Activation of the hedgehog pathway in advanced prostate cancer
- Tao Sheng†1,
- Chengxin Li†1, 2,
- Xiaoli Zhang†1,
- Sumin Chi1, 2,
- Nonggao He1,
- Kai Chen1,
- Frank McCormick3,
- Zoran Gatalica4 and
- Jingwu Xie1Email author
© Sheng et al; licensee BioMed Central Ltd. 2004
Received: 22 September 2004
Accepted: 13 October 2004
Published: 13 October 2004
The hedgehog pathway plays a critical role in the development of prostate. However, the role of the hedgehog pathway in prostate cancer is not clear. Prostate cancer is the second most prevalent cause of cancer death in American men. Therefore, identification of novel therapeutic targets for prostate cancer has significant clinical implications.
Here we report that activation of the hedgehog pathway occurs frequently in advanced human prostate cancer. We find that high levels of hedgehog target genes, PTCH1 and hedgehog-interacting protein (HIP), are detected in over 70% of prostate tumors with Gleason scores 8–10, but in only 22% of tumors with Gleason scores 3–6. Furthermore, four available metastatic tumors all have high expression of PTCH1 and HIP. To identify the mechanism of the hedgehog signaling activation, we examine expression of Su(Fu) protein, a negative regulator of the hedgehog pathway. We find that Su(Fu) protein is undetectable in 11 of 27 PTCH1 positive tumors, two of them contain somatic loss-of-function mutations of Su(Fu). Furthermore, expression of sonic hedgehog protein is detected in majority of PTCH1 positive tumors (24 out of 27). High levels of hedgehog target genes are also detected in four prostate cancer cell lines (TSU, DU145, LN-Cap and PC3). We demonstrate that inhibition of hedgehog signaling by smoothened antagonist, cyclopamine, suppresses hedgehog signaling, down-regulates cell invasiveness and induces apoptosis. In addition, cancer cells expressing Gli1 under the CMV promoter are resistant to cyclopamine-mediated apoptosis. All these data suggest a significant role of the hedgehog pathway for cellular functions of prostate cancer cells.
Our data indicate that activation of the hedgehog pathway, through loss of Su(Fu) or overexpression of sonic hedgehog, may involve tumor progression and metastases of prostate cancer. Thus, targeted inhibition of hedgehog signaling may have significant implications of prostate cancer therapeutics.
The hedgehog (Hh) pathway plays a critical role in embryonic development and tissue polarity . Secreted Hh molecules bind to the receptor patched (PTC-PTCH1, PTCH2), thereby alleviating PTC-mediated suppression of smoothened (SMO), a putative seven-transmembrane protein. SMO signaling triggers a cascade of intracellular events, leading to activation of the pathway through GLI-dependent transcription . The hedgehog receptor PTCH1 is also a target gene of this pathway, which forms a negative feedback mechanism to maintain the pathway activity at an appropriate level in a given cell. Activation of Hh signaling through loss-of-function somatic mutations of PTCH1 in human basal cell carcinomas (BCCs) disrupts this feedback regulation, leading to uncontrolled SMO signaling. Activating mutations of SMO in BCCs, on the other hand, are resistant to PTCH1-mediated inhibition, leading to an outcome similar to PTCH1 inactivation [3–6]. More recently, abnormal activation of the sonic hedgehog pathway, through over-expression of sonic hedgehog, has been implicated in the development of subsets of medulloblastomas, small cell lung cancer and gastrointestinal tract (GI) cancers [7–10].
Development of prostate requires hedgehog signaling. Although the initial formation of prostate buds does not require sonic hedgehog signaling (shh), shh is critical for maintaining appropriate prostate growth, proliferation and tissue polarity [11–14]. In the adult prostate, however, the activity of the hedgehog pathway is quite low. It remains to be tested whether this hedgehog pathway is activated during development of prostate cancer, the second most prevalent cause of cancer death in American men. Activation of the hedgehog pathway is often indicated by elevated levels of PTCH1 and HIP. In addition to PTCH1 mutation, SMO activation and hedgehog over-expression, loss of Su(Fu) can result in activation of the hedgehog pathway. In the human, the Su(Fu) gene is localized at chromosome 10q24, a region with LOH in several types of cancer including prostate cancer, lung cancer, breast cancer and medulloblastomas [15, 16]. As a negative regulator of the hedgehog pathway, Su(Fu) inhibits the function of Gli molecules, leading to inactivation of this pathway [17–19]. Su(Fu) is also reported to affect beta-catenin function . In addition, over-expression of sonic hedgehog is shown to be involved in the development of GI cancers [9, 10]. Here we report our findings that activation of the hedgehog pathway occurs frequently in advanced prostate cancers, possibly through loss of Su(Fu) protein or over-expression of sonic hedgehog.
Elevated expression of hedgehog target genes in prostate cancer specimens
As an important regulator of tissue polarity, active hedgehog signaling is required for ductual morphogenesis and proliferation during prostate development [11–14]. The adult prostate, on the other hand, does not contain active hedgehog signaling. Because hedgehog signaling is an important regulator for epithelial-mesenchymal interaction, an event critical during prostate cancer development, we examined whether the hedgehog-signaling pathway is activated in prostate cancer.
Altered expression of Su(Fu) and Shh in prostate cancer specimens
The role for activated hedgehog signaling for cellular functions of prostate cancer
Following treatment with 5 μM cycloapmine in PC3 cells, expression of hedgehog target genes were dramatically inhibited (data not shown here), which was accompanied with a significant reduction of BrdU positive cells (see Fig. 6B for details). This effect is specific because addition of tomotidine, a non specific compound with a similar structure to cycloapmine, had no effects on either target gene expression or DNA synthesis (indicated by BrdU labeling in Fig. 6B and 6C). The prostate epithelial RWPE-1 cells which have no activated hedgehog signaling, on the other hand, were not sensitive to cyclopamine (data not shown here), indicating that cyclopamine specifically affects cells with elevated hedgehog signaling. LN-CAP, Du145 and TSU cells, like PC3 cells were also sensitive to cyclopamine treatment (Fig. 6C).
All these data indicate that the hedgehog pathway is activated in advanced prostate cancers, as indicated by high expression of PTCH1 and HIP. Our results also indicate that hedgehog signaling is required for cell proliferation and cell invasion of prostate cancer cells. Thus, targeted inhibition of the hedgehog pathway may be effective in future prostate cancer therapeutics.
Hedgehog signaling pathway regulates cell proliferation, tissue polarity and cell differentiation during normal development. Abnormal signaling of this pathway has been reported in a variety of human cancers, including basal cell carcinomas, medulloblastomas, small cell lung cancer and GI cancers [3, 4, 6–10, 22, 23]. Our findings in this report indicate a role of the sonic hedgehog pathway in prostate cancer. We detected a high expression of hedgehog target genes, PTCH1 and HIP, in advanced or metastatic prostate cancers. In contrast, only 22% of prostate tumors with Gleason scores 3–6 have elevated expression of PTCH1 and HIP. While our manuscript is being reviewed, three independent groups have recently reported similar results [24–26]. Thus, the hedgehog signaling pathway is frequently activated in advanced or metastatic prostate cancers.
Alterations of genes in the hedgehog pathway in prostate cancer
In our studies, we found that some prostate tumors had no detectable Su(Fu) protein expression while others contained high levels of Shh protein expression. We further identified inactivated mutations of Su(Fu) in two prostate cancers. In addition to inactivated mutations in the coding region, Su(Fu) may be inactivated through promoter methylation. The heterogeneous nature of prostate cancer makes it difficult to screen prostate cancer specimens for Su(Fu) mutations since the tumor content is often less than 5% of the specimens. Future improvement can be achieved using microdissection techniques for collecting pure population of tumor cells in gene mutation analysis.
Since all available prostate cancer cell lines express Su(Fu) at a high level, the role of Su(Fu) on cellular functions of prostate cancer cannot be investigated in these cells. It appears that over-expression of sonic hedgehog may be responsible for hedgehog signaling activation in these cell lines [our unpublished data and [24–26]]. After screening over 30 human cancer cell lines, we identified non-prostate cancer cell line with elevated hedgehog target genes and no detectable Su(Fu) expression (data not shown here). The growth suppression effects of Su(Fu) was demonstrated in this cell line, in which Su(Fu) expression down-regulated hedgehog target genes, inhibited DNA synthesis and cell growth (data not shown here). Thus, inactivation of Su(Fu) can contribute to active hedgehog signaling in prostate cancer.
Su(Fu) is reported to affect β-catenin signaling [27, 28]. We analyzed expression of β-catenin and E-cadherin in our prostate cancer array and detected cytoplasmic distribution of E-cadherin and β-catenin only in PC51 (data not shown), indicating that Su(Fu) may be able to affect both the wnt pathway and the hedgehog pathway in prostate cancer.
In addition to Su(Fu) inactivation, over-expression of Shh is another mechanism by which the hedgehog pathway is activated in cancer [7–10]. We noticed that sonic hedgehog expression varies from tumor to tumor, which may be resulted from the heterogeneity of prostate cancer. Our immunohistostaining also revealed that three tumors (PC14, PC20 and PC36) expressed PTCH1 and HIP at high levels, but had no alterations of Shh and Su(Fu). This could be due to elevated expression of indian hedgehog, or even alterations of other components of the pathway (such as Rab23 or Fused).
Once hedgehog pathway is activated, the target gene expression will be up-regulated. Thus, analysis of target gene expression using immunohistochemistry will be an effective way to detect hedgehog pathway activation in prostate cancer. Currently, PTCH1, Gli1 and HIP are good markers for the hedgehog pathway.
Perspectives on prostate cancer therapy
Our findings not only provide novel basic understanding of prostate cancer, but also allow us to design new ways to treat prostate cancer. With a specific SMO antagonist, cyclopamine, it will be possible in the future to treat prostate cancers, which have over-expressed sonic hedgehog. However, as a downstream molecule, tumors with Su(Fu) inactivation may not respond to cyclopamine treatment. Therefore, additional small molecule inhibitors appear to be necessary to treat Su(Fu) inactivated prostate cancer. One possibility is to use Gli1 SiRNA since we have indicated that down-regulation of Gli1 may be an important mechanism by which inhibition of the hedgehog pathway by cyclopamine induces apoptosis (Fig. 8). Sanchez et al also indicated that Gli1 SiRNA down-regulated DNA synthesis in prostate cancer cells .
Taken together, our findings suggest that activation of the hedgehog pathway involves prostate cancer progression. There might be several mechanisms by which the hedgehog pathway is activated in advanced prostate cancers, including loss of Su(Fu) protein expression, over-expression of sonic hedgehog or other alterations. We demonstrate that activation of the hedgehog pathway is associated with DNA synthesis and cell invasiveness in prostate cancer cells. Inhibition of the hedgehog pathway, on the other hand, causes apoptosis possibly through down-regulation of Gli1. Our studies predict that targeted inhibition of the hedgehog pathway may be an effective way to prevent prostate cancer progression.
Materials and methods
Tissue Microarray of Prostate Cancer
A total of 55 paraffin-embedded tissue blocks from patients with prostate cancer were obtained from UTMB Surgical pathology with approval from the Institutional Review Board (IRB). Pathological reports, H#E staining of each specimen were reviewed to determine the nature of the disease and the Gleason scores. Of 55 specimens, 18 were from tumors with Gleason scores 3–6, 15 with Gleason score 7 and 22 with Gleason scores 8–10. The tumor area was first identified before tissue microarray (1.5 mm in diameter for specimens) was assembled with Beecher's Tissue arrayer-I® according to manufacturer's instruction http://www.beecherinstruments.com.
Immunohistochemistry and Western blotting
A standard avidin-biotin immunostaining technique was performed using a kit from Vector laboratories using specific antibodies to Su(Fu) (Santa Cruz Biotechnology Cat# 10933), PTCH1 (Santa Cruz Biotechnology Cat# 6149), HIP (R&D systems Cat# AF1568) and Shh (Santa Cruz Biotechnology Cat# 9024) and PSA (Vector laboratories). Positive staining was in red or brown. The specificity of antibodies was tested using the very peptide used for raising the antibodies, which abolished the specific staining. Hematoxylin was used for counterstaining (in blue). Protein was analyzed by Western analysis with appropriate antibodies [Su(Fu) antibodies were from Santa Cruz, beta-actin antibody was purchased from Sigma). The signals were visualized with the enhanced chemiluminescence detection system (Amersham).
Cell lines and Cell invasion assay
Cell lines (RWPE-1, Du145, PC3, LN-CAP were purchase from ATCC and cultured according to the suggested conditions. TSU was kindly provided by Dr. Allen Gao. Cell invasion assay was performed with BD Bio-coat cell invasion chambers according to manufacturer's instruction (BD Bioscience, Inc., Franklin Lakes, NJ), with triplicates for each sample and the experiment was repeated three times with the similar results. Cell were treated with 5 μM cyclopamine (or tomatidine) before (for 12 h) and during cell invasion assay (for 24 h). The rate of cell invasion was calculated by dividing cell numbers penetrated the matrigels by the number of cell in the control chambers (without matrigels).
RT-PCR and sequencing analysis
Total RNA was isolated using Trizol® reagent (Invitrogen), and RT-PCR was performed using Promega's RT-PCR system according to the manufacturer's protocol. Two pairs of Su(Fu) primers were used (the first set with the forward primer 5'-cctacgcaccccgatggcg-3" and the reverse primer 5'-agccaaaaccactacctcca-3'; the second set with the forward primer 5'-tccaggttaccgctatcgtc-3' ad the reverse primer 5'-tagtgtagcggactgtcg-3'). PCR products were first purified using Qiagen's Gel Extraction Kit. Due to existence of possible Su(Fu) splicing isoforms in humans, Su(Fu) genetic mutations were screened after the PCR products were cloned into TOPO® TA cloning vectors (Invitrogen). Several independent clones (from three experiments) of each PCR product were selected for sequencing analysis in UTMB sequencing facility. All mutations were confirmed by at least six independent clones.
Real-time PCR We used Applied Biosystems' assays-by-demand 20× assay mix of primers and TaqMan probes (FAM™ dye-labeled) for the target genes (human Gli and PTCH1, the sequences have been patented by Applied Biosystems, Foster City, CA) and pre-developed 18S rRNA (VIC™-dye labled probe) TaqMan® assay reagent (P/N 4319413E) for an internal control. The primers are designed to span exon-exon junctions so as not to detect genomic DNA and the primers and probe sequences were searched against the Celera database to confirm specificity. To obtain the relative quantitation of gene expression, a validation experiment was performed to test the efficiency of the target amplification and the efficiency of the reference amplification. All absolute values of the slope of log input amount vs. ΔCT were <0.1. Separate tubes (singleplex) one-step RT-PCR was performed with 20 ng RNA for both target genes and endogenous control. The reagent we used was TaqMan one-step RT-PCR master mix reagent kit (P/N 4309169). The cycling parameters for one-step RT-PCR was: reverse transcription 48°C for 30 min, AmpliTaq activation 95°C for 10 min, denaturation 95°C for 15 sec and annealing/extension 60°C for 1 min (repeat 40 times) on ABI7000. Triplicate CT values were analyzed in Microsoft Excel using the comparative CT(ΔΔCT) method as described by the manufacturer(Applied Biosystems, Foster City, CA). The amount of target (2-ΔΔCT) was obtained by normalization to an endogenous reference (18sRNA) and relative to a calibrator.
BrdU labeling and TUNEL assay
BrdU labeling was performed using an in situ cell proliferation kit (Roche Molecular Biochemicals) . Cells were treated with 5μM cyclopamine (or tomatidine) for 12 h before BrdU labeling (1 h at 37°C). The percentage of BrdU positive cells was obtained by counting over 1000 cells under microscope, and the experiment was repeated twice with similar results. TUNEL assay was performed using an in situ cell death kit (Roche Molecular Biochemicals) [21, 29]. Cells were treated with 5 μM cyclopamine (or tomatidine) for 36 h before TUNEL assay).
List of abbreviations
prostate specific antigen
suppressor of fused
human homologue of patched 1
basal cell carcinoma.
we thank Dr. Huiping Guo for technical support of real-time PCR. J.X. was supported by grants from a NIH R01 grant, a DOD grant and the Sealy Foundation for biomedical Sciences.
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