Oncogenic KRAS Activates Hedgehog Signaling Pathway in Pancreatic Cancer Cells

Abstract

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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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 N. I. T. Buchler H. J. Cancer. 110: PubMed Scopus Google Scholar). Hh signaling is essential for and cell maintenance in P.W. McMahon Genes Dev. 2001; 15: PubMed Scopus Google Scholar). Hh to of a which in activates downstream that the protein in the GLI L. Beachy P.A. PubMed Scopus Google Scholar). of the Hh signaling including the and the are in human PDA and cell Hh activity a that inhibits Hh signaling through with J. Beachy P.A. Genes Dev. 2002; 16: PubMed Scopus Google in PDA cells with Hh signaling, cell in and tumor in in the and 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 N. I. T. Buchler H. J. Cancer. 110: PubMed Scopus Google Scholar, G. S. V. D. R. M. C. H. C. A. W. Maitra A. Cancer Res. PubMed Scopus Google Scholar). The of activation of the RAS and Hh pathways in the of PDA that between these two pathways may be a important for the and development of PDA. the effects between KRAS and Hh signaling in pancreatic tumorigenesis are not results from that expression of Hedgehog is sufficient to the signaling pathway by a mutation in the 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 and a that activation of the Hedgehog pathway is not sufficient to mutations in the gene to downstream of di Magliano M. S. A. J. Hebrok M. Genes Dev. PubMed Scopus Google Scholar). In addition, although expression of endogenous of oncogenic leads to pancreatic intraepithelial neoplasia to all found in the human and PDA in 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 activation of Hh signaling is not sufficient to pancreatic intraepithelial neoplasia and PDA in a mouse model in which Hh signaling is in the pancreatic epithelium di Magliano M. S. A. J. Hebrok M. Genes Dev. PubMed Scopus Google Scholar). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in pancreatic we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh signaling pathway in PDA. that oncogenic transformation of human pancreatic ductal epithelial cells by oncogenic KRAS is by GLI activation and that specific of oncogenic KRAS activity inhibits Hh signaling in PDA cell lines with KRAS These results demonstrate that oncogenic KRAS is involved in activation of the pathway in PDA cells and that between the oncogenic KRAS and Hh pathways may play an important role in cancer development pancreatic PD98059, and from was by J. and from was from kinase and from was from was from and from Jackson and from RNA including and H. C. M. H. 1997; PubMed Google Scholar). for expression of from and by A. 1999; PubMed Google Scholar). was from C. M.S. C. T. C.M. D.M. D.S. Nature. 2005; PubMed Scopus Google Scholar). and an pancreatic ductal epithelial cell was by of and in with and cells by of cells with a and cells from the and in with was to the of to was by cells with the and the and with the cells in the of cells to all the cells RAS of RAS was a of the RAS of as G. J. Cheng Cancer Res. PubMed Scopus Google Scholar). in of cell with and the was on of of at for cell at in a and was between and a The of was from and the was for two was used to the between the two less than was as of cell was with the protein of protein and to in in with for by for detected by cells with the other and the 6 the was with and the cells in at with U0126 other the cells and activity was with the to the the cells in the with of and the was was by at for in a of the was with of and the was of was to the activity The from the was by the from the of to was at with similar and by H. H. C. M. H. 1997; PubMed Google Scholar). RNA was from cells as J. Cheng J. PubMed Scopus Google Scholar). was an and gene expression for of GLI1 and protein in the as by the The of was by to endogenous and to a Oncogenic of by the of oncogene pancreatic we a KRAS human PDA model an cell cell is a human pancreatic duct epithelial cell from normal by genes of human is and of tumor in T. L. J. D.A. M.S. Am. J. Pathol. 1996; Google Scholar, H. L. C. N. J. J. M.S. Am. J. Pathol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). expression of in cells a expression to transformation of the cell all in we used of the of cells to and The cells, increased of RAS activity, and in In addition, expression of in cells to the activation of downstream as and The and in the cells with the cells. These observations are in with results from an KRAS human PDA model the cell J. J. M. M.S. 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P. Y. 1999; PubMed Scopus Google Scholar). in of with to an of and endogenous GLI1 suggest that activates Hh signaling in cells as GLI1 is not a downstream effector but a gene and a of Hh signaling pathway M. G. A.M. F. J. Cancer. Full Text Full Text PDF PubMed Scopus Google Scholar). Oncogenic KRAS for GLI1 in PDA oncogenic KRAS is essential for GLI1 activation in PDA cells, we the expression of the oncogenic KRAS in human PDA cell lines and that mutant RNA that in and cells was used T.B. Hahn J. Cancer Res. 2005; PubMed Scopus Google Scholar). The of KRAS oncogene by the in and cells to RAS with the KRAS the in and cells with cells with the siRNA, the and of of in and cells to and cell death Therefore, to between and cells be knockdown by in a of activity and endogenous of GLI1 in results in cells the that of by was to we used a PDA cell cells not KRAS to the of the activity and GLI1 in cells not These results demonstrate that effects of in and cells are specific to the mutant data that oncogenic KRAS plays an important role in Hh signaling in PDA cells. but the for the of Hh which downstream of the oncogene KRAS the activation of Hh signaling, we the of Hh activity in cells in to specific inhibitors that the RAS downstream MEK and of MEK by U0126 and PD98059 to a of activity and endogenous GLI1 a PI3K-specific These results that the kinase pathway was directly for the activation of Hh signaling in cells. The inhibitory effect of U0126 and PD98059 was in PDA cells activating KRAS and cells with the inhibitors for U0126 and PD98059 inhibited expression as as endogenous GLI1 that the of is not to a of of the we the of and in cells with in U0126 and PD98059 the of the of of which is essential for the but not the is for the activation of Hh signaling, we that a constitutively active but not a constitutively active the inhibitory effect of oncogene KRAS knockdown on Hh we BRAFE600, a constitutively active a constitutively active form of with the and subsequently the in expression of of MEK activity and the inhibitory effect of on Hh signaling the other although the activity inhibited by as by kinase expression of to activity by results cells not These results that the RAF/MEK/MAPK signaling, but not the is critical for GLI activation in PDA. KRAS, through the RAF/MEK/MAPK GLI1 the by which oncogene KRAS activation of Hh signaling, we oncogenic KRAS GLI1 at the protein level. Because of the was specific to the endogenous GLI protein we an GLI1 expression T. S. J. J. Full Text Full Text PDF PubMed Scopus Google and and cells and subsequently the expression of GLI1 in the expression of GLI1 in both and cells with oncogenic KRAS activity by of new protein by we that of MEK by U0126 to a of GLI1 in cells The of GLI1 protein in cells was the of GLI1 protein in cells was estimated to be less than GLI1 protein was by the inhibitor in the of these results suggest the oncogene KRAS, through the blocks GLI1 and leads to the activation of Hh signaling in pancreatic cancer cells. of GLI the of KRAS the and of Hh signaling activation in pancreatic cancer development, we GLI1 expression an P. A.M. B. A.J. A.M. A. M. S. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of GLI1 has been P. A.M. B. A.J. A.M. A. M. S. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the of the GLI1 siRNA, we a GLI1 with GLI1 and cells. GLI1 GLI1 protein expression in cells GLI1 not of and cells GLI1 gene to a attenuation of of cells as by the results cells not we a in and cells. specific has been to effects on Hh signaling both in and in A. 1999; PubMed Google Scholar). of to Hh signaling as by the of activity in cells expression of of in a similar to that of GLI1 similar results in cells not In GLI1 gene effect on of cells, which KRAS mutation these data suggest that activation of Hh signaling is important for KRAS oncogenic transformation in pancreatic cancer cells. active KRAS mutations are one of the earliest and most common genetic alterations in pancreatic (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). between KRAS and other oncogenic pathways in PDA have not been In we the between oncogenic KRAS and the Hh signaling which has been to be in human PDA and cell lines (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 N. I. T. Buchler H. J. Cancer. 110: PubMed Scopus Google Scholar). In the human pancreatic ductal epithelial cell we that to increased endogenous GLI1 and transcriptional activity, suppressing oncogenic KRAS expression by inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS we found that Hh activation was by inhibitors specific for MEK, but not PI3K, in and mutant KRAS PDA cell of active form of BRAFE600, but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. suggest that oncogenic KRAS, through the RAF/MEK/MAPK GLI1 protein and plays an important role in activating the Hh signaling pathway in the of Hh pancreatic and genetic suggest that GLI and play as as in Hh development E. Clin. 2005; PubMed Scopus Google the of and in pancreatic tumorigenesis are not of with other Hh signaling as Sonic hedgehog SMO, and have been in human pancreatic cancer and pancreatic cancer cell lines (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 N. I. T. Buchler H. J. Cancer. 110: PubMed Scopus Google Scholar). the other the of in human pancreatic cancer has not been of a on a active form of the suggest that although activation is sufficient to drive pancreatic not human pancreatic di Magliano M. S. A. J. Hebrok M. Genes Dev. PubMed Scopus Google Scholar). the and between GLI1 and is that these are by signaling pathway in a similar suggest that expression of is inhibited by KRAS knockdown and MEK GLI1 expression in both and cells that oncogenic KRAS activates Hh signaling through of GLI in PDA. of signaling in in the pancreatic epithelium has been to expression 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 we that activation of the Hh pathway represents important by which oncogenic KRAS promotes tumor The of oncogenic KRAS to Hh signaling in the of Hh an for than of PDA cells lines with Hh signaling activity are to (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). The with an that Hh activation activates and the RAS pathway J. M. C. E. F. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google suggest that RAS and Hh signaling pathways form a to tumorigenesis in pancreatic and are essential for signaling in the of in and of cells, activation of the pathway is not sufficient to drive activation Lu K. G.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that activation of the RAS pathway is sufficient to drive Hh signaling activation in PDA cells through of GLI expression the addition of Hh and the effect of RAS is mediated by the RAF/MEK/MAPK pathway of of GLI1 expression in oncogenic PDA cells leads to a attenuation of in that activation of the Hh signaling pathway may represent an important for pancreatic is with a report by Hebrok and di Magliano M. S. A. J. Hebrok M. Genes Dev. PubMed Scopus Google that although Hh signaling not human pancreatic of Hh and signaling the of in active and with results are in with an in which through protein kinase and the signal-regulated kinase transcriptional activity in cells in a G.M. Cancer Res. PubMed Scopus Google Scholar). these observations and suggest that activation of the by and at the of GLI is an of the Hh signaling. The of between and Hh signaling pathways in pancreatic that the RAS and Hh pathways may represent a new for PDA. of for cells, of for J. for FTS, A. for the Cancer for Cheng for of and of for critical of the with