Hedgehog pathway by lCKz4bE6

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									    Hedgehog Pathway in Cancer
        -Pancreatic cancer as an example
          Veratrum californicum                Desert



Sonic




                                             Indian




                                  R6楊士弘
                                  2007.1.4
              Introduction
• Human Sonic Hedgehog (SHH) mutants:
  holoprosencephaly(前腦發育畸形症)
• first discovered in Drosophila
• paramount role in the proper development of
  the embryo
• involved in basal cell carcinoma
• Gorlin Syndrome: PTCH1 mutation


                            Nat Rev Drug Discov 2006;5:1026-33
                       獨眼畸形


Holoprosencephaly & Cyclopia




                   www.thefetus.net/page.php
           Gorlin Syndrome
        (basal cell nevus syndrome)
• Autosomal dominant
• Incidence: 1 in 50,000-150,000
• Gene: PTCH1 (9q22.3-q31), defective
• Multiple basal cell carcinomas in lifetime
  (>90%)
• Predisposing to other cancers, especially
  medulloblastoma (10%), rhabdomyosarcoma
• Pancreatic cancer? (no data)
              Hedgehog Pathway

                                                 Gene
                                                 PTCH1/Ptch1/ptc: patched
                                                           (-) feedback
                                                 SMOH/Smo: smoothened
                                                 Sufu: suppressor of fused
                                                 Gli/Ci: cubitus interruptus
                                                 KIF7/Cos2
                                                 Hip: Hh-interacting protein
                                                      (-) feedback



Ligand                        Transcriptional factor
Shh: Sonic Hedgehog (音速小子)    Gli-1: activator & (+) feedback in vertebrates
Dhh: Desert Hedgehog (沙漠刺蝟)   Gli-2: mainly activator
Ihh: Indian Hedgehog (印度刺蝟)   Gli-3: mainly repressor
                                                   Nat Rev Cancer 2003;3:903-11
Hedgehog Pathway


          N-palmitoylation


                               autocatalytic




              C: cholesterol
              Disp: Dispatched


                  BioEssays 26:387–394, 2004
          Cell. Mol. Life Sci. 63 (2006) 642–652
                   Patched vs Smoothened




PLoS Biol. 2006 Jul;4(8):e232    Nat Rev Drug Discov 2006;5:1026-33
             Hedgehog Pathway
Drosophila



                                      Cos-2: Costal-2
                                      dpp: decapentaplegic
                                      iro: iroquois
                                      col: collier
                                      en: engrailed
                                      PKA: protein kinase A
                                      CKI: casein kinase I
                                      GSK-3: glycogen
                                          synthase kinase-3β




             Gli-1, 2, 3 in mammals
                                       NRMCB 2005;6:306
                                 Cancer Res 2006; 66(13): 6463-7

      HH & Cilia
(IFT88, IFT172) IFT: intraflagellar transport protein
HH-GLI in Cancer




         TRENDS in Cell Biology 2007;17(9):438
1996-06-14
                  HH Pathway in BCC




                                                     Cell 85, 841–851 (1996)


             BCNS (n=2)               Sporadic BCC (n=1)
             Exon 15, duplication     Exon 3, C→T



             Exon 15, 11bp deletion
                                              Science 272, 1668–1671 (1996)
HH Pathway in Medulloblastoma
                 Tomatidine: analogue of cyclopamine
                 without inhibition of HH pathway




      (1μM)




                      Science 297, 1559–1561 (2002)
      HH Pathway in Other Cancers
•   Melanoma
•   Oral SCC
•   Esophageal cancer
•   SCLC
•   GI cancers
•   Prostate cancer
•   Rhabdomyosarcoma
•   Colon cancer
•   B cell lymphoma
•   HCC
          Cyclopamine(環杷明)
• Isolated from corn lily (加州藜蘆)
   NIH-3T3 cell




 Forskolin: adenylyl cyclase activator
                                         Nature 406, 1005–1009 (2000)
Hh & Cell Cycle Regulation
Dosophila




            Molly Duman-Scheel-Nature 2002;417, 299–304
              Shh Induction by NF-κB
 AsPC-1 & SUIT-2: pancreatic cancer


Anti-pp65




Anti-Shh
                                                                       (mutant)


       PDTC: pyrrolidine dithiocarbamate ODN: oligodeoxynucleotides
             (NF-κB inhibitor)
                             Hiroshi Nakashima-Cancer Res 2006; 66(14): 7041-9
HPDE-c7: pancreatic ductal cell immotalized by E6/E7 of HPV-16
                                                          U0126: MEK1/2 inhibior

        Transfection
                              HH & K-ras                  PD98059: MEK1 inhibitor
                                                          Wortmannin: PI3K inhibitor

mRNA




                                                             Ras
                                        cyclopamine
  Gli                                                                    PI3K
                                                       MEK1/2
                                                                           melanoma
                                                           ?
                                        Hh      Ptch    Smo        Gli     Hhip



                                                         PNAS 2007;104, 5895–5900
                                              J. Biol. Chem. 2007;282, 14048–14055
              HH & TGF-β
PANC-1

                                mRNA            Hh    TGF-β

          CPN
                                               Ptch    Smad
              SB
     CPN+SB
                                               Smo

  mRNA     mRNA
                                                Gli
     CPN: cyclopamine
     SB431542: TβRI inhibitor




                                       Cancer Res 2007;67(14):6981–6
                Shh in Angiogenesis
                     Corneal neovascularization
Fibroblast




Ang: angiopoietin




                                ROBERTO POLA-Nature Med. 7, 706–711 (2001)
Hedgehog in Pancreas Development




                  Cell. Mol. Life Sci. 63 (2006) 642–652
Ihh & Shh in Pancreatic Cancer
   Ihh




   Shh




                      Pancreas 2006;32:119-129
IHC Results of HH




            SPT: solid and pseudopapillary tumor
            MCN: mucinous cystic neoplasm
            IPMN: intraductal papillary mucinous neoplasm
            PA: pancreatic ductal adenocarcinoma


                       Pancreas 2007;34:340-346
                Sonic Hedgehog (Shh)
Shh-Normal                  Shh-PanIN-1              Shh-PanIN-2




Shh-PanIN-3                  Shh-Cancer (70%)         Ptc1-Cancer




PanIN: pancreatic intraepithelial neoplasia


                                                Sarah P. Thayer-Nature 2003;425:851-56
            Animal Model & Cell Lines
Pdx1 (pancreatic duodenal homeobox 1)-Shh mice

 •   Epithelium dysplasia, resemble PanIN-1,2
 •   Her-2/neu overexpression          Smo-Cancer

 •   K-ras codon 12 mutation
 •   Ptc1 expression
 •   Smo expression
 •   26 pancreatic cancer cell lines hh+
 •   Cyclopamine induced apoptosis and tumor
     reduction in Smo-expressed xenograft models.
 Cyclopamine: Smo inhibitor
                                            Sarah P. Thayer-Nature 2003;425:851-56
Xenograft




       Sarah P. Thayer-Nature 2003;425:851-56
  Shh in Multistep Tumorigenesis
• Epithelial proliferation, possibly through ↑
  cyclin D1 & ↓ p21
• Protect epithelium from apoptosis, possibly
  through ↑ PI3K/Akt, Bcl-2 and Bcl-XL
• Shh/K-ras activating cells resistant to PI3K,
  MAPK, mTOR inhibition, but responsive to
  simultaneous Shh and PI3K inhibition


                           Jennifer P. Morton-PNAS 2007;104:5103-08
 Silencing of HHIP by Methylation
RT-PCR




                    Cancer Biol. Ther. 2005 4: 728–733
Blockage of Shh




      Georg Feldmann-Cancer Res 2007;67(5):2187–96
Modified Boyden chamber invasion/migration assays




                       Georg Feldmann-Cancer Res 2007;67(5):2187–96
E3LZ10.7 Xenograft




          Georg Feldmann-Cancer Res 2007;67(5):2187–96
Georg Feldmann-Cancer Res 2007;67(5):2187–96
Human autopsy




                  ALDH inhibitor diethylaminobenzaldehyde




                   P<0.05
                Georg Feldmann-Cancer Res 2007;67(5):2187–96
MiaPaCa-2 cells
                  Synergism




                  Z. Shafaee-Cancer Chemother Pharmacol (2006) 58: 765–770
human primary tumor or metastases

          Pancreatic
           Cancer
          Stem Cell




Chenwei Li-Cancer Res 2007;67(3):1030–7
                             (mRNA)




Chenwei Li-Cancer Res 2007;67(3):1030–7
    HH Inhibitor
                                                 Johns Hopkins University




                                                  Smo inhib. Kd = 1.2 nM




                                                   Smo inhib. Kd = 12 nM




                                                   Smo inhib. Kd = 44 nM


Nat Rev Drug Discov 2006;5:1026-33   Curr Top Med Chem. 2005;5(2):147-57
        HH Inhibitor



                 Smo inhib. Kd = 71 nM




Curr Top Med Chem. 2005;5(2):147-57      Nat Rev Drug Discov 2006;5:1026-33
         Hh-Antag-691



                            Ptc1+/-p53 -/- mice

                                     Hh-Antag
 Gli




                                                  D50

Cancer Cell 2004;6:229–40                          Cancer Res 2005; 65: 4975–4978
                About Curis Inc.
• In January 2007, Genentech treated the first patient in
  a phase I clinical trial of a systemically administered
  small molecule Hedgehog antagonist for the treatment
  of cancer.
• The phase I trial is designed as an open-label study of a
  systemic Hedgehog antagonist in patients with locally
  advanced or metastatic cancers that are refractory to
  standard therapy or for whom no standard therapies
  exist.
• The trial is expected to enroll approximately 50
  patients spread across several dose-escalating cohorts.
   GANT61 & GANT58
                                                 human, prostate, carcinoma




                                       no adverse side effects, such as weight
                                       loss, ulcerations, or general non-well being
Matthias Lauth-PNAS 2007;104;8455-8460 of the animals
                 Summary
• Hedgehog pathway plays a role in many
  cancer types.
• Other signaling pathways related to cancer
  modulate the hedgehog pathway.
• Blockage with antagonists of the pathway can
  inhibit tumor growth.
• The pathway is probably involved in the
  maintenance of cancer stem cells.
Thanks for your attention!
                  HH in Cancer Stem Cell
     MM (NCI-H929)                       Breast cancer (isolated from xenograft)
                                                      CD44+CD24-/low lineage-




PNAS 2007 Mar 6;104(10):4048-53   Cancer Res. 66, 6063–6071 (2006)
    Cyclopamine in GBM Stem Cell

    U87-MG




ALDH: aldehyde dehydrogenase




                               Stem Cells. 2007 Jul 19; [Epub ahead of print]
Antisense Smo




         Gene Therapy (2006) 13, 1587–1594

								
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