Expression Of Smoothened Research Articles

MiR-20a regulates proliferation, differentiation and apoptosis in P19 cell model of cardiac differentiation by targeting Smoothened

ABSTRACTMicroRNA (miR)-20a, a member of the miR-17-92 cluster related to cardiac development, was obviously downregulated in myocardially differentiated P19 cells compared with normal P19 cells. Smoothened (SMO) is a member of the Hh pathway. Hh signaling induces cardiac differentiation in P19 cells, and SMO mediates the Hh pathway during embryonic development. Using bioinformatic prediction software Targetscan (http://www.targetscan.org/), PicTar (http://pictar.bio.nyu.edu), and miRBase (http://microrna.sanger.ac.uk/), miR-20a and the 3′-untranslated region (3′-UTR) of SMO mRNA were predicted to have complementary binding regions. Accordingly, we inferred that miR-20a might act as a regulator of SMO, and regulate proliferation, differentiation and apoptosis in P19 cells. We determined the expression of miR-20a, SMO and marker proteins of cardiomyocytes (cTnT, GATA4 and desmin) by quantitative real-time PCR (qRT-PCR) and western blot assays, and found that P19 cells had differentiated into cardiomyocytes successfully at differentiation day 10, and downregulation of miR-20a and upregulation of SMO existed in myocardially differentiated P19 cells. Cell proliferation, differentiation and apoptosis detection showed that miR-20a upregulation inhibited proliferation and differentiation and enhanced apoptosis in P19 cells. Moreover, we verified that miR-20a directly targeted SMO and knockdown of SMO and miR-20a overexpression had similar effects on P19 cell proliferation, differentiation and apoptosis, which verified the speculation that miR-20a inhibits proliferation and differentiation and enhances apoptosis in P19 cells by directly targeting SMO. Our results suggest that miR-20a may be a potential target against congenital heart diseases.

Prognostic significance of WNT and hedgehog pathway activation markers in cancer of unknown primary.

Cancer of unknown primary (CUP) possesses distinct biology and peculiar natural history, in which the roles of the winged and hedgehog signalling pathways are unclear. We constructed tissue microarrays and studied the immunohistochemical (IHC) expression of β-catenin, smoothened (SMO) and the transcription factors TCF, LEF, GLI1 in 87 CUP cases for prognostic significance. A low rate of IHC expression of proteins was seen, the cut-off used being any expression in ≥1% of tumour cells. At univariate analysis, only nuclear IHC SMO expression displayed a statistically significant association with favourable outcome [median Overall survival (OS) of 19months in SMO-positive vs. 12months in SMO-negative cases, P=0·01]. An activated Wnt pathway, defined as IHC expression of any of nuclear β-catenin, TCF and LEF, was significantly associated with favourable progression free survival (median 9 vs. 5months, P=0·037) and OS (median 19 vs. 13months, P=0·04). This prognostic impact on OS was mainly driven by nuclear expression of TCF and/or LEF (P=0·03). No prognostic significance of the hedgehog pathway activation status, defined as IHC expression of SMO or nuclear GLI1, could be established. A favourable prognostic impact of the concurrent activation of both pathways was observed. A trend for association of activated Wnt with response to chemotherapy (responders 67% among activated Wnt cases vs. 35% among nonactivated Wnt cases, P=0·07) was observed in CUP adenocarcinomas. Activation of the Wnt pathway was a positive prognostic factor in a small CUP series, possibly via enhanced chemosensitivity. Independent validation is warranted.

The Hedgehog signalling pathway mediates drug response of MCF-7 mammosphere cells in breast cancer patients.

BCSCs (breast cancer stem cells) have been shown to be resistant to chemotherapy. However, the mechanisms underlying BCSC-mediated chemoresistance remain poorly understood. The Hh (Hedgehog) pathway is important in the stemness maintenance of CSCs. Nonetheless, it is unknown whether the Hh pathway is involved in BCSC-mediated chemoresistance. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain BCSC-enriched MCF-7 MS (MCF-7 mammosphere) cells. We showed that MCF-7 MS cells are sensitive to salinomycin, but not paclitaxel, distinct from parent MCF-7 cells. The expression of the critical components of Hh pathway, i.e., PTCH (Patched), SMO (Smoothened), Gli1 and Gli2, was significantly up-regulated in MCF-7 MS cells; salinomycin, but not paclitaxel, treatment caused a remarkable decrease in expression of those genes in MCF-7 MS cells, but not in MCF-7 cells. Salinomycin, but not paclitaxel, increased apoptosis, decreased the migration capacity of MCF-7 MS cells, accompanied by a decreased expression of c-Myc, Bcl-2 and Snail, the target genes of the Hh pathway. The salinomycin-induced cytotoxic effect could be blocked by Shh (Sonic Hedgehog)-mediated Hh signalling activation. Inhibition of the Hh pathway by cyclopamine could sensitize MCF-7 MS cells to paclitaxel. In addition, salinomycin, but not paclitaxel, significantly reduced the tumour growth, accompanied by decreased expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Furthermore, the expression of SMO and Gli1 was positively correlated with the expression of CD44+ / CD24-, and the expression of SMO and Gli1 in CD44+ / CD24- tissues was associated with a significantly shorter OS (overall survival) and DFS (disease-free survival) in breast cancer patients receiving chemotherapy.

MicroRNA-370 Attenuates Hepatic Fibrogenesis by Targeting Smoothened.

Recent research shows that abnormal expression of microRNA plays an important role in the process of hepatic fibrosis . miR-370 has been reported to be involved in liver function and is suppressed during hepatic carcinogenesis. The aim of this study was to investigate the role of miR-370 in hepatic fibrosis. The expression levels of miR-370 in rat fibrotic livers and activated hepatic stellate cells (HSCs) were evaluated by quantitative real-time PCR. The effect of miR-370 on the activation of HSCs was analyzed by flow cytometric analyses, real-time PCR and Western blot. Adenovirus carrying miR-370 was injected through the tail vein to access the effect of miR-370 on hepatic fibrosis induced by CCl4 in rats. The downstream targets of miR-370 were predicted by the Target Scan database and verified by luciferase assays, real-time PCR and Western blot in HSCs and were further confirmed by immunohistochemistry in vivo. Real-time PCR showed that miR-370 expression was significantly reduced in rat fibrotic livers and TGFβ1-stimulated HSCs. Overexpression of miR-370 inhibited the proliferation of HSC-T6 cells via inducing cell apoptosis and suppressed the activation of HSCs. Upregulation of miR-370 obviously attenuated the CCl4-induced liver fibrosis in rats. miR-370 was directly bound to the 3'UTR of Smoothened (SMO) and suppressed the expression of SMO in HSCs and fibrotic livers. Our study demonstrated that miR-370 plays an inhibitory role in hepatic fibrogenesis by targeting SMO. Restoration of miR-370 may have beneficial effects on the treatment of liver fibrosis.

Targeting the SMO oncogene by miR-326 inhibits glioma biological behaviors and stemness.

Few studies have associated microRNAs (miRNAs) with the hedgehog (Hh) pathway. Here, we investigated whether targeting smoothened (SMO) with miR-326 would affect glioma biological behavior and stemness. To investigate the expression of SMO and miR-326 in glioma specimens and cell lines, we utilized quantitative real-time (qRT)-PCR, Western blot, immunohistochemistry, and fluorescence in situ hybridization. The luciferase reporter assay was used to verify the relationship between SMO and miR-326. We performed cell counting kit-8, transwell, and flow cytometric assays using annexin-V labeling to detect changes after transfection with siRNA against SMO or miR-326. qRT-PCR assays, neurosphere formation, and immunofluorescence were utilized to detect the modification of self-renewal and stemness in U251 tumor stem cells. A U251-implanted intracranial model was used to study the effect of miR-326 on tumor volume and SMO suppression efficacy. SMO was upregulated in gliomas and was associated with tumor grade and survival period. SMO inhibition suppressed the biological behaviors of glioma cells. SMO expression was inversely correlated with miR-326 and was identified as a novel direct target of miR-326. miR-326 overexpression not only repressed SMO and downstream genes but also decreased the activity of the Hh pathway. Moreover, miR-326 overexpression decreased self-renewal and stemness and partially prompted differentiation in U251 tumor stem cells. In turn, the inhibition of Hh partially elevated miR-326 expression. Intracranial tumorigenicity induced by the transfection of miR-326 was reduced and was partially mediated by the decreased SMO expression. This work suggests a possible molecular mechanism of the miR- 326/SMO axis, which can be a potential alternative therapeutic pathway for gliomas.

Effect of Substance P on type II alveolar epithelial cells exposed to hyperoxia and its regulation of the Sonic hedgehog signaling pathway.

Oxidative stress injury and cell death in alveolar epithelial cells may lead to abnormal repair, further resulting in acute and chronic pulmonary diseases. Substance P (SP) has multiple biological activities. The Sonic hedgehog (SHH) pathway is important in lung development and decreasing epithelial injury. To investigate the effects of SP on alveolar epithelial type II cells (AEC IIs), AEC IIs were exposed to 95% oxygen and the SHH signaling pathway was examined. Primary AEC IIs were isolated and purified from premature rats. The cells were divided into four groups: The air (21% oxygen) group, hyperoxia (95% oxygen) group, hyperoxia + SP group and hyperoxia + SP + L703.606 group. The activity of AEC IIs was examined using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. The apoptotic rate of AEC IIs was analyzed by flow cytometry. The oxidative damage was evaluated by flow cytometry and reactive oxygen species (ROS) were detected using a 2',7'‑dichlorodihydrofluorescein diacetate probe. Quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression of the SHH signaling molecule Smoothened (SMO). The results demonstrated that exposure to 95% oxygen for 24 h significantly increased the level of ROS, contributed to apoptosis and markedly decreased the proliferation of AEC IIs. Compared with hyperoxia exposure, SP treatment decreased the level of ROS, reduced AEC II apoptosis and improved the cell survival sequentially. SMO was found to be expressed in AEC IIs and its expression increased when the cells were in hyperoxic conditions. These effects were enhanced by treatment with SP, which was able to significantly increase the expression of SMO. The aforementioned protective effect was weakened following treatment with L703.606. These findings suggested that SP was a protective regulatory factor that was able to decrease the hyperoxia‑induced cell injury and death, and improve the survival of AEC IIs exposed to hyperoxia, which may be associated with the activation of the SHH signaling pathway.

Hedgehog-signaling is upregulated in non-producing human adrenal adenomas and antagonism of hedgehog-signaling inhibits proliferation of NCI-H295R cells and an immortalized primary human adrenal cell line

Hedgehog (Hh)-signaling pathway is important in embryonic development. Activation of Hh-signaling is associated with tumorigenesis. Recent studies demonstrate that Hh-signaling is involved in the development of the adrenal gland in mice and is important in regulating adrenal proliferation.We studied the expression of Sonic hedgehog (SHH), Smoothened (SMO), Patched1 (PTCH1) and GLI family zinc finger 1 (GLI1) in human adrenal and in adrenocortical tumors using immunohistochemistry and semi-quantitative reverse transcriptase-polymerase chain reaction. Modulation of GLI1 and SMO messenger ribonucleic acid (mRNA) expression was investigated with forskolin. The role of Hh-signaling was studied in NCI-H295R cells and in an immortalized primary cell line using the Hh-agonist smoothened agonist (SAG) and the Hh-antagonist cyclopamine.The Hh-pathway components SHH, GLI1, PTCH1 and SMO were detectable in all adrenal glands. While in cortisol-producing adenomas (CPA), Hh-signaling expression levels were comparable to that in normal adrenal cortex, a much higher mRNA expression of GLI1, SMO and SHH was observed in non-producing adenomas (NPA). Interestingly, stimulation of cultured adrenal cells with forskolin led to a decrease in expression of GLI1 and SMO mRNAs. Antagonism of Hh-signaling resulted in a lower proliferation rate of adrenocortical cells, while Hh-agonism had no significant effect on adrenal cell proliferation.Our data show Hh-signaling activity in adult adrenal glands. Activation of the PKA pathway results in lower expression of Hh-signaling proteins. This might explain the lower expression of the Hh components GLI1 and SMO in CPA in comparison to the higher expression in NPA. Hh-signaling might be involved in the tumorigenesis of NPA.

Down-regulation of Smoothened gene expression inhibits proliferation of breast cancer stem cells

To investigate the influence of down-regulating Smoothened (SMO) gene expression through short hairpin RNA (shRNA) on the proliferation of breast cancer stem cells. Human SMO shRNA was designed, synthesized chemically, and transfected into MCF-7 cells to down-regulate SMO gene. By using G418, stable cells with down-regulated SMO were selected. In vitro proliferation of these cells was measured by CCK8 assay. The proportion of CD44(+)/CD24(-) cells was detected by flow cytometry and the mammospheres formation was determined by suspension sphere culture. The expression of SMO, GLI1 and Oct4 was detected by Western blot. In vivo, the volume of tumor was measured every 3 days and the expression of SMO, GLI1 and Oct4 detected by Western blot. In vitro, the cells were transfected with SMO-shRNA and selected by G418 after 21 days. SMO-shRNA effectively down-regulated the expression of SMO gene and protein, and inhibited the proliferation of MCF-7 and markedly reduced the proportion of CD44(+)/CD24(-) cells and mammospheres. In vivo, SMO-shRNA treatment of MCF-7 significantly inhibited the volume of tumor. The positive rate of SMO in negative control and SMO-shRNA group was 5/5 and 2/5, respectively. The expression of SMO, GLI1 and Oct4 in different groups were 0.72 ± 0.17 and 0.21 ± 0.09, 1.21 ± 0.21 and 0.47 ± 0.12, 0.83 ± 0.13 and 0.25 ± 0.07. SMO, GLI1 and Oct4 down-regulation significantly suppressed at protein levels (P < 0.05). The shRNA by chemical synthesis can effectively down-regulate SMO gene expression and inhibit the proliferation of breast cancer stem cells.

541 SONIC HEDGEHOG SIGNALING IN NORMAL HUMAN BLADDER DEVELOPMENT

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology (II)1 Apr 2013541 SONIC HEDGEHOG SIGNALING IN NORMAL HUMAN BLADDER DEVELOPMENT Guodong Zhu, Haiyen E. Zhau, Dapeng Wu, Linlin Zhang, Lei Li, Dalin He, and Leland W.K. Chung Guodong ZhuGuodong Zhu Xi'an, China, People's Republic of More articles by this author , Haiyen E. ZhauHaiyen E. Zhau Los Angeles, CA More articles by this author , Dapeng WuDapeng Wu Xi'an, China, People's Republic of More articles by this author , Linlin ZhangLinlin Zhang Xi'an, China, People's Republic of More articles by this author , Lei LiLei Li Xi'an, China, People's Republic of More articles by this author , Dalin HeDalin He Xi'an, China, People's Republic of More articles by this author , and Leland W.K. ChungLeland W.K. Chung Los Angeles, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1936AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Sonic hedgehog (SHH) signaling is important in bladder development. Mice with defective hedgehog signaling develop bladder anomalies. Clinically, urinary tract malformations are reported in human fetuses and infants with mutations of SHH and related signaling pathway genes. Information on the expression of SHH and associated signaling genes in normal human bladder development is fragmentary. This study determined the temporal and spatial expression patterns of SHH signaling pathway components in human fetal bladders by immunohistochemistry (IHC). METHODS Twenty-two archival bladder specimens from 16 male and 6 female human fetuses aged 12- to 36-week (wk) were obtained from the First Affiliated Hospital of Medical College of Xi'an Jiaotong University with the approval of Institutional Review Board. All specimens were processed for IHC staining with SHH, Patched1 (PTC1), Patched2 (PTC2), Smoothened (SMO), GLI1 and proliferating cell nuclear antigen (PCNA) protein expression, and analyzed by semi-quantitative H-Score methodology (Zhu G, et al., Prostate, 2007). IHC H-Score in tissues was standardized by quantitative real-time polymerase chain reaction and western blot analyses using cell lines and human fetal prostate tissues. RESULTS High intensity of SHH and SMO expression was detected in developing bladder urothelial cells, with no staining in lamina propria (LP), but with minimal expression of SMO in differentiating smooth muscle (SM) layers. The spatial distribution pattern of PTC1 and GLI1 was more complex with minimal expression in the LP layer, moderate expression in the SM layer, and high expression in the urothelium. PTC2 expression was mainly localized in the urothelium and LP, but no expression in the SM layer. All SHH signaling components were detected in fetal bladder tissues throughout the development, with expression peaks at 12- and 23-wk, coinciding with high cell proliferation as indicated by PCNA staining in the cell nuclei of urothelium and SM. CONCLUSIONS Autocrine SHH signaling in urothelium, and paracrine SHH signaling in SM, mediated by SMO, PTC1 and GLI1, were shown during human bladder development. Expression of SHH signaling components peaked, at 12-and 23-wk. The first expression peak at 12-wk may relate to urothelium growth, SM induction, and dilation of the bladder cavity. The second expression peaked at 23-wk may relate to urothelium and SM layer differentiation. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e222 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guodong Zhu Xi'an, China, People's Republic of More articles by this author Haiyen E. Zhau Los Angeles, CA More articles by this author Dapeng Wu Xi'an, China, People's Republic of More articles by this author Linlin Zhang Xi'an, China, People's Republic of More articles by this author Lei Li Xi'an, China, People's Republic of More articles by this author Dalin He Xi'an, China, People's Republic of More articles by this author Leland W.K. Chung Los Angeles, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Activation of the Sonic Hedgehog pathway in thyroid neoplasms and its potential role in tumor cell proliferation

The sonic hedgehog (SHH) pathway is activated in several types of malignancy and plays an important role in tumor cell proliferation and tumorigenesis. SHH binding to a 12-pass transmembrane receptor, Patched (PTCH), leads to freeing of Smoothened (SMO) and subsequent activation of GLI transcription factors. In the present study, we analyzed the expression of SHH, PTCH, SMO, and GLI1 in 31 follicular thyroid adenomas (FTA), 8 anaplastic thyroid carcinomas (ATC), and 51 papillary thyroid carcinomas (PTC) by immunohistochemical staining. More than 65% of FTA, PTC, and ATC specimens stained positive for SHH, PTCH, SMO, and GLI. However, the expression of the genes encoding these four molecules did not correlate with any clinicopathologic parameters, including the age, gender, the status of BRAF gene mutation, tumor stage, local invasion, and metastasis. Three thyroid tumor cell lines (KAT-18, WRO82, and SW1736) all expressed the genes encoding these four molecules. 5-Bromo-2-deoxyuridine labeling and MTT cell proliferation assays revealed that cyclopamine (CP), an inhibitor of the SHH pathway, was able to inhibit the proliferation of KAT-18 and WRO82 cells more effectively than SW1736 cells. CP led to the arrest of cell cycle or apoptosis. Knockdown of SHH and GLI expression by miRNA constructs that target SHH or GLI mRNA in KAT-18 and SW1736 cells led to the inhibition of cell proliferation. Our results suggest that the SHH pathway is widely activated in thyroid neoplasms and may have potential as an early marker of thyroid cancer or as a potential therapeutic target for thyroid cancer treatment.

Regulatory mechanisms of Hedgehog signaling pathway for epithelial-mesenchymal transition in pancreatic cancer cells

To explore the blocking effects of hedgehog signaling pathway on the processes of cell migration, invasion and epithelial-mesenchymal transition (EMT) in human pancreatic cancer cells and elucidate its possible mechanisms. The lentiviral expression vector for RNA interference of human Smoothened (SMO) gene was constructed to silence the expression of SMO. And RNAi against SMO was used to suppress the hedgehog signaling pathway in human pancreatic cancer Panc-1 cells. The in vitro invasion capacity in Panc-1 cells was assessed by Matrigel/Transwell chamber assay. Real-time PCR (polymerase chain reaction) and Western blot were used to detect the expressions of such EMT markers as E-cadherin, N-cadherin, β-catenin, vimentin and fibronectin and such transcription factors as Snail, Slug, Twist1 and Sip1. The stable interference of SMO could suppress the hedgehog signaling activity in Panc-1 cells. The inhibition of hedgehog signaling reduced the in vitro invasion capacity significantly in Panc-1 cells. The expression of E-cadherin significantly increased while N-cadherin, vimentin and fibronectin were significantly down-regulated in the RNAi group. Compared to the control group, the expressions of Snail and Slug were significantly reduced in the SMO knock-down group. The inhibition of hedgehog signaling pathway reduces the in vitro invasion capacity in human pancreatic cancer cells. And the EMT process is significantly suppressed. The mechanism is partially correlated with the down-regulations of Snail and Slug.

Overexpression of Hedgehog signaling molecules and its involvement in triple-negative breast cancer

The purpose of this study was to investigate the activation of Hedgehog (Hh) signaling molecules and its involvement in triple-negative breast cancer (TNBC). A total of 123 cases of paraffin blocks, including 83cases of primary breast carcinoma, 30 cases of mammary hyperplasia and 10cases of normal breast tissue, were immunohistochemically analyzed for Sonic Hedgehog (SHH), Patched-1 (PTCH1), Smoothened (SMO) and glioma-associated oncogene homoglog1 (GLI1) expression. The expression of SMO and GLI1 in TNBC was significantly increased in comparison to non-triple-negative breast cancer (nTNBC). GLI1 expression manifested an inverse association with the estrogen receptor. The levels of GLI1 expression were increased in lymph node-positive cases. The expression of SHH and SMO was increased in high histological grades. Furthermore, the expression of SMO and GLI1 was correlated with superior tumor stage. The expression of SHH, SMO and GLI1 was significantly increased in breast cancer and mammary hyperplasia. PTCH1 expression was significantly decreased in breast cancer compared to mammary hyperplasia and normal breast tissue. For the first time, clinical evidence has been provided in support of significant roles of Hh signaling in TNBC. Hh signaling is involved in breast ductal changes and malignant transformation. Measures to inhibit Hh activity may improve the prognosis of TNBC patients.

Abstract 275: Burkitt lymphoma cells express GLI1 but are not responsive to Hedgehog signaling

Squamous cell carcinoma (SCC) is an epithelial malignancy involving many anatomical sites and is the most common cancer capable of metastatic spread. Development of early diagnosis methods and novel therapeutics are important for prevention and mortality reduction. In this effort, numerous molecular alterations have been described in SCCs. SCCs share many phenotypic and molecular characteristics, but they have not been extensively compared. This article reviews SCC as a disease, including: epidemiology, pathology, risk factors, molecular characteristics, prognostic markers, targeted therapy, and a new approach to studying SCCs. Through this comparison, several themes are apparent. For example, HPV infection is a common risk factor among the four major SCCs (NMSC, HNSC, ESCC, and NSCLC) and molecular abnormalities in cell-cycle regulation and signal transduction predominate. These data reveal that the molecular insights, new markers, and drug targets discovered in individual SCCs may shed light on this type of cancer as a whole.

Abstract 3117: Expression of Hedgehog pathway molecules in patient-derived pancreatic adenocarcinoma xenograft models

Abstract Introduction. Pancreatic cancer (PC) is a devastating disease characterized by high failure rates of the conventional therapies. Increasing evidence suggest that PC resistance to therapies is due to their intrinsic ability to develop high contents of fibrotic stroma. It was recently shown that Hedgehog (HH) pathway can influence PC sensitivity to gemcitabine by modulating stroma production via paracrine mechanisms in between tumor cells and its stroma environment. Taking advantage that the stroma in engrafted human tumors in nude mice is produced by the host, we investigated the differential expression of HH pathway molecules by human PC cells and associated murine stroma in PC xenograft models. For this purpose we developed a new species-discriminating qRT-PCR methodology and analyzed a comprehensive collection of 27 patient-derived PC xenograft models previously characterized for their chemosensitivity. Methods. RNA was extracted from snap frozen PC xenograft samples. After reverse transcription, the samples were assessed using human and mouse species-specific qPCR assays for Tata Binding Protein (TBP), Sonic (SHH), Indian (IHH) and Desert (DHH) Hedgehog ligands, the Hedgehog receptor PATCH1 (PTCH1) and the G-coupled downstream receptor Smoothened (SMO), and for the Zn finger transcription factors GLI1, GLI2, GLI3. Data from human and mouse TBP assays were used for normalization and as surrogate markers of content of human tumor cells vs murine stroma cells in the models. Results. As measured using TBP, the amount of murine stroma in the PC xenograft models is comparable to that in primary PC tumors and could be as high as 50%. The PC xenograft models with the highest amount of stroma were also most resistant to standard therapies. Among the 27 PC xenograft models the expression of the HH molecules was highly variable. However, we consistently evidenced that the human PC cells preferentially express the HH ligands with much lower or neclectable expression of PTCH1, SMO and GLI transcription factors. SHH and at a lower level IHH were found to be predominant compared to DHH. Of note, tumors cells of few selected xenograft models express however a consistent level of HH receptors and GLI transcription factors. On the opposite, the stroma cells were shown to preferentially express the receptors PTCH1 and SMO as well as GLI transcription factors, but not the HH ligands. In nearly all xenograft models, the transcription factor GLI1 and at lower levels GLI2 were more highly expressed than GLI3. Conclusion. In PC xenograft models, HH ligands are expressed by tumor cells, whereas respective receptors and downstream signaling molecules are predominantly expressed in the murine stroma. This result argues in favor of a predominantly paracrine mechanism of HH in most of the PC xenograft models investigated. The activity of HH pathway inhibitors to modulate the tumor stroma and affect tumor growth will be investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2011-3117

Indian Hedgehog and Its Targets in Human Endometrium: Menstrual Cycle Expression and Response to CDB-2914

Progesterone is critical for secretory endometrial differentiation in women, but its downstream mediators are poorly understood. Our objective was to investigate endometrial expression of Indian Hedgehog (IHH) and genes involved in its signaling [smoothened (SMO), patched-1 (PTCH1), glioma-associated oncogene homolog 1 (GLI1), and GLI2] during the menstrual cycle and the effects of the selective progesterone receptor modulator CDB-2914 on its expression. Comparisons between normally cycling volunteers and women with symptomatic fibroids who received CDB-2914 or placebo were made at a clinical research center. Endometrial biopsy was performed on 34 volunteers, 17 additional women with fibroids. Endometrial expression of IHH, SMO, PTCH1, GLI1, and GLI2 by in situ hybridization and/or RT-PCR and IHH, GLI1, and PTCH1 immunohistochemistry were evaluated. RT-PCR showed expression of IHH, SMO, PTCH1, GLI1, and GLI2, with significant increases in IHH (5.2-fold) and GLI1 (3.6-fold) in endometrium exposed to CDB-2914 compared with placebo. In situ hybridization showed IHH mRNA expression in glands and stroma that was stronger in secretory samples. Among volunteers, IHH and GLI1 immunohistochemistry scores were higher in the secretory than proliferative phase in the nuclei and cytoplasm of glands and stroma (P=0.0002-0.04). Compared with follicular-phase controls, women exposed to CDB-2914 showed increased IHH expression in all compartments except stromal cytoplasm (P=0.0199-0.0423); GLI1 was up-regulated in glandular nuclei and cytoplasm compared with both volunteers and women receiving placebo (P≤0.0416). The temporal increase in endometrial IHH and GLI1 during the secretory phase, and their modulation by CDB-2914, suggests progestin regulation and a potential role in endometrial differentiation and implantation.

Immunohistochemical expression of SHH, PTC, SMO and GLI1 in glandular odontogenic cysts and dentigerous cysts

To investigate expression of the sonic hedgehog (SHH) signaling pathway components in glandular odontogenic cysts (GOCs), and dentigerous cysts (DCs). Immunohistochemical staining for SHH, patched (PTC), smoothened (SMO), and the transcriptional factor GLI1 were investigated in the 12 GOCs specimens and 20 DCs. In GOCs and DCs, immunoreactivity for SHH, PTC, SMO, and GLI1 were detected in the epithelial cytoplasm. Each of the genes of the SHH signaling pathway was expressed in similar patterns in the epithelial lining of the cysts. The expression of SHH, PTC, SMO, and GLI1 was significantly higher in epithelia than that of subepithelial fibroblasts (P < 0.01). No statistical difference among the labeling index of the epithelial lining among the different cyst types could be revealed. The findings suggest that the proteins of the SHH signaling pathway are predominantly located within the epithelial components of GOCs and DCs. SHH signaling pathway may play a role in epithelial lining formation.

PTC gene mutations and expression of SHH, PTC, SMO, and GLI-1 in odontogenic keratocysts

The Patched ( PTC) gene is responsible for basal cell nevus syndrome (BCNS) accompanied by multiple odontogenic keratocysts (OKCs), and its product plays a role in the Sonic hedgehog (SHH) signaling pathway involving smoothened (SMO) and GLI-1. To clarify the role of SHH signaling in OKCs, the expression of SHH, PTC, SMO, and GLI-1 and mutations of PTC were examined in 18 sporadic, 4 BCNS-associated OKCs and 7 control gingivae. SHH, PTC, SMO, and GLI-1 were detected in all OKC and gingiva samples by reverse transcriptase-polymerase chain reaction (RT-PCR). Immunoreactivity for SHH and GLI-1 was markedly higher in epithelial components than in subepithelial cells, while immunoreactivity for PTC and SMO was similar in epithelial components and subepithelial cells in OKCs. The positive rate of PTC and SMO expression in subepithelial cells of OKCs was significantly higher than that in gingivae. The positive rate of GLI-1 expression in subepithelial cells of BCNS-associated OKCs was significantly higher than that in primary OKCs. These results suggest that the SHH signaling might be involved in the pathophysiologic nature of OKCs. While mutations of the PTC gene could not be detected in 4 BCNS-associated OKCs by direct DNA sequencing, 3 of 5 primary and 4 of 4 recurrent OKCs had several mutations of this gene. These results suggest that PTC mutations are probably related not only to BCNS-associated OKCs but also to sporadic OKCs.

Expression of Sonic hedgehog (SHH) signaling molecules in ameloblastomas.

To clarify the roles of Sonic hedgehog (SHH) signal transduction in oncogenesis and cytodifferentiation of odontogenic tumors, expression of SHH, Patched (PTC), Smoothened (SMO), and GLI1 was analyzed in ameloblastomas as well as in tooth germs. Tissue specimens of 9 tooth germs, 36 benign ameloblastomas, and 1 malignant ameloblastoma were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry for the expression of SHH, PTC, SMO, and GLI1. Expression of SHH, PTC, SMO, and GLI1 mRNA was detected in all tooth germ and ameloblastoma samples. Immunohistochemical reactivity for SHH, PTC, SMO, and GLI1 was detected in both normal and neoplastic odontogenic tissues. Expression of SHH, PTC, and GLI1 was more evident in epithelial cells than in mesenchymal cells, whereas SMO reactivity was marked in both epithelial and mesenchymal components in tooth germs and ameloblastomas. In ameloblastomas, these SHH signaling molecules were expressed more intensely in peripheral columnar or cuboidal cells than in central polyhedral cells; keratinizing cells and granular cells showed no or little reactivity. Expression of SHH, PTC, SMO, and GLI1 in tooth germs and ameloblastomas suggests that these SHH signaling molecules might play a role in epithelial-mesenchymal interactions and cell proliferation in tooth development as well as in growth of these epithelial odontogenic tumors.

Functional Smoothened is required for expression of GLI3 in colorectal carcinoma cells

We investigated a role for Hedgehog signalling in colon cancer by studying transcription of members of the pathway in human colorectal carcinoma cell lines. We determined the methylation status and screened the gene encoding the Hedgehog receptor-associated protein Smoothened (SMO) for putative mutations. In three cell lines lacking SMO expression the SMO promoter was fully methylated and the transcription factor GLI3 was not expressed. Two additional cell lines both having one methylated SMO allele and expressing mutant SMO did not express GLI3. Our results suggest that expression of wild-type SMO is required for expression of GLI3 by a mechanism that is independent of conventional Hedgehog signalling.