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Oncogenic KRAS Activates Hedgehog Signaling Pathway in Pancreatic Cancer Cells

2007; Elsevier BV; Volume: 282; Issue: 19 Linguagem: Inglês

10.1074/jbc.m611089200

ISSN

1083-351X

Autores

Zhenyu Ji, Fang Mei, Jingwu Xie, Xiaodong Cheng,

Tópico(s)

Epigenetics and DNA Methylation

Resumo

Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAFE600, but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression. Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAFE600, but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression. Pancreatic ductal adenocarcinoma (PDA) 2The abbreviations used are: PDA, pancreatic ductal adenocarcinoma; FTS, S-trans,transfarnesylthiosalicylic acid; GLI, glioblastoma gene product; Hh, hedgehog; HPDE, human pancreatic ductal epithelium; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; Shh, Sonic hedgehog; SMO, smoothened gene product; siRNA, small interfering RNA. is the fourth leading cause of cancer-related death for both men and women in the United States. It was estimated that in 2006 33,730 new cases would be diagnosed, and 32,300 would die from the disease (American Cancer Society Cancer Facts and Figures 2006). Therefore, PDA is one of the most lethal human diseases, with a 5-year survival rate of less than 4% and a median survival of less than 6 months. PDA is one of the better-characterized neoplasms at the genetic level. There are now sufficient clinical, genetic, and pathological data to support a tumor progression model for PDA in which the pancreatic ductal epithelium progresses from normal to increased grades of pancreatic intraepithelial neoplasia to invasive cancer (1Hruban R.H. Goggins M. Parsons J. Kern S.E. Clin. Cancer Res. 2000; 6: 2969-2972PubMed Google Scholar, 2Klein W.M. Hruban R.H. Klein-Szanto A.J. Wilentz R.E. Mod. Pathol. 2002; 15: 441-447Crossref PubMed Scopus (105) Google Scholar). Accompanying the progressive morphological changes is the sequential accumulation of genetic alterations in the KRAS oncogene and the tumor suppressors INK4A, p53, and SMAD4/DPC4, although these alterations have not been linked to the acquisition of specific histopathological attributes (3Moskaluk C.A. Hruban R.H. Kern S.E. Cancer Res. 1997; 57: 2140-2143PubMed Google Scholar, 4Yamano M. Fujii H. Takagaki T. Kadowaki N. Watanabe H. Shirai T. Am. J. 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Cancer Res. 1993; 53: 2704-2707PubMed Google Scholar, 9Cheng J.Q. Ruggeri B. Klein W.M. Sonoda G. Altomare D.A. Watson D.K. Testa J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3636-3641Crossref PubMed Scopus (700) Google Scholar, 10Ruggeri B.A. Huang L. Wood M. Cheng J.Q. Testa J.R. Mol. Carcinog. 1998; 21: 81-86Crossref PubMed Scopus (288) Google Scholar, 11Sahin F. Maitra A. Argani P. Sato N. Maehara N. Montgomery E. Goggins M. Hruban R.H. Su G.H. Mod. Pathol. 2003; 16: 686-691Crossref PubMed Scopus (96) Google Scholar). Aberrant RAS activation plays a critical role in tumorigenesis; activating RAS mutations are found in 30% of all human cancers (12Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar). Of all human cancers, PDA has the highest incidence of activating KRAS mutations (13Almoguera C. Shibata D. Forrester K. Martin J. Arnheim N. Perucho M. Cell. 1988; 53: 549-554Abstract Full Text PDF PubMed Scopus (1904) Google Scholar). Activating KRAS mutations, representing the earliest genetic changes associated with the transformation of normal ductal epithelium and PDA development, have been detected in pancreatic duct lesions with minimal cytological and architectural atypia and occasionally in histologically normal pancreas (3Moskaluk C.A. Hruban R.H. Kern S.E. Cancer Res. 1997; 57: 2140-2143PubMed Google Scholar, 14Klimstra D.S. Longnecker D.S. Am. J. Pathol. 1994; 145: 1547-1550PubMed Google Scholar, 15Luttges J. Schlehe B. Menke M.A. Vogel I. Henne-Bruns D. Kloppel G. Cancer. 1999; 85: 1703-1710Crossref PubMed Scopus (154) Google Scholar, 16Caldas C. Hahn S.A. Hruban R.H. Redston M.S. Yeo C.J. Kern S.E. Cancer Res. 1994; 54: 3568-3573PubMed Google Scholar, 17Tada M. Ohashi M. Shiratori Y. Okudaira T. Komatsu Y. Kawabe T. Yoshida H. Machinami R. Kishi K. Omata M. Gastroenterology. 1996; 110: 227-231Abstract Full Text PDF PubMed Scopus (285) Google Scholar). The frequency of KRAS mutations correlates with disease progression, reaching almost 100% in pancreatic adenocarcinomas. Targeted endogenous expression of an oncogenic KRAS allele in the mouse pancreas is sufficient to drive the development of pancreatic intraepithelial neoplasia and subsequently at low frequency the progression to both locally invasive adenocarcinoma and metastatic disease with sites of spread exactly as found in human pancreatic cancer (18Aguirre A.J. Bardeesy N. Sinha M. Lopez L. Tuveson D.A. Horner J. Redston M.S. DePinho R.A. Genes Dev. 2003; 17: 3112-3126Crossref PubMed Scopus (806) Google Scholar, 19Hingorani S.R. Petricoin E.F. Maitra A. Rajapakse V. King C. Jacobetz M.A. Ross S. Conrads T.P. Veenstra T.D. Hitt B.A. Kawaguchi Y. Johann D. Liotta L.A. Crawford H.C. Putt M.E. Jacks T. Wright C.V. Hruban R.H. Lowy A.M. Tuveson D.A. Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1806) Google Scholar, 20Hingorani S.R. Wang L. Multani A.S. Combs C. Deramaudt T.B. Hruban R.H. Rustgi A.K. Chang S. Tuveson D.A. Cancer Cell. 2005; 7: 469-483Abstract Full Text Full Text PDF PubMed Scopus (1705) Google Scholar). These observations suggest that KRAS plays an essential role in the initiation, development, and maintenance of PDA. Recently, the hedgehog (Hh) signaling pathway has been implicated as playing an important role in the progression and maintenance of PDA (21Thayer S.P. di Magliano M.P. Heiser P.W. Nielsen C.M. Roberts D.J. Lauwers G.Y. Qi Y.P. Gysin S. Fernandez-del Castillo C. Yajnik V. Antoniu B. McMahon M. Warshaw A.L. Hebrok M. Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar, 22Berman D.M. Karhadkar S.S. Maitra A. Montes D.O. Gerstenblith M.R. Briggs K. Parker A.R. Shimada Y. Eshleman J.R. Watkins D.N. Beachy P.A. Nature. 2003; 425: 846-851Crossref PubMed Scopus (1129) Google Scholar, 23Kayed H. Kleeff J. Keleg S. Guo J. Ketterer K. Berberat P.O. Giese N. Esposito I. Giese T. Buchler M.W. Friess H. Int. J. Cancer. 2004; 110: 668-676Crossref PubMed Scopus (98) Google Scholar). Hh signaling is essential for morphogenesis, tissue patterning, and stem cell maintenance in metazoan embryos (24Ingham P.W. McMahon A.P. Genes Dev. 2001; 15: 3059-3087Crossref PubMed Scopus (2489) Google Scholar). Hh binds to its membrane receptor Patched (PTC), releasing PTC inhibition of a seven-transmembrane protein, Smoothened (SMO), which in turn activates downstream cytoplasmic transcription factors; that is, the CI protein in Drosophila or the mammalian homologue GLI proteins (25Lum L. Beachy P.A. Science. 2004; 304: 1755-1759Crossref PubMed Scopus (695) Google Scholar). Components of the Hh signaling pathway, including the ligand and the receptors, are overexpressed in human PDA tissues and cell lines. Suppressing Hh activity using cyclopamine, a steroidal alkaloid that inhibits Hh signaling through direct interaction with SMO (26Chen J.K. Taipale J. Cooper M.K. Beachy P.A. Genes Dev. 2002; 16: 2743-2748Crossref PubMed Scopus (1224) Google Scholar), in some PDA cells with activated Hh signaling, can inhibit cell growth in vitro and reduce tumor growth in vivo in the xenograft and orthotopic mouse model (21Thayer S.P. di Magliano M.P. Heiser P.W. Nielsen C.M. Roberts D.J. Lauwers G.Y. Qi Y.P. Gysin S. Fernandez-del Castillo C. Yajnik V. Antoniu B. McMahon M. Warshaw A.L. Hebrok M. Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar, 22Berman D.M. Karhadkar S.S. Maitra A. Montes D.O. Gerstenblith M.R. Briggs K. Parker A.R. Shimada Y. Eshleman J.R. Watkins D.N. Beachy P.A. Nature. 2003; 425: 846-851Crossref PubMed Scopus (1129) Google Scholar, 23Kayed H. Kleeff J. Keleg S. Guo J. Ketterer K. Berberat P.O. Giese N. Esposito I. Giese T. Buchler M.W. Friess H. Int. J. Cancer. 2004; 110: 668-676Crossref PubMed Scopus (98) Google Scholar, 27Feldmann G. Dhara S. Fendrich V. Bedja D. Beaty R. Mullendore M. Karikari C. Alvarez H. Iacobuzio-Donahue C. Jimeno A. Gabrielson K.L. Matsui W. Maitra A. Cancer Res. 2007; 67: 2187-2196Crossref PubMed Scopus (613) Google Scholar). The coincidence of uncontrolled activation of the RAS and Hh pathways in the early stages of PDA suggests that cross-talk between these two pathways may be a very important mechanism for the initiation and development of PDA. However, the causal effects between KRAS and Hh signaling in pancreatic tumorigenesis are not clear. Earlier results from Pdx-Shh mice had suggested that ectopic expression of Hedgehog ligands is sufficient to activate the Ras signaling pathway by inducing a mutation in the Kras gene (21Thayer S.P. di Magliano M.P. Heiser P.W. Nielsen C.M. Roberts D.J. Lauwers G.Y. Qi Y.P. Gysin S. Fernandez-del Castillo C. Yajnik V. Antoniu B. McMahon M. Warshaw A.L. Hebrok M. Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar), and a recent study indicates that cell-autonomous activation of the Hedgehog pathway is not sufficient to induce mutations in the Kras gene or to activate MAPK downstream of Ras (28Pasca di Magliano M. Sekine S. Ermilov A. Ferris J. Dlugosz A.A. Hebrok M. Genes Dev. 2006; 20: 3161-3173Crossref PubMed Scopus (260) Google Scholar). In addition, although expression of endogenous level of oncogenic Kras, KrasG12D, leads to pancreatic intraepithelial neoplasia identical to all three stages found in the cognate human condition and eventually PDA in mice (19Hingorani S.R. Petricoin E.F. Maitra A. Rajapakse V. King C. Jacobetz M.A. Ross S. Conrads T.P. Veenstra T.D. Hitt B.A. Kawaguchi Y. Johann D. Liotta L.A. Crawford H.C. Putt M.E. Jacks T. Wright C.V. Hruban R.H. Lowy A.M. Tuveson D.A. Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1806) Google Scholar), activation of Hh signaling alone is not sufficient to induce pancreatic intraepithelial neoplasia and PDA in a mouse model in which Hh signaling is activated specifically in the pancreatic epithelium (28Pasca di Magliano M. Sekine S. Ermilov A. Ferris J. Dlugosz A.A. Hebrok M. Genes Dev. 2006; 20: 3161-3173Crossref PubMed Scopus (260) Google Scholar). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in pancreatic adenocarcinomas, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh signaling pathway in PDA. Our study shows that oncogenic transformation of human pancreatic ductal epithelial (HPDE) cells by oncogenic KRAS is indeed accompanied by enhanced GLI activation and that specific down-regulation of oncogenic KRAS activity inhibits Hh signaling in PDA cell lines with KRAS mutations. These results demonstrate that oncogenic KRAS is involved in activation of the Hh/GLI pathway in PDA cells and that cross-talk between the oncogenic KRAS and Hh pathways may play an important role in promoting cancer development during pancreatic tumorigenesis. Reagents—Wortmannin, PD98059, U0126, and MG132 were purchased from Calbiochem. S-Trans,transfarnesylthiosalicylic acid (FTS) was provided by Dr. Victor J. Bauer (Concordia Pharmaceuticals). Cyclopamine and tomatidine were purchased from Toronto Research Chemicals (North York, Ontario, Canada). Cycloheximide was from Sigma. Mouse anti-RAS antibodies, rabbit anti-MAPK, anti-phospho-MAPK, anti-AKT, anti-phospho-AKT, anti-phospho-glycogen synthase kinase 3β, and anti-Myc antibodies were obtained from Cell Signaling Inc. (Beverly, MA). Mouse anti-KRAS antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-α-tubulin was from Molecular Probe (Eugene, OR). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were from Jackson Immunolabs (West Grove, PA). KRASD12-specific siRNA duplex and control siRNA were obtained from Dharmacon RNA Technologies (Lafayette, CO). Gli luciferase reporter constructs including 8 × 3′Gli BSwt-luc and 8 × 3′Gli BSmut-luc were described previously (29Sasaki H. Hui C. Nakafuku M. Kondoh H. Development. 1997; 124: 1313-1322Crossref PubMed Google Scholar). Plasmids for expression of Renilla luciferase (pRL-SV40-luc) were from Promega (Madison, WI). pCS2-MT and pCS2-MT-Gli3C′ΔClaI were provided by Dr. Altaba (30Altaba A. Development. 1999; 126: 3205-3216Crossref PubMed Google Scholar). pBabe-BRAFE600 was obtained from Dr. Daniel Peeper (31Michaloglou C. Vredeveld L.C. Soengas M.S. Denoyelle C. Kuilman T. van der Horst C.M. Majoor D.M. Shay J.W. Mooi W.J. Peeper D.S. Nature. 2005; 436: 720-724Crossref PubMed Scopus (1688) Google Scholar). Cell Culture and Transfection—HPDE-c7, an immortalized pancreatic ductal epithelial cell line, was provided by Dr. Ming-Sound Tsao (University of Toronto, Canada) and cultured in keratinocyte serum-free (KSF) medium supplemented with bovine pituitary extract and epidermal growth factor (Invitrogen). KRASV12-transformed HPDE-c7 cells were produced by retroviral infection of HPDE-c7 cells with a KRASV12 construct. Panc-1 and AsPC-1 cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium (AsPC-1) or Dulbecco's modified Essential medium (Panc-1) supplemented with 10% fetal bovine serum (Invitrogen). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions (the ratio of plasmid (μg) to lipid (μl) was 1:3). Retroviral Infection—Retroviruses were generated by transfecting amphotropic Phoenix packaging cells with the retroviral vector pBabepuro-KRAS4BG12V and the corresponding empty vector pBabepuro using Lipofectamine Plus reagent (Invitrogen). Retroviral supernatants were collected, filtered, and incubated with the target cells in the presence of 4 μg/ml Polybrene (Sigma). After 48 h, cells were subjected to selection using 0.5 μg/ml puromycin (ICN Biomedicals, Irvine, CA) until all the untransduced cells had died. RAS Activity Assay—The GTP loading status of RAS was assessed using a glutathione S-transferase (GST) fusion of the RAS binding domain (RBD) of RAF (GST-RAF-RBD) as described earlier (32Young T.W. Mei F.C. Yang G. Thompson-Lanza J.A. Liu J. Cheng X. Cancer Res. 2004; 64: 4577-4584Crossref PubMed Scopus (107) Google Scholar). Anchorage-independent Cell Growth Assay—Cells (1 × 103–5 × 105) were suspended in 2 ml of HPDE cell medium with 0.35% agarose (Invitrogen), and the suspension was placed on top of 5 ml of solidified 0.7% agarose. Triplicate cultures at three different dilutions for each cell type were maintained at 37 °C in a 5% CO2 atmosphere, and fresh medium was added after 1 week. Colonies were photographed between 14 and 24 days under a phase contrast microscope. The number of colonies was counted from each experiment, and the experiment was reproduced twice. Student's t test for two samples was used to determine the statistical significance between the two groups. A p value less than 0.05 was regarded as statistically significant. Immunoblotting Analysis—Protein concentration of cell lysates was assayed with the Bio-Rad protein assay reagent. Equal amounts of protein (5–30 μg) were loaded onto 10% SDS-polyacrylamide mini-gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes after electrophoresis. After being blocked overnight in 5% milk in Tris-buffered saline-Tween, blots were incubated with primary antibodies for 1.5 h followed by horseradish peroxidase-conjugated secondary antibody (1:4000) for 45 min. Antigen-antibody complexes were detected by enhanced chemiluminescence. Luciferase Reporter Gene Assay—For luciferase assay, cells were transfected with the Gli or other luciferase reporter construct (0.5 μg/well) and the SV40-Renilla control plasmid (2 ng/well) using Lipofectamine 2000. After 6 h the medium was replaced with fresh growth medium, and the cells were incubated in 5% CO2 at 37 °C overnight. After treatment with U0126 or other compounds, the cells were harvested, and luciferase activity was measured with the dual luciferase reporter assay system (Promega) according to the manufacturer's instruction. Briefly, the transfected cells were lysed in the 12-well plates with 100 μl of reporter lysis buffer, and the lysate was transferred into Eppendorf tubes. Cell debris was removed by centrifugation at top speed for 30 s in a refrigerated microcentrifuge. 20 μl of the supernatant was mixed with 100 μl of LAR II buffer, and the luminescence was immediately measured (first reading). After 20 s, 100 μl of Stop & Glo® reagent was added to measure the Renilla luciferase activity (second reading). The value from the first reading was divided by the value from the second reading of each sample to obtain normalized luciferase activity. Each experiment was repeated at least three times with similar results. Gli luciferase reporter constructs 8 × 3′Gli BSwt-luc and 8 × 3′Gli BSmut-luc were provided by Dr. H. Sasaki (29Sasaki H. Hui C. Nakafuku M. Kondoh H. Development. 1997; 124: 1313-1322Crossref PubMed Google Scholar). Quantitative Real-time PCR—Total RNA was isolated from cultured cells as previously described (33Mei F.C. Young T.W. Liu J. Cheng X. FASEB J. 2006; 20: 497-499Crossref PubMed Scopus (36) Google Scholar). Real-time PCR analysis was conducted using an Applied Biosystems Prism 7000 sequence detection system and TaqMan® gene expression assays for relative quantification of GLI1 (Hs00171790_ m1) and hedgehog-interacting protein (Hs00368450_m1) mRNA. Duplicate CT values were analyzed in Microsoft Excel using the comparative CT(ΔΔCT) method as described by the manufacturer (Applied Biosystems). The amount of target (2–ΔΔCT) was obtained by normalized to endogenous reference (18 S) and relative to a calibrator. Oncogenic Transformation of HPDE by KRASV12—To probe the mechanism of oncogene KRAS-mediated pancreatic tumorigenesis, we established a KRAS oncogene-based human PDA model using an immortalized primary HPDE cell line, HPDE-c7. This well characterized cell line is a near-diploid human pancreatic duct epithelial cell line originally derived from normal pancreas. Although immortalized by E6/E7 genes of human papilloma virus-16, HPDE-c7 is non-tumorigenic and incapable of inducing tumor growth in nude mice (34Furukawa T. Duguid W.P. Rosenberg L. Viallet J. Galloway D.A. Tsao M.S. Am. J. Pathol. 1996; 148: 1763-1770PubMed Google Scholar, 35Ouyang H. Mou L. Luk C. Liu N. Karaskova J. Squire J. Tsao M.S. Am. J. Pathol. 2000; 157: 1623-1631Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Stable expression of KRASV12 in HPDE-c7 cells using a retroviral expression vector led to transformation of the cell line. For all experiments performed in this study, we used early passages of the same puromycin-selected pool of KRASV12-transformed HPDE-c7 cells to eliminate potential clonal and passage variations. The resultant cells, HPDE-c7-KRASV12, expressed increased levels of total RAS protein, showed high RAS-GTP activity, and grew anchorage-independently in soft agar. In addition, expression of KRASV12 in HPDE-c7 cells also led to the activation of its downstream effectors, such as MAPK and AKT. The basal phospho-MAPK and phospho-AKT levels were enhanced in the HPDE-c7-KRASV12 cells compared with the parental cells. These observations are in complete agreement with results obtained from an independently established KRAS human PDA model using the same HPDE-c7 parental cell line (36Qian J. Niu J. Li M. Chiao P.J. Tsao M.S. Cancer Res. 2005; 65: 5045-5053Crossref PubMed Scopus (105) Google Scholar). Activation of Hh Signaling by KRASV12 in HPDE—To examine the roles of the activating KRAS oncogene in controlling Hh signaling during the oncogenic transformation of HPDE, we compared the activities of Hh signaling in parental, vector control, and KRASV12-expressing HPDE cells. As shown in Fig. 1A, KRASV12 expression led to a significantly increased activation of Gli-mediated luciferase activity, whereas the expression of the Myc reporter and a mutant Gli reporter that is incapable of binding GLI transcription factors were not affected. Moreover, the KRASV12-induced Gli-luciferase activity was insensitive to cyclopamine treatment (5 μm), whereas the same treatment resulted in significant inhibition of Gli-luciferase activity in an SMO-expressing 10T1/2 cell line (Fig. 1B). These results suggest that KRASV12 activates Hh signaling in HPDE cells in a ligand-independent manner. Further supporting our results based on the luciferase assay, oncogenic KRAS also up-regulated endogenous levels of GLI1 and hedgehog-interacting protein mRNAs, both of which are Hh target genes, in immortalized HPDE cells as measured by real-time PCR (Fig. 1C). To ensure that the apparent activation of Hh signaling observed in HPDE-c7-KRASV12 cells was caused specifically by activation of the KRAS pathway rather than an indirect effect associated with the transformation of HPDE, we suppressed the oncogenic KRAS activity in HPDE-c7-KRASV12 cells using a specific nontoxic RAS antagonist, FTS, which dislodges RAS from its membrane anchorage domains and accelerates its degradation (37Marom M. Haklai R. Ben Baruch G. Marciano D. Egozi Y. Kloog Y. J. Biol. Chem. 1995; 270: 22263-22270Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 38Jansen B. Schlagbauer-Wadl H. Kahr H. Heere-Ress E. Mayer B.X. Eichler H. Pehamberger H. Gana-Weisz M. David E. Ben Kloog Y. Wolff K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14019-14024Crossref PubMed Scopus (88) Google Scholar). FTS has been shown to act as a functional KRAS inhibitor in human pancreatic cell lines that express activated KRAS (39Weisz B. Giehl K. Gana-Weisz M. Egozi Y. Baruch G. Ben Marciano D. Gierschik P. Kloog Y. Oncogene. 1999; 18: 2579-2588Crossref PubMed Scopus (103) Google Scholar). As shown in Fig. 1D, treatment of HPDE-c7-KRASV12 with FTS led to an inhibition of Gli-luciferase and endogenous GLI1 mRNA. Taken together, our studies suggest that KRASV12 specifically activates Hh signaling in HPDE cells as GLI1 is not only a downstream effector but also a direct target gene and a reliable marker of Hh signaling pathway activities (40Kasper M. Regl G. Frischauf A.M. Aberger F. Eur. J. Cancer. 2006; 42: 437-445Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Oncogenic KRAS Is Responsible for GLI1 Activation in PDA Cells—To further test whether oncogenic KRAS is essential for GLI1 activation in PDA cells, we knocked down the expression of the oncogenic KRAS in human PDA cell lines (AsPC-1 and Panc-1) that express mutant KRASD12 using RNA interference. A previously reported siRNA duplex that specifically knocks down KRASD12 in AsPC-1 and Panc-1 cells was used (41Brunner T.B. Cengel K.A. Hahn S.M. Wu J. Fraker D.L. McKenna W.G. Bernhard E.J. Cancer Res. 2005; 65: 8433-8441Crossref PubMed Scopus (67) Google Scholar). The inactivation of KRAS oncogene by the KRASD12-specific siRNA duplex in AsPC-1 and Panc-1 cells led to decreased cellular RAS levels (Fig. 2A). Consistent with the reduced KRAS level, the phospho-MAPK levels were suppressed in KRASD12-knockdown AsPC-1 and Panc-1 cells compared with cells treated with the control siRNA, confirming the specificity and effectiveness of KRASD12-siRNA. Gene silencing of KRASD12 in AsPC-1 and Panc-1 cells eventually led to growth inhibition and cell death 96 h after siRNA transfection. Therefore, experiments related to KRASD12 siRNA were performed 24 h after transfection when no noticeable difference between control and KRASD12 siRNA-transfected cells could be observed. KRASD12 knockdown by siRNA resulted in a dose-dependent inhibition of Gli-luciferase activity and endogenous levels of GLI1 mRNA in AsPC-1 (Fig. 2B). Similar results were also observed in Panc-1 cells (Fig. 2C). To exclude the possibility that our observed inactivation of Gli-luciferase by KRASD12-specific siRNA was due to potential off-target effects, we used a PDA cell line, BxPC-3 cells (which does not contain KRAS mutation), to confirm the specificity of KRASD12 siRNA. As expected, the Gli-luciferase activity and GLI1 mRNA level in BxPC-3 cells did not change significantly after introducing KRASD12-specific siRNA (Fig. 2D). These results demonstrate that effects of KRASD12 siRNA observed in AsPC-1 and Panc-1 cells are specific to the KRASD12 mutant allele. Taken together, our data indicate that oncogenic KRAS plays an important role in regulating Hh signaling in PDA cells. RAF/MEK/MAPK, but Not the PI3K/AKT Pathway, Is Required for the KRAS-mediated Activation of Hh Signaling—To further determine which downstream effectors of the oncogene KRAS mediate the activation of Hh signaling, we examined the levels of Hh activity in HPDE-c7-KRASV12 cells in response to specific inhibitors that target the RAS downstream effectors MEK and PI3K. Inhibition of MEK by U0126 (20 μm) and PD98059 (40 μm) led to a significant reduction of Gli-luciferase activity and endogenous GLI1 mRNA levels (Fig. 3A), whereas wortmannin (100 nm), a PI3K-specific inhibitor, had little effect. These results suggested that the RAF/MEK/MAP kinase pathway was directly responsible for the KRAS-mediated activation of Hh signaling in HPDE-c7-KRASV12 cells. The inhibitory effect of U0126 and PD98059 was also observed in PDA cells expressing activating KRAS mutant. When AsPC-1 and Panc-1 cells were treated with the various inhibitors for 24 h, U0126 and PD98059 inhibited Gli1-luciferase expression (Fig. 3B) as well as endogenous GLI1 mRNA levels (Fig. 3C), whereas wortmannin had no effects. To ensure that the apparent non-effect of wortmannin is not due to a lack of efficacy of the compound, we determined the phosphorylation status of MAPK and AKT in Panc-1 cells treated with various pharmacological inhibitors. As shown in Fig. 3D, U0126 and PD98059 significantly suppressed the phosphorylation levels of MAPK. Wortmannin abolished the phosphorylation of Ser-473 of AKT, which is essential for its activation. If the RAF/MEK/MAPK, but not the PI3K/AKT pathway, is indeed responsi

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