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Constitutive Hedgehog Signaling in Chondrosarcoma Up-Regulates Tumor Cell Proliferation

2006; Elsevier BV; Volume: 168; Issue: 1 Linguagem: Inglês

10.2353/ajpath.2006.050001

1525-2191

Tri Dung Tiet, Sevan Hopyan, Puviindran Nadesan, Nalan Gökgöz, Raymond Poon, Alvin C. Lin, Taiqiang Yan, Irene L. Andrulis, Benjamin A. Alman, Jay S. Wunder,

Bone Tumor Diagnosis and Treatments

Chondrosarcoma is a malignant cartilage tumor that may arise from benign precursor lesions, such as enchondromas. Some cases of multiple enchondromas are caused by a mutation that results in constitutive activation of Hedgehog-mediated signaling. We found that chondrosarcomas expressed high levels of the Hedgehog target genes PTCH1 and GLI1. Treatment with parathyroid hormone-related protein down-regulated Indian Hedgehog (IHH) expression in normal growth plates but not in chondrosarcoma or enchondroma organ cultures. Treatment of the chondrosarcoma organ cultures with Hedgehog protein increased cell proliferation rate, whereas addition of chemical inhibitors of Hedgehog signaling decreased the proliferation rate. Chondrosarcoma xenografts from 12 different human tumors were established in NOD-SCID mice. Treatment with triparanol, an inhibitor of Hedgehog signaling, resulted in a 60% decrease in tumor volume, a 30% decrease in cellularity, and a 20% reduction in proliferation rate. These results show that Hedgehog signaling is active in chondrosarcoma and benign cartilage tumors and regulates tumor cell proliferation. Our data raise the intriguing possibility that Hedgehog blockade could serve as an effective treatment for chondrosarcoma, a tumor for which there are currently no universally effective nonsurgical management options. Chondrosarcoma is a malignant cartilage tumor that may arise from benign precursor lesions, such as enchondromas. Some cases of multiple enchondromas are caused by a mutation that results in constitutive activation of Hedgehog-mediated signaling. We found that chondrosarcomas expressed high levels of the Hedgehog target genes PTCH1 and GLI1. Treatment with parathyroid hormone-related protein down-regulated Indian Hedgehog (IHH) expression in normal growth plates but not in chondrosarcoma or enchondroma organ cultures. Treatment of the chondrosarcoma organ cultures with Hedgehog protein increased cell proliferation rate, whereas addition of chemical inhibitors of Hedgehog signaling decreased the proliferation rate. Chondrosarcoma xenografts from 12 different human tumors were established in NOD-SCID mice. Treatment with triparanol, an inhibitor of Hedgehog signaling, resulted in a 60% decrease in tumor volume, a 30% decrease in cellularity, and a 20% reduction in proliferation rate. These results show that Hedgehog signaling is active in chondrosarcoma and benign cartilage tumors and regulates tumor cell proliferation. Our data raise the intriguing possibility that Hedgehog blockade could serve as an effective treatment for chondrosarcoma, a tumor for which there are currently no universally effective nonsurgical management options. Cartilaginous tumors are the most common neoplasms effecting bone.1Spjut HJ Dorfman HD Fechner RE Ackerman LV Tumours of cartilaginous origin.in: Dorfman HD Fechner RE Ackerman LV Tumours of Bone and Cartilage. Armed Forces Institute of Pathology, Washington DC1983: 77-110Google Scholar, 2Levesque J Marx R Bell RS Wunder JS Kandel R White LA Enchondroma.in: Levesque J Marx R Bell RS Wunder JS Kandel R White L A Clinical Guide to Primary Bone Tumors. Williams and Wilkins, Philadelphia1998: 98-107Google Scholar They range from benign lesions, such as enchondromas and osteochondromas, to malignant chondrosarcomas. The benign lesions cause disability because of pain, limb deformity, and pathological fracture. Enchondromas and osteochondromas have a potential for malignant change, greater than 50% in some cases of multiple enchondromatosis (ie, Maffucci syndrome).3Schwartz HS Zimmerman NB Simon MA Wroble RR Millar EA Bonfiglio M The malignant potential of enchondromatosis.J Bone Joint Surg Am. 1987; 69: 269-274PubMed Google Scholar Chondrosarcomas can be difficult to treat as neither chemotherapy nor radiotherapy is typically effective. Radical surgery is the mainstay of treatment.4Fiorenza F Abudu A Grimer RJ Carter SR Tillman RM Ayoub K Mangham DC Davies AM Risk factors for survival and local control in chondrosarcoma of bone.J Bone Joint Surg Br. 2002; 84: 93-99Crossref PubMed Scopus (252) Google Scholar The growth plate, located between the cartilaginous epiphysis and the newly generated bone in the metaphysis, is responsible for longitudinal bone growth. Within the growth plate, chondrocytes proliferate and then proceed through an orderly differentiation process eventually resulting in programmed cell death. Indian Hedgehog (IHH) and parathyroid hormone-related protein (PTHrP) are part of a negative feedback loop that tightly regulates the fate of growth plate chondrocytes. Prehypertrophic and hypertrophic chondrocytes express IHH, which stimulates growth plate chondrocytes to proliferate. Hedgehog (HH) activates GLI-mediated transcription through the transmembrane proteins patched (PTCH) and smoothened (SMO). GLI transcription factors contain activator and repressor domains, and the cleavage and removal of the activation domain results in transcriptional repression. GLI-mediated transcriptional activation results in the up-regulation of target genes including the transmembrane protein PTCH1 and the transcription factor GLI1. During bone development, IHH also stimulates the expression of PTHrP, which inhibits chondrocyte differentiation by preventing proliferating chondrocytes from entering the prehypertrophic stage. PTHrP additionally inhibits IHH expression to complete the negative feedback loop (Figure 1). Thus, IHH and PTHrP signaling and feedback pathways regulate the rate of chondrocyte proliferation, differentiation, as well as normal longitudinal bone development.5Vortkamp A Lee K Lanske B Segre GV Kronenberg HM Tabin CJ Regulation of rate of cartilage differentiation by Indian Hedgehog and PTH-related protein.Science. 1996; 273: 613-622Crossref PubMed Scopus (1615) Google Scholar, 6Kronenberg HM Lee K Lanske B Segre GV Parathyroid hormone-related protein and Indian Hedgehog control the pace of cartilage differentiation.J Endocrinol. 1997; 154: S39-S45PubMed Google Scholar, 7Lanske B Karaplis AC Lee K Luz A Vortkamp A Pirro A Karperien M Defize LH Ho C Mulligan RC Abou-Samra AB Juppner H Segre GV Kronenberg HM PTH/PTHrP receptor in early development and Indian Hedgehog-regulated bone growth.Science. 1996; 273: 663-666Crossref PubMed Scopus (1103) Google Scholar, 8Amizuka N Henderson JE Hoshi K Warshawsky H Ozawa H Goltzman D Karaplis AC Programmed cell death of chondrocytes and aberrant chondrogenesis in mice homozygous for parathyroid hormone-related peptide gene deletion.Endocrinology. 1996; 137: 5055-5067Crossref PubMed Scopus (121) Google Scholar, 9Amizuka N Karaplis AC Henderson JE Warshawsky H Lipman ML Matsuki Y Ejiri S Tanaka M Izumi N Ozawa H Goltzman D Haploinsufficiency of parathyroid hormone-related peptide (PTHrP) results in abnormal postnatal bone development.Dev Biol. 1996; 175: 166-176Crossref PubMed Scopus (208) Google Scholar, 10Amizuka N Warshawsky H Henderson JE Goltzman D Karaplis AC Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation.J Cell Biol. 1995; 126: 1611-1623Crossref Scopus (414) Google Scholar, 11Schipani E Lanske B Hunzelman J Luz A Kovacs CS Lee K Pirro A Kronenberg HM Juppner H Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormone-related peptide.Proc Natl Acad Sci USA. 1997; 94: 13689-13694Crossref PubMed Scopus (321) Google Scholar, 12Kobayashi T Chung UI Schipani E Starbuck M Karsenty G Katagiri T Goad DL Lanske B Kronenberg HM PTHrP and Indian Hedgehog control differentiation of growth plate chondrocytes at multiple steps.Development. 2002; 129: 2977-2986PubMed Google Scholar, 13Chung UI Schipani E McMahon AP Kronenberg HM Indian Hedgehog couples chondrogenesis to osteogenesis in endochondral bone development.J Clin Invest. 2001; 107: 295-304Crossref PubMed Scopus (331) Google Scholar The IHH-PTHrP signaling pathway is implicated in the pathogenesis of many types of benign cartilage tumors. Enchondromas, osteochondromas and chondroblastomas all express PTHrP and its receptor PTHR1.14Bovee JV van den Broek LJ Cleton-Jansen AM Hogendoorn PC Up-regulation of PTHrP and Bcl-2 expression characterizes the progression of osteochondroma towards peripheral chondrosarcoma and is a late event in central chondrosarcoma.Lab Invest. 2000; 80: 1925-1934Crossref PubMed Scopus (117) Google Scholar, 15Romeo S Bovee J Jadnanansing N Taminiau A Hogendoorn P Expression of cartilage growth plate signalling molecules in chondroblastoma.J Pathol. 2004; 202: 113-120Crossref PubMed Scopus (35) Google Scholar, 16Pateder DB Gish MW O'Keefe RJ Hicks DG Teot LA Rosier RN Parathyroid hormone-related peptide expression in cartilaginous tumors.Clin Orthop. 2002; 403: 198-204Crossref PubMed Scopus (21) Google Scholar, 17Kunisada T Moseley JM Slavin JL Martin TJ Choong PF Co-expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in cartilaginous tumours: a marker for malignancy?.Pathology. 2002; 34: 133-137Crossref PubMed Scopus (29) Google Scholar PTHrP expression levels correlate with the proliferative rate of cartilage tumors.16Pateder DB Gish MW O'Keefe RJ Hicks DG Teot LA Rosier RN Parathyroid hormone-related peptide expression in cartilaginous tumors.Clin Orthop. 2002; 403: 198-204Crossref PubMed Scopus (21) Google Scholar, 17Kunisada T Moseley JM Slavin JL Martin TJ Choong PF Co-expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in cartilaginous tumours: a marker for malignancy?.Pathology. 2002; 34: 133-137Crossref PubMed Scopus (29) Google Scholar We previously identified a PTHR1 mutation (R150C PTHR1) that leads to excessive Hedgehog signaling in multiple enchondromatosis.18Hopyan S Gokgoz N Poon R Gensure RC Yu C Cole WG Bell RS Juppner H Andrulis IL Wunder JS Alman BA A mutant PTH/PTHrP type I receptor in enchondromatosis.Nat Genet. 2002; 30: 306-310Crossref PubMed Scopus (208) Google Scholar In addition, mice overexpressing either R150C PTHR1 or the Hedgehog activated transcription factor Gli-2 in the growth plate developed a phenotype similar to enchondromatosis, providing an in vivo model supporting the role of constitutive Hedgehog signaling in the pathogenesis of enchondromatosis.18Hopyan S Gokgoz N Poon R Gensure RC Yu C Cole WG Bell RS Juppner H Andrulis IL Wunder JS Alman BA A mutant PTH/PTHrP type I receptor in enchondromatosis.Nat Genet. 2002; 30: 306-310Crossref PubMed Scopus (208) Google Scholar Similar to enchondromatosis, osteochondromas also exhibit defective Hedgehog signaling. Mutations in the EXT genes, which regulate Hedgehog diffusion, are frequently associated with osteochondromas.19Stickens D Brown D Evans GA EXT genes are differentially expressed in bone and cartilage during mouse embryogenesis.Dev Dyn. 2000; 218: 452-464Crossref PubMed Scopus (63) Google Scholar Holoprosencephaly, a disorder in which there is malformation of the central portions of the brain and head, can be caused by mutations in Sonic Hedgehog (SHH). A variety of teratogens also cause this disorder.20Bale AE Sheep, lilies and human genetics.Nature. 2000; 406: 944-945Crossref PubMed Scopus (13) Google Scholar, 21Cooper MK Porter JA Young KE Beachy PA Teratogen-mediated inhibition of target tissue response to Shh signaling.Science. 1998; 280: 1603-1607Crossref PubMed Scopus (760) Google Scholar Cell culture studies have verified that many of the compounds that cause holoprosencephaly also block HH signaling. These compounds include Veratrum alkaloids (eg, cyclopamine) and inhibitors of 7-dehydrocholesterol reductase (eg, triparanol), an enzyme in the distal cholesterol synthesis pathway. In this study we investigated IHH-PTHrP signaling in cartilage tumors. Although previous studies examined PTHrP signals in cartilage neoplasia, the role of HH signals has received little attention. Here we show that HH signals are active in chondrosarcoma and benign cartilage tumors and that feedback inhibition of PTHrP on IHH expression is absent. Blockade of HH signaling in chondrosarcoma explants and xenografts attenuated the neoplastic phenotype by decreasing tumor cell proliferation, tumor cellularity, and tumor volume. Targeting HH signaling may be an effective chemotherapeutic strategy for patients with chondrosarcoma. Tissues were obtained from the Sarcoma Tumor Banks at Mount Sinai Hospital and the Hospital for Sick Children, as well as the Mount Sinai Hospital Bone Bank. Consent was obtained for each specimen according to each institution's policies. Frozen specimens were processed immediately after surgical excision, cryopreserved, and stored at −70°C. Samples used for explant organ cultures and xenografts were obtained directly from the operating room. Sufficient case material for all studies was not available from each tumor. Single-strand conformation polymorphism analysis was used to screen for mutations in the PTHR1 gene. Genomic DNA was extracted from specimens using Qiagen reagents (QIAamp DNA Mini kit; Mississauga, ON, Canada) and used as template for polymerase chain reaction (PCR) amplification of fragments containing an exon and its adjacent intron boundaries. The 33P-ATP incorporated PCR product was denatured and electrophoresed on a native polyacrylamide gel containing 10% glycerol. Band shifts were further analyzed by sequencing (Thermo Sequenase sequencing kit; Amersham Life Science, Cleveland, OH). DNA samples from cases of enchondromatosis known to harbor the R150C variant were used as a positive control.18Hopyan S Gokgoz N Poon R Gensure RC Yu C Cole WG Bell RS Juppner H Andrulis IL Wunder JS Alman BA A mutant PTH/PTHrP type I receptor in enchondromatosis.Nat Genet. 2002; 30: 306-310Crossref PubMed Scopus (208) Google Scholar Total RNA was extracted from ∼50 mg of each specimen (Trizol Reagent; Life Technologies, Inc., Grand Island, NY) and examined for expression of select genes using semiquantitative reverse-transcriptase (RT)-PCR. Two hundred ng of RNA template were reverse-transcribed into cDNA (Perkin-Elmer/Roche, Branchburg, NJ). One μl of cDNA from the above RT reaction was used for each PCR reaction. Each primer pair was designed to flank at least one intron, to prevent and distinguish amplification of contaminating genomic DNA. Genes of interest were matched to internal control genes that had similar PCR kinetics, and both were analyzed over a range of cycles to ensure the result was obtained from the logarithmic phase of PCR amplification to allow direct comparison of gene expression levels.22Wunder JS Eppert K Burrow SR Gokgoz N Bell RS Andrulis IL Co-amplification and overexpression of CDK4, SAS and MDM2 occurs frequently in human parosteal osteosarcomas.Oncogene. 1999; 18: 783-788Crossref PubMed Scopus (130) Google Scholar, 23Hopyan S Gokgoz N Bell RS Andrulis IL Alman BA Wunder JS Expression of osteocalcin and its transcriptional regulators core-binding factor alpha 1 and MSX2 in osteoid-forming tumours.J Orthop Res. 1999; 17: 633-638Crossref PubMed Scopus (43) Google Scholar PCR products were electrophoresed on agarose gels, stained with ethidium bromide, and photographed for densitometry (ImageQuant Software v3.0; Molecular Dynamics, Sunnyvale, CA). Relative levels of band intensity of the gene of interest were compared to the internal control gene within the linear range of amplification and normalized to a positive control sample on each gel. Appropriate negative controls were included in each RT-PCR experiment. Each sample was analyzed in triplicate over at least three different numbers of cycles. Gene-specific TaqMan Assay-on-Demand (PE/Applied Biosystems, Foster City, CA) was used, consisting of a fluorogenic probe and a pair of oligonucleotides. We performed standard quantitative RT-PCR reactions for IHH, PTCH1, and GLI1 on the ABI Prism 7900HT (Applied Biosystems, Foster City, CA) sequence detection system. Asparagine synthetase (AS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal control genes. The reactions were performed in duplicate in a 20-μl reaction volume using TaqMan Universal PCR Master Mix (ABI) on a 96-well plate format. We used the absolute standard curve method to determine the copy number of the transcript of interest by relating the PCR signal to a standard curve that was first constructed from RNA of known concentration. This curve was then used as a reference standard for extrapolating quantitative information for mRNA targets of unknown concentrations. cDNA from colon cell lines SW1417 and RW948 were used as reference standards for IHH and PTCH1, respectively. cDNA from a pool of 11 tumor cell lines was used as a reference standard for GLI1. Primary tissue specimens were cut into 2-mm-thick sections and maintained in Dulbecco's modified Eagle's medium with high glucose, as previously described for 4 days.24Caron JP Toppin DS Block JA Effect of polysulfated glycosaminoglycan on osteoarthritic equine articular cartilage in explant culture.Am J Vet Res. 1993; 54: 1116-1121PubMed Google Scholar Cultures were treated with either 0.1% fetal bovine serum (agonist control), 5 μg/ml Ihh-N (R&D Systems, Minneapolis, MN), 10−7 mol/L PTHrP (Bachem, King of Prussia, PA), 10−4 mol/L cyclopamine (a gift from William Gaffield, U.S. Department of Agriculture, Albany, CA), 10−4 mol/L tomatidine (Sigma, St. Louis, MO), or 10 μg/ml of neutralizing 5E1 anti-SHH monoclonal antibody (Hybridoma Bank, University of Iowa, Iowa City, IA). Proliferation was measured using thymidine incorporation and normalized to the total number of cells. Explant specimens were incubated with 10 μCi/ml tritiated (3H-T) thymidine for the final 20 hours of culture, then digested with 0.5% pronase (Boehringer Mannheim, Laval, Quebec, Canada) for 1 hour, followed by 0.125% collagenase A (Boehringer Mannheim) for 8 hours. Cells were counted and resuspended in 0.5 ml of 1% sodium dodecyl sulfate/0.005 mol/L ethylenediamine tetraacetic acid. Nucleic acids were precipitated with 1 ml of ice-cold 25% trichloroacetic acid and pipetted onto 2.4-cm glass microfiber filters (Whatman GF/C; Whatman, Maidstone, UK) over a suction apparatus. The filters were washed twice with 2 ml of 5% trichloroacetic acid, air-dried, and placed in scintillation vials with 5 ml of scintillation cocktail, and counts per minute averaged over 5 minutes. Semiquantitative RT-PCR was used to measure expression of type X collagen (COLX), a marker of terminally differentiated growth plate chondrocytes, relative to β2-microglobulin (β2M), an internal control gene. Twelve human chondrosarcoma tumor samples were obtained fresh from the operating room after surgical removal, divided into 120 explants each 5 mm × 5 mm × 5 mm, and xenografted into the subcutaneous tissues on the back of NOD-SCID mice. By gavage, one group of mice was treated with triparanol, a Hedgehog-blocking agent, three times a week at a dose of 400 mg/kg. The control mice were treated with the carrier, olive oil. Both groups of mice were sacrificed after 15 weeks of treatment. The amplex red cholesterol assay (Molecular Probes, Burlington, Ontario, Canada) was used to measure serum cholesterol to determine whether the mice were able to absorb triparanol.25von Mering C Basler K Distinct and regulated activities of human Gli proteins in Drosophila.Curr Biol. 1999; 9: 1319-1322Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 26Pathi S Pagan-Westphal S Baker DP Garber EA Rayhorn P Bumcrot D Tabin CJ Blake Pepinsky R Williams KP Comparative biological responses to human Sonic, Indian, and Desert Hedgehog.Mech Dev. 2001; 106: 107-117Crossref PubMed Scopus (160) Google Scholar To determine whether treatment with triparanol blocked HH signaling in vivo, the level of expression of two pathway target genes, Gli-1 and Ptch, was measured in the ends of the long bones by RT-PCR. After sacrifice and removal of the xenograft, tumor volume was calculated using three-dimensional measurements as previously described.27Poon R Smits R Li C Jagmohan-Changur S Kong M Cheon S Yu C Fodde R Alman BA Cyclooxygenase-two (COX-2) modulates proliferation in aggressive fibromatosis (desmoid tumor).Oncogene. 2001; 20: 451-460Crossref PubMed Scopus (90) Google Scholar Tumors were placed in 4% paraformaldehyde, and sections were stained with hematoxylin and eosin. For 12 tumor explants, the number of nuclei in 10 high-power fields of views was counted. Immunohistochemistry was performed using antibodies against the N-terminus of PTCH (G-19; Santa Cruz Biotechnology, Santa Cruz, CA). Sections were blocked for endogenous peroxidase, followed by pepsin treatment and incubation with primary antibody at 4°C overnight. Primary antibody was detected using a 3,3′-diaminobenzidine color substrate immunostaining kit (Vector Laboratories, Burlingame, CA). IgG antibody was used instead of the anti-PTCH antibody as a negative control. Mice were treated intravenously with BrdU 2 hours before sacrifice for assessment of xenograft proliferation. Immunohistochemical detection of BrdU using anti-BrdU antibodies was performed (Roche Applied Science, Laval, Quebec, Canada). Sections were counterstained with Light Green. For each section 10 high-power fields of view were analyzed. The number of BrdU-positive cells was divided by the total number of cells to determine the percent proliferation. DNA damaged cells were detected using the terminal dUTP nick-end labeling (TUNEL) assay for in situ end-labeling. Recombinant terminal deoxynucleotidyl transferase (Tdt) (Life Technologies, Inc., Gaithersburg, MD) was used to detect 3′ ends of cleaved DNA. Biotin 16-dUTP (Roche Diagnostics, Laval, Quebec, Canada), avidin-horseradish peroxidase, and 3,3′-diaminobenzidine detection method was performed. The slides were counterstained with hematoxylin. For each section 10 fields of view were analyzed. The number of TUNEL-positive cells was divided by the total number of cells to determine the percentage of apoptosis. Because each tumor provided multiple explants, statistical analysis was based on the number of patient samples (ie, n = 12) and used to compare tumor volume, cellularity, proliferation, and apoptosis rate between triparanol-treated and control explants. Data in plots are presented as mean values with 95% confidence intervals. The P values were calculated using the two-tailed t-test. We examined the expression of key members of the IHH/PTHrP signaling pathway using RT-PCR analysis for samples in which sufficient high-quality RNA was available. IHH and PTHrP were both expressed in all 4 solitary enchondromas and all 23 chondrosarcoma specimens, although the levels were variable (Table 1). IHH was not expressed in any of the four osteochondromas and in only two of seven cases of chondroblastoma. PTHR1, the IHH receptor PTCH, and the Hedgehog responsive transcription factors25von Mering C Basler K Distinct and regulated activities of human Gli proteins in Drosophila.Curr Biol. 1999; 9: 1319-1322Abstract Full Text PDF PubMed Scopus (48) Google Scholar GLI1, GLI2, and GLI3 were consistently expressed in all tumors. SHH transcripts were not detected in any cartilage tumors.Table 1Expression of IHH/PTHrP Members in Cartilage Neoplasia and Normal TissuesIHHPTCH1GLI1GLI2GLI3PTHrPPTHR1Growth plate4/44/44/44/44/44/44/4Articular cartilage0/44/42/42/44/42/44/4Cortical bone2/1111/1111/1111/1111/1111/1111/11Osteochondroma0/44/44/44/44/44/44/4Chondroblastoma2/77/77/77/77/72/77/7Enchondroma4/44/44/44/44/44/44/4Chondrosarcoma23/2323/2323/2323/2323/2323/2323/23The fraction of each specimen type for which transcripts were detectable by RT-PCR is listed. Enchondromas and chondrosarcomas all expressed each of the factors (except for Sonic Hedgehog, which was not expressed in any of the tumors). IHH, Indian hedgehog; PTCH1, patched-one; PTHrP, parathyroid hormone-related protein; PTHR1, type 1 PTH/PTHrP receptor. Open table in a new tab The fraction of each specimen type for which transcripts were detectable by RT-PCR is listed. Enchondromas and chondrosarcomas all expressed each of the factors (except for Sonic Hedgehog, which was not expressed in any of the tumors). IHH, Indian hedgehog; PTCH1, patched-one; PTHrP, parathyroid hormone-related protein; PTHR1, type 1 PTH/PTHrP receptor. To determine whether the HH signal is active in the various cartilage lesions, we used real-time PCR for quantitative assessment of expression of IHH as well as target genes known to be up-regulated by HH signaling (Figure 2). The level of expression of IHH was significantly lower in normal articular cartilage than in any of the other samples (P < 0.01). Enchondromas and low- and high-grade chondrosarcomas expressed IHH at levels similar to that of the growth plate. There was a significantly higher level of expression in intermediate grade chondrosarcomas (P < 0.05). However, there were only three cases each of low- and high-grade chondrosarcomas compared to 23 intermediate-grade tumors that makes it difficult to interpret the significance of the differences in the levels of expression for these grades of tumors. The levels of expression of PTCH1 and GLI1, Hedgehog target genes, were significantly increased in intermediate grade chondrosarcomas and in enchondromas, compared to the other cartilaginous lesions (P < 0.05). The small numbers of low- and high-grade tumors and the large 95% confidence interval ranges also makes the meaning of the differences in PTCH1 and GLI1 expression in these tumor grades uncertain. However, the elevated levels of IHH and the Hedgehog target genes suggest that Hedgehog-mediated signaling is activated in chondrosarcomas and enchondromas. We tested whether the PTHrP/IHH signaling pathway is functional in cartilage tumors by establishing short term primary organ cultures of enchondromas from 2 patients, chondrosarcomas from 10 patients, and normal growth plates from 4 patients. Treatment of enchondromas, chondrosarcomas as well as growth plates for 4 days with 10−7 mol/L PTHrP (Figure 3A) resulted in delayed hypertrophic differentiation based on decreased expression of Type X collagen (COLX) mRNA, an exclusive marker of hypertrophic chondrocytes (P < 0.01 for all three explant types).5Vortkamp A Lee K Lanske B Segre GV Kronenberg HM Tabin CJ Regulation of rate of cartilage differentiation by Indian Hedgehog and PTH-related protein.Science. 1996; 273: 613-622Crossref PubMed Scopus (1615) Google Scholar PTHrP lead to a decrease in proliferation in growth plate explants based on BrdU incorporation (P < 0.05), as would be expected based on its normal physiological role (Figure 3B), but had no effect on normal articular cartilage (data not shown). Surprisingly, PTHrP treatment did not significantly effect neoplastic chondrocyte proliferation for any of the 2 enchondromas or 10 chondrosarcomas, as measured by tritiated thymidine (3H-T) uptake and BrdU incorporation (P = 0.5; Figure 3B). In comparison, treatment of cultures with recombinant Ihh-N increased 3H-T uptake (P = 0.02) as well as BrdU incorporation (P = 0.02; Figure 3C) in all three tissue types suggesting a positive proliferative effect. Treatment also decreased the level of COLX expression (relative expression of COLX compared to β2M, as measured using semiquantitative RT-PCR, decreased from a mean of 1.3 to 0.4; P = 0.03). Hedgehog stimulation of cartilage tumor explants increased PTCH1 and GLI1 expression by 35% (P < 0.05) but had no significant effect on IHH (P = 0.15). Blocking Hedgehog signaling in tumor explant and growth plate cultures with cyclopamine, a known HH pathway antagonist, decreased tumor cell proliferation based on 3H-T uptake (P < 0.01) and BrdU incorporation (P < 0.02; Figure 3D). The structurally related but inactive compound tomatidine21Cooper MK Porter JA Young KE Beachy PA Teratogen-mediated inhibition of target tissue response to Shh signaling.Science. 1998; 280: 1603-1607Crossref PubMed Scopus (760) Google Scholar served as a negative control and did not significantly affect tumor cell proliferation according to 3H-T results (1120 versus 1080 counts per minute; P = 0.9 for chondrosarcoma samples). Similar to the effect of cyclopamine, treatment with triparanol diminished tumor cell proliferation (1190 versus 530 counts per minute; P < 0.05 for chondrosarcoma samples) as did the HH-neutralizing antibody 5E1,28Ericson J Morton S Kawakami A Roelink H Jessell TM Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity.Cell. 1996; 87: 661-673Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar according to partially blocked 3H-T uptake (1160 versus 780 counts per minute; P < 0.05 for chondrosarcoma samples). Western blot confirmed binding of the anti-HH antibody 5E1 to human Ihh-N. Hedgehog blockade of cartilage tumor explants decreased PTCH1 and GLI1 expression by a mean of 25% (P < 0.05) but had no significant effect on IHH (P = 0.1). These findings are consistent with the known consequences of IHH and PTHrP signaling on normal growth plate chondrocyte proliferation and differentiation, respectively.26Pathi S Pagan-Westphal S Baker DP Garber EA Rayhorn P Bumcrot D Tabin CJ Blake Pepinsky R Williams KP Comparative biological responses to human Sonic, Indian, and Desert Hedgehog.Mech Dev. 2001; 106: 107-117Crossref PubMed Scopus (160) Google Scholar, 28Ericson J Morton S Kawakami A Roelink H Jessell TM Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity.Cell. 1996; 87: 661-673Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar, 29Guo J Lanske B Liu BY Divieti P Kronenberg H