated as myc collaborator by retrovirally the Raf/MAP kinase pathway for pharmacological accelerated lymphomagenesis in E mu-myc transgenic mice. intervention. Trends Mol Med 2002, 8:S27-31. Cell 1991, 65:753-763. 4. Chang F, Steelman LS, Lee JT, Shelton JG, Navolanic PM, Blalock WL, 24. van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Gulden Franklin RA, McCubrey JA: Signal transduction mediated by the H, Berns A: Identification of cooperating oncogenes in E mu- Ras/Raf/MEK/ERK pathway from cytokine receptors to tran- myc transgenic mice by provirus tagging. Cell 1991, scription factors: potential targeting for therapeutic 65:737-752. intervention. Leukemia 2003, 17:1263-1293. 25. Bea S, Tort F, Pinyol M, Puig X, Hernandez L, Hernandez S, Fernandez 5. Kerkhoff E, Rapp UR: The Ras-Raf relationship: an unfinished PL, van Lohuizen M, Colomer D, Campo E: BMI-1 gene amplifica- puzzle. Adv Enzyme Regul 2001, 41:261-267. tion and overexpression in hematological malignancies 6. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, occur mainly in mantle cell lymphomas. Cancer Res 2001, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, 61:2409-2412. Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, 26. Vonlanthen S, Heighway J, Altermatt HJ, Gugger M, Kappeler A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake Borner MM, van Lohuizen M, Betticher DC: The bmi-1 oncopro- H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones tein is differentially expressed in non-small cell lung cancer K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri and correlates with INK4A-ARF locus expression. Br J Cancer G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, 2001, 84:1372-1376. Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA: Mutations of the BRAF Pre-publication history gene in human cancer. Nature 2002, 417:949-954. 7. Lyons JF, Wilhelm S, Hibner B, Bollag G: Discovery of a novel Raf The pre-publication history for this paper can be accessed kinase inhibitor. Endocr Relat Cancer 2001, 8:219-225. here: 8. Allen LF, Sebolt-Leopold J, Meyer MB: CI-1040 (PD184352), a tar- geted signal transduction inhibitor of MEK (MAPKK). Semin Oncol 2003, 30:105-116. http://www.biomedcentral.com/1471-2407/4/24/prepub 9. Rapp UR, Fensterle J, Albert S, Gotz R: Raf kinases in lung tumor development. Adv Enzyme Regul 2003, 43:183-195. 10. Kerkhoff E, Fedorov LM, Siefken R, Walter AO, Papadopoulos T, Rapp UR: Lung-targeted expression of the c-Raf-1 kinase in Publish with Bio Med Central and every transgenic mice exposes a novel oncogenic character of the scientist can read your work free of charge wild-type protein. Cell Growth Differ 2000, 11:185-190. 11. Fedorov LM, Tyrsin OY, Papadopoulos T, Camarero G, Gotz R, Rapp "BioMed Central will be the most significant development for UR: Bcl-2 determines susceptibility to induction of lung can- disseminating the results of biomedical researc h in our lifetime." cer by oncogenic CRaf. Cancer Res 2002, 62:6297-6303. Sir Paul Nurse, Cancer Research UK 12. Fedorov LM, Papadopoulos T, Tyrsin OY, Twardzik T, Gotz R, Rapp UR: Loss of p53 in craf-induced transgenic lung adenoma Your research papers will be: leads to tumor acceleration and phenotypic switch. Cancer Res available free of charge to the entire biomedical community 2003, 63:2268-2277. 13. Mallon R, Feldberg LR, Kim SC, Collins K, Wojciechowicz D, Hol- peer reviewed and published immediately upon acceptance lander I, Kovacs ED, Kohler C: An enzyme-linked immunosorb- cited in PubMed and archived on PubMed Central ent assay for the Raf/MEK1/MAPK signaling cascade. Anal Biochem 2001, 294:48-54. yours — you keep the copyright 14. Afify S, Rapp UR, Högger P: Validation of a simple chromatogra- BioMedcentral phy assay for the quantification of the Raf kinase inhibitor Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 6 of 6 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Cancer Springer Journals

Use of mitogenic cascade blockers for treatment of C-Raf induced lung adenoma in vivo: CI-1040 strongly reduces growth and improves lung structure

Kramer, Boris; Götz, Rudolf; Rapp, Ulf
BMC Cancer , Volume 4 (1) – Jun 1, 2004
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Copyright © 2004 by Kramer et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine general; Medicine/Public Health, general
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10.1186/1471-2407-4-24
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Abstract

Background: Signaling networks promoting cell growth and proliferation are frequently deregulated in cancer. Tumors often are highly dependent on such signaling pathways and may become hypersensitive to downregulation of key components within these signaling cascades. The classical mitogenic cascade transmits stimuli from growth factor receptors via Ras, Raf, MEK and ERK to the cell nucleus and provides attractive molecular targets for cancer treatment. For example, Ras and Raf kinase inhibitors are already in a number of ongoing phase II and phase III clinical trials. In this study the effect of the Raf kinase inhibitor BAY 43-9006 and of the MEK inhibitor CI-1040 (PD184352) on a Raf dependent lung tumor mouse model was analyzed in detail. Methods: We have generated a lung cancer mouse model by targeting constitutively active C-Raf kinase to the lung. These mice develop adenomas within 4 months of life. At this time-point they received daily intraperitoneal injections of either 100 mg/kg BAY 43-9006 or CI-1040 for additional 21 days. Thereafter, lungs were isolated and the following parameters were analyzed using histology and immunohistochemistry: overall lung structure, frequency of adenoma foci, proliferation rate, ERK activity, caspase-3 activation, and lung differentiation. Results: Both inhibitors were equally effective in vitro using a sensitive Raf/MEK/ERK ELISA. In vivo, the systemic administration of the MEK inhibitor CI-1040 reduced adenoma formation to a third and significantly restored lung structure. The proliferation rate of lung cells of mice treated with CL-1040 was decreased without any obvious effects on differentiation of pneumocytes. In contrast, the Raf inhibitor BAY 43-9006 did not influence adenoma formation in vivo. Conclusion: The MEK inhibitor CI-1040 may be used for the treatment of Ras and/or Raf- dependent human malignancies. Background tin for the treatment of advanced breast cancer and Oncogene-based therapeutics is a novel approach to Gleevec for chronic myelogenous leukemia have proven inhibit proteins, which are essential for the initiation and that cancer therapies targeting specific molecular altera- maintenance of malignancies [1]. Agents such as Hercep- tions in signaling pathways are successful [2]. The Ras- Page 1 of 6 (page number not for citation purposes) BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 MAP kinase pathway has a central role in regulating tumor 1040: daily intraperitoneal injections at a dose of 100 mg/ cell growth and survival, differentiation and angiogenesis kg from 4 months of age over a period of 21 days, n = 12 and has been targeted for therapeutic intervention in the each group). At this dose both drugs were well tolerated past [3,4]. C-Raf kinase and MEK are downstream effec- (data not shown). Serum concentration of BAY 43-9006 tors of the Ras signaling cascade. Both kinases are essential was determined by liquid-liquid extraction and consecu- for cellular homeostasis and induce both proliferation tive HPLC analysis [14]. Lungs were isolated and analyzed and survival by suppression of apoptosis [5]. Raf and Ras at the end of the treatment period. Animals were com- mutations found in human malignancies convey constitu- pared to non-treated or placebo-treated transgenic mice of tive activity to these signaling molecules thereby convert- the same litter. Placebo treatment did not cause any ing them into an oncogenic state [6]. changes in comparison to untreated mice (data not shown). In this study the Raf inhibitor BAY 43-9006 [7] and the MEK inhibitor CI-1040 [8] were tested as potential drugs Histology and immunohistochemistry in a transgenic mouse lung cancer model [9]. We have pre- Lungs were fixed under 25 cm water pressure with forma- viously established this mouse model by expressing lin. Histology was done on formalin-fixed, paraffin- mutated, constitutively active C-Raf kinase (C-Raf BxB) embedded lung specimen. 4 µm-cut sections were depar- under the control of the human surfactant protein C (SP- affinized, rehydrated in graded alcohols and hematoxylin C) promoter [10]. C-Raf BxB lacks the regulatory NH -ter- and eosin stained. Antigen retrieval was performed with minal sequences including the Ras interaction domain. citrate buffer, pH 6.0, and microwave treatment. Endog- Lung targeted expression of constitutively active C-Raf enous peroxidase activity was blocked by incubation with induced lung adenomas within 4 months of life [10]. 3% methanol. Unspecific binding was blocked by normal Although these adenomas are stable for more than one goat serum. The primary antibody for p-ERK (Cell signal- year, deficiencies in other genes such as Bcl-2 or p53 were ing 9101), PCNA (DAKO M 0879), for Ki-67 (DAKO M found to modulate adenoma growth or even switch the 7249) and Bmi-1 (Santa Cruz sc-10745) were detected by phenotype of tumor cells, respectively [11,12]. The effects a peroxidase labelled secondary antibody (DAKO P0450). of BAY 43-9006 and CI-1040 were assessed in this lung Corresponding secondary antibodies were used to detect adenoma model by daily intraperitoneal injections of activated caspase-3 primary antibody (Cell signaling these drugs at concentrations of 100 mg per kg body 9664) and pro-SP-C antibody (generous gift of Dr. Jeffrey weight over a period of three weeks to four months old C- A. Whitsett, Cincinnati Children's Hospital Medical Raf-BxB mice. Center, OH/USA). Staining was performed using diami- nobenzidine and hematoxylin counterstaining. Slides were analyzed in a blinded fashion. Random pictures Methods ELISA were taken of each specimen. Adenoma foci were counted Enzyme-linked immunosorbent assay for the MEK signal- per mm . Positive cells in adenoma foci were counted and ing cascade was done as previously described [13]. In expressed as percentage of all adenoma cells. brief, plates were coated with an anti ERK-antibody (Santa Cruz, sc-94). A kinase reaction was performed in the pres- Results ence of ERK, Raf, MEK and the respective inhibitor. Phos- BAY 43-9006 and CI-1040 are equally effective in vitro but differ in their in vivo properties phorylated ERK was detected by a sandwich technique using a secondary antibody (BioLabs, #9106L) and a per- To determine whether the inhibitors used in our study are oxidase-linked species-specific tertiary antibody (Amer- active we performed at first an in vitro assay. The activity sham Pharmacia Biotech, #NA 931). Similar results were of BAY 43-9006 and CI-1040 was measured using an obtained with constitutively active C-Raf BxB (data not ELISA method that detects phosphorylation of ERK that is shown). dependent on Raf and MEK activity [13]. Both substances were equally effective in vitro with complete inhibition of Animals ERK phosphorylation at concentrations of less than 1 µM Lung targeted expression of constitutively active C-Raf (Fig. 1a). The IC values for inhibition of C-Raf by BAY (SP-C C-Raf BxB), lacking the regulatory N-terminal 43-9006 and by CI-1040 in our assays were 16 nM and 12 sequences, which comprise the Ras interaction domain, nM, respectively. In addition, the effect of both inhibitors induced lung adenomas within 4 months of life [10]. The was also tested on cell lines. At a concentration of 0,01 µM adenomas were indistinguishable from those induced by the inhibitory effect of CI-1040 on ERK phosphorylation expression of wild type C-Raf (data not shown). Litters of was more than 90 %, whereas BAY 43-9006 at 4 µM lead the transgenic mice were randomly assigned to the control to an inhibition in the range between 60 – 80 % as deter- groups (no treatment, n = 11, respectively placebo treat- mined by Western blotting and immunohistochemistry ment, n = 12) and to the study group (BAY 43-9006 or CI- (data not shown). Page 2 of 6 (page number not for citation purposes) BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 BAY 43-9006 BAY 43-9006 P<0.05 A CI-1040 P<0.05 CI-1040 0 0 -3 -2 -1 0 1 2 10 10 10 10 10 10 BAY 43-9006 CI-1040 Placebo µM E F CI-1040 Placebo BAY 43-9006 MEK inh Figure 1 ibitor CI-1040 but not Raf inhibitor BAY 43-9006 reduces lung adenomas in vivo MEK inhibitor CI-1040 but not Raf inhibitor BAY 43-9006 reduces lung adenomas in vivo. (A) MEK activity was completely inhibited in vitro by BAY 43-9006 and CI-1040 as determined by ERK activation enzyme-linked immunosorbent assay. IC50 values for BAY 43-9006 and CI-1040 were 16 nM and 12 nM respectively. (B, C) The amount of P-ERK and the number of P-ERK positive cells were reduced in vivo by treatment with CI-1040 but not with BAY 43-9006. (D, E, F) Treat- ment with CI-1040 but not with BAY 43-9006 reduced adenomas and improved lung structure after 21d (hematoxylin and eosin staining; scale bars = 60 µm). Lung adenomas as well as the overall lung morphology was indistinguishable in placebo treated and untreated animals (data not shown). At next, the in vivo effects of a systemic administration of phosphorylated ERK was clearly reduced as determined by the inhibitors were analyzed using our Raf dependent immunohistochemistry (Fig. 1b) or Western blotting lung tumor mice. Comparable serum concentrations of (data not shown). The number of cells that were stained both inhibitors were reached after intraperitoneal injec- by the Phospho-ERK antibody was reduced almost three- tion as determined by liquid-liquid extraction of mouse fold (Fig. 1c). In contrast, phosphorylation of ERK in lung serum and consecutive HPLC analysis [14] and Sirrenberg adenomas in vivo was virtually not affected by systemic unpublished data). Lung sections of transgenic mice that treatment of mice with BAY 43-9006 (Fig. 1b,1c) or in have been treated for three weeks with either CI-1040 or controls (data not shown). Moreover, treatment with CI- BAY 43-9006 were examined for the amount of phospho- 1040 did not only reduce adenomas but also improve the rylated ERK. After treatment with CI-1040 the amount of overall lung structure with thin alveolar walls, whereas Page 3 of 6 (page number not for citation purposes) Percentage Inhibition P-ERKpositive Cells% BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 BAY 43-9006 again has no detectable effect (Fig. not be useful as anticancer drugs because inhibition of Raf 1d,1e,1f). is always counterbalanced by reactivation [16]. Finally, it was recently suggested that C-Raf has signaling properties The number of adenoma foci per mm was determined that are independent of its protein kinase activity [17]. and found to be reduced more than 75 % after CI-1040 Therefore, it has also to be considered that BAY 43-9006- treatment in comparison to BAY 43-9006 treated or pla- bound kinase-inactive C-Raf has still transforming activity cebo treated animals (Fig. 2a). This treatment results in a in pneumocyte type II cells. But this possibility appears reduced ratio of lung to body weight of lungs obtained unlikely in the light of earlier results demonstrating the from mice after CI-1040 treatment (7.5 ± 0.3 mg/g) in loss of transforming activity and concomitant gain of comparison to lungs isolated from mice after BAY 43- dominant-negative function of c-Raf genes with muta- 9006 treatment (10.4 ± 0.6 mg/g, P < 0.05). tions that inactivate the protein kinase activity of C-Raf [18-20]. Moreover, there is a direct correlation between CI-1040 reduces proliferation in lung adenomas but has no reversal of a transformed phenotype by BAY43-9006 and influence on apoptosis or lung specific differentiation Phospho-ERK suppression (Sirrenberg and Rapp, unpub- The adenoma foci were further analyzed for proliferation, lished data). apoptosis and differentiation to decipher the mechanism of action by which CI-1040 reduced adenomas. We Previous work on CI-1040 has already established that assessed proliferation by immunohistochemistry for pro- this drug has a cytostatic effect on subcutaneously liferating cellular antigen (PCNA), Ki-67 and Bmi-1. The implanted colon tumors of human and mouse origin har- percentage of PCNA positive cells was clearly reduced by boring RAS mutations [21]. In addition, in a mouse half after CI-1040 treatment (Fig. 2b). The percentage of model of metastatic melanoma that was dependent on a Ki-67 and Bmi-1 positive cells was reduced to a third after B-Raf mutation CI-1040 was preventing the formation of treatment with CI-1040 (Fig. 2c,2d). Apoptotic cells were new pulmonary metastases and caused rapid regression of detected by immunohistochemistry for activated caspase- already established pulmonary metastases [22]. Both pub- 3 and no difference was detected after BAY 43-9006 or CI- lications as well as our report indicate that CI-1040 has a 1040 treatment (Fig. 2e). Adenoma cells were analyzed broad spectrum of antitumor activity and will be useful for the expression of pro-SP-C as a marker of alveolar type for the treatment of cancers that are dependent on muta- II cell differentiation. The percentage of pro-SP-C positive tions or overexpression of members of the mitogenic cas- cells did not change after BAY 43-9006 or CI-1040 treat- cade upstream of ERK. ment (Fig. 2f). We also determined the effects of both inhibitors on expression of Bmi-1. The number of Bmi-1 positive cells Discussion We were able to show inhibition of ERK phosphorylation in lung sections was reduced more than twofold by CI- in vitro by both, the MEK inhibitor CI-1040 and the Raf 1040, whereas BAY 43-9006 had no effect. The observed inhibitor BAY 43-9600. In addition, both drugs inhibited effects of CI-1040 may be limited to proliferation as nei- ERK phosphorylation to a comparable level when tested ther apoptosis nor alveolar differentiation was directly on cell lines in tissue culture (data not shown). Although affected. Bmi-1 was originally cloned as c-Myc cooperat- both inhibitors reached comparable serum concentra- ing oncogene in murine lymphomas [23,24]. A role for tions after intraperitoneal administration (data not Bmi-1 in human tumorigenesis was recently suggested by shown), the MEK inhibitor was active in mice whereas the the discovery of Bmi-1 amplification in mantle cell lym- Raf inhibitor was not. Why BAY 43-9006 is not active in phomas [25] and of Bmi-1 overexpression in a variety of our Raf dependent lung tumor mouse model and whether tumors including non-small cell lung cancer [26]. Since Raf and MEK might fulfill different functional roles with CI-1040 might be more effective in inhibiting the prolif- respect to lung adenoma formation needs to be further eration of Bmi-1 positive adenoma cells, we want to addressed in detail in future. address in future whether Bmi-1 is a self-renewal factor of lung stem cells or lung adenoma stem cells. One possible explanation for the observed effects may relate to differences in the in vivo accessibility of these Conclusions inhibitors to sub-cellular sites where C-Raf is localized. These experiments demonstrate an essential role of MEK/ Alternatively, inhibition of Raf kinases might be more dif- ERK signaling for the maintenance of Raf induced trans- ficult to achieve due to multiple feedback mechanisms formation in vivo. Effects on the ERK pathway in vivo may [15], whereas the regulation of MEK is less complex and be predictive for effective tumor drug design and therapy inhibition of MEK is easier achieved in the living organ- of Raf driven tumors. CI-1040 is currently evaluated in ism. Along this line it was already discussed previously phase II trials and may be a promising anti-proliferative that inhibitors targeting the kinase activity of Raf might drug for Ras and/or Raf-dependent human malignancies. Page 4 of 6 (page number not for citation purposes) BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 7 P<0.05 BAY 43-9006 CI-1040 Placebo BAY 43-9006 CI-1040 Placebo MEK Figure 2 inhibitor CI-1040 reduces proliferation in lung adenomas but has no influence on apoptosis or lung specific differentiation MEK inhibitor CI-1040 reduces proliferation in lung adenomas but has no influence on apoptosis or lung spe- cific differentiation. (A) The number of adenoma foci per mm was reduced after treatment with CI-1040 but not with BAY 43-9006. (B) CI-1040 reduced the PCNA positive cells in lung adenomas by half but not BAY 43-9006. (C, D) Ki-67 and Bmi-1 positive cells were reduced to a third in lung adenomas after CI-1040 treatment. (E) Neither BAY 43-9006 nor CI-1040 acti- vated the caspase-3 mediated apoptotic pathway in lung adenomas. (F) BAY 43-9006 or CI-1040 treatment did not change cell differentiation in lung adenomas, which was assessed by expression of pro-SP-C, a marker for alveolar type II cells. Page 5 of 6 (page number not for citation purposes) PCNA positive Cells Ki-67 positive Cells Number of Adenoma Foci per mm Pro SP-C positive Cells Bmi-1 positive Cells Activ. Caspase 3 positive Cells ‰ BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/24 BAY 43-9006 in small samples of serum. J Chromatogr B Biomed Competing interests Appl in press. None declared. 15. Cohen PT, Browne GJ, Delibegovic M, Munro S: Assay of protein phosphatase 1 complexes. Methods Enzymol 2003, 366:135-144. 16. Hall-Jackson CA, Eyers PA, Cohen P, Goedert M, Boyle FT, Hewitt N, Authors' contributions Plant H, Hedge P: Paradoxical activation of Raf by a novel Raf BWK was primarily involved and responsible for work inhibitor. Chem Biol 1999, 6:559-568. 17. Huser M, Luckett J, Chiloeches A, Mercer K, Iwobi M, Giblett S, Sun related to histology and immunohistochemistry. RG car- XM, Brown J, Marais R, Pritchard C: MEK kinase activity is not ried out or supervised all work related to the ELISA and necessary for Raf-1 function. Embo J 2001, 20:1940-1951. the drug administration. URR conceived the study and 18. Kolch W, Heidecker G, Lloyd P, Rapp UR: Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature 1991, participated in its design and coordination. All authors 349:426-428. were involved in drafting the manuscript and all approved 19. Bruder JT, Heidecker G, Rapp UR: Serum-, TPA-, and Ras- the final manuscript. induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev 1992, 6:545-556. 20. Troppmair J, Bruder JT, Munoz H, Lloyd PA, Kyriakis J, Banerjee P, Acknowledgements Avruch J, Rapp UR: Mitogen-activated protein kinase/extracel- We thank Ralf Schreck for assistance in preparation of the manuscript, lular signal-regulated protein kinase activation by onco- genes, serum, and 12-O-tetradecanoylphorbol-13-acetate Tamara Potapenko for expert help with the mouse work and Ludmilla Wix- requires Raf and is necessary for transformation. J Biol Chem ler and Renate Metz for Phospho-ERK assays. Furthermore, we thank 1994, 269:7030-7035. Christian Sirrenberg (Merck KGaA, Darmstadt) for the collaboration and 21. Sebolt-Leopold JS, Dudley DT, Herrera R, Van Becelaere K, Wiland his support. 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Fedorov LM, Tyrsin OY, Papadopoulos T, Camarero G, Gotz R, Rapp "BioMed Central will be the most significant development for UR: Bcl-2 determines susceptibility to induction of lung can- disseminating the results of biomedical researc h in our lifetime." cer by oncogenic CRaf. Cancer Res 2002, 62:6297-6303. Sir Paul Nurse, Cancer Research UK 12. Fedorov LM, Papadopoulos T, Tyrsin OY, Twardzik T, Gotz R, Rapp UR: Loss of p53 in craf-induced transgenic lung adenoma Your research papers will be: leads to tumor acceleration and phenotypic switch. Cancer Res available free of charge to the entire biomedical community 2003, 63:2268-2277. 13. Mallon R, Feldberg LR, Kim SC, Collins K, Wojciechowicz D, Hol- peer reviewed and published immediately upon acceptance lander I, Kovacs ED, Kohler C: An enzyme-linked immunosorb- cited in PubMed and archived on PubMed Central ent assay for the Raf/MEK1/MAPK signaling cascade. Anal Biochem 2001, 294:48-54. yours — you keep the copyright 14. Afify S, Rapp UR, Högger P: Validation of a simple chromatogra- BioMedcentral phy assay for the quantification of the Raf kinase inhibitor Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 6 of 6 (page number not for citation purposes)

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Published: Jun 1, 2004

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