Activation of the hedgehog pathway in advanced prostate cancer

Tao Sheng 2 Chengxin Li 1 2 Xiaoli Zhang 2 Sumin Chi 1 2 Nonggao He 2 Kai Chen 2 Frank McCormick 0 Zoran Gatalica 3 Jingwu Xie 2 0 UCSF Cancer Center , 2340 Sutter Street, San Francisco, CA 94115 , USA 1 Department of Dermatology, Xijing hospital , Xi'an 710032 , China 2 Sealy Centers for Cancer Cell Biology and Environmental Health, Department of Pharmacology and Toxicology, University of Texas Medical Branch , Galveston, Texas, 77555-1048 , USA 3 Department of Pathology, Creighton University Medical Center , 601 N 30th St. Omaha, NE 68131 , USA Background: 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. Results: 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 hedgehoginteracting 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. Conclusion: 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. - Background The hedgehog (Hh) pathway plays a critical role in embryonic development and tissue polarity [1]. 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 GLIdependent transcription [2]. 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 overexpression, 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 [20]. 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. Results 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. Activation of hedgehog signaling causes elevated expression of target genes PTCH1 and HIP. Thus, increased protein expression of PTCH1 and HIP indicates activation of the hedgehog pathway. Using PTCH1 antibodies [10], we examined 59 prostate cancer samples for hedgehog signaling activation (see Table 1, Additional file 1 for details). We first tested the specificity of the PTCH1 antibodies in MEF cells. Ptch1 null MEF cells have no active Ptch1 gene, thus should not have positive staining with PTCH1 antibodies. Indeed, no staining was seen in Ptch1 null MEF cells (Fig. 1A). After transfection of PTCH1 expressing plasmid, transfected cells showed positive staining (Fig. 1A), indicating that the PTCH1 antibodies are specific to PTCH1. Furthermore, PTCH1 immunohistostining was abolished after addition of the specific peptide, from which the antibodies were raised (Fig. 1B,1c). We found that percentage of PTCH1 positive staining tumors increased in high grade tumors (Table 1, Additional file 1). In prostate cancers with Gleason scores 36, 4 out of 18 specimens were positive for PTCH1 (22%), whereas 16 out of 22 undifferentiated carcinomas (Gleason Scores of 810) expressed (...truncated)