Hh Inhibitors Research Articles

Abstract 2242: Inhibition of acetyl-CoA carboxylase attenuates lipidation and activity of oncogenic signaling pathways in pancreatic cancer

Abstract Normal cells can use circulating lipids, while some tumor cells require de novo biogenesis of fatty acids for survival irrespective of existing exogenous lipids. Acetyl-CoA Carboxylase (ACC) is the rate-limiting enzyme in the process of de novo fatty acid synthesis and one of its isoforms (ACC1) is overexpressed in various cancer types. We have developed a class of novel, potent small molecule inhibitors of ACC, which preferentially inhibit human ACC1 activity. Interestingly, in vitro the compounds are active in a selective subset of cancer cells, regardless of their proliferation rate, and pancreatic cancer cells are among the most sensitive cell lines to ACC1 inhibition both in vitro and in vivo. Notably, pancreatic cancer is characterized by alterations in several oncogenic pathways: KRAS, Hedgehog (Hh) and Wnt. One mechanism that modulates the activity of the above-mentioned oncogenic signaling molecules is protein lipidation. We hypothesized that in pancreatic cancer cells de novo fatty acid synthesis is an important source of lipids necessary for fully acting KRAS, Hh and Wnt molecules, and therefore the process is essential for cell proliferation and survival. To investigate this as potential novel anti-tumor mechanism of action, we conducted a series of experiments to assess the effect of ACC inhibition on these oncogenic pathways in vitro and in vivo. In vitro we first investigated the effect of ACC inhibition on Wnt secretion and activity, a process requiring lipidation of Wnt. Treatment of a Wnt3A-overexpressing cell line, L-Wnt3A, with the ACC inhibitor, indeed, leads to reduced Wnt3A protein levels secreted in the media. This result was further confirmed by the Super8xTOPFlash reporter assay in HEK293 cells incubated with conditional media from L-Wnt3A cells, with or without treatment with the ACC inhibitor. Furthermore, we showed that in Capan-2 cells, in which ligand-dependent autocrine Wnt signaling is active, treatment with the ACC inhibitor decreased the expression of β-catenin target genes and cell proliferation. In vivo profiling of the ACC inhibitor in a set of patient derived xenograft (PDX) models of pancreatic cancer indicated a correlation between the sensitivity to ACC inhibition and aberrant activation of Hh and Wnt signaling in the tumors, as shown by immunohistochemical detection of downstream targets of the two pathways. Currently, we are investigating the specific changes in Hh and Wnt signaling, following ACC inhibition in both a PDX model of pancreatic cancer, as well as in a Capan-2 xenograft mouse model. Together, these studies are building strong support for the hypothesis that tumor sensitivity to de novo fatty acid synthesis inhibition is linked to the dependence on multiple oncogenic signaling pathways that are regulated by protein lipidation, and highlight a potential new general approach for targeting ACC for the treatment of cancers. Citation Format: Elissaveta Petrova, Arne Scholz, Andrea Sturz, Juliane Paul, Franziska Siegel, Dominik Mumberg, Ningshu Liu. Inhibition of acetyl-CoA carboxylase attenuates lipidation and activity of oncogenic signaling pathways in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2242. doi:10.1158/1538-7445.AM2015-2242

Abstract 5423: Identification of immunotargets in Hedgehog medulloblastoma for immunotherapy

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Although approximately 75% of patients survive with current therapies, the associated complications drastically affect their quality of life, including severe neurological impairment and secondary cancers. Approximately 30% of MB cases exhibit activated hedgehog signaling pathway (HH-MB). Recent clinical trials using small molecular HH inhibitors exhibit promising results. Unfortunately, there are also indications of high rates of resistance and relapses, affirming the pressing need to develop complimentary therapies. Although immunotherapy has previously been proposed to treat MB, unique antigens are rare and monoclonal antibody therapies may not ensure tumor cytotoxicity, without additional adjuvants and associated severe side effects. However, recent successful developments of various cancer immunotherapies, especially the chimeric antigen receptor modified T-cell therapy, have rekindled interest in MB immunotherapies. To address the heterogeneity of MB and to develop a precision immunotherapy, we propose to identify uniquely or differentially overexpressed cell surface proteins in HH-MB, using human MB microarray and RNA-Seq data. Our preliminary studies have identified 39 potential candidates that are differentially expressed in HH-MB. We are currently confirming the presence and cellular localization of these candidate proteins using human MB tissue microarrays. This work is supported by NIH (T32 HL07915 [GST]) and Stand Up To Cancer - St. Baldrick's Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. Citation Format: Grace S. Tan, Tom Curran. Identification of immunotargets in Hedgehog medulloblastoma for immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5423. doi:10.1158/1538-7445.AM2015-5423

Abstract 3666: Synthesis and evaluation of itraconazole analogues for hedgehog pathway inhibition

Abstract The hedgehog (Hh) pathway is a developmental signaling pathway that plays a key role in embryonic development. While the pathway is generally quiescent during adult life, dysregulation of the Hh pathway can lead to cell proliferation and tumor growth. This abnormal Hh signaling is typically responsible for tumors associated with basal cell carcinoma (BCC) and medulloblastoma (MB) and inhibiting the Hh pathway is a valid target to develop novel chemotherapeutic agents. Most Hh inhibitors identified to date target the protein smoothened (SMO), a crucial component of the Hh pathway. Vismodegib is a SMO inhibitor that was recently approved by the FDA for the treatment of advanced BCC. However, there has been an emergence of resistance to Vismodegib; this resistance is a result of a point mutation in SMO. This occurrence of Vismodegib-resistant SMO mutations highlights the therapeutic need for new Hh pathway inhibitors. Itraconazole (ITZ), a well-known antifungal agent, was recently determined to be a potent inhibitor of the Hh pathway in vitro and in vivo; ITZ was also found to be a potent inhibitor of wild type SMO and Vismodegib-resistant SMO. ITZ is well-characterized and there is a good understanding of its potential side effects. This well-known safety profile makes ITZ a desirable drug candidate. Our ongoing studies on the ITZ scaffold are focused on exploring the regions of ITZ that are required for potent Hh pathway inhibition. The design, synthesis, and preliminary in vitro evaluation of these ITZ analogues will be reported herein. Citation Format: Jennifer Pace, M. Kyle Hadden. Synthesis and evaluation of itraconazole analogues for hedgehog pathway inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3666. doi:10.1158/1538-7445.AM2015-3666

Abstract 3631: A novel combination of Hedgehog inhibitors with carboplatin exhibits synergy in ovarian cancer treatment by altered regulation of DNA repair networks

Abstract Ovarian cancer (OVCA) is the deadliest of all the reproductive cancers, affecting over 22,000 lives of women annually in the USA alone. In spite of their initial promising response rates to platinum drugs, more than 70% of patients relapse and die with the advanced disease. An important factor contributing to the poor outcomes in OVCA is platinum resistance (PR) and disease recurrence. Therefore, it is vital to understand the molecular mechanisms contributing to tumor resistance to platinum drugs and disease recurrence. Our preliminary studies identified aberrant activation of Hedgehog (Hh) signaling in advanced stage tumors and in OVCA cells, particularly those that are resistant to platinum agents. Further genetic and biochemical studies revealed altered expression of several cell cycle checkpoint and DNA repair genes involved in repair of platinum drug-induced DNA damage in these tumors and cell lines. Aberrant Hh signaling is implicated in the regulation of several signaling pathways including cell cycle, differentiation and DNA repair networks. Hence, we hypothesized that inhibition of Hh signaling could affect the expression of altered DNA damage response and repair networks and sensitize OVCA cells to platinum therapy. To this end, we evaluated two small molecule inhibitors of Hh signaling that target SMO (BMS-833923) and GLI transcription factors (GANT61) either alone or in combination with carboplatin in several OVCA cell lines by clonogenic and MTS cell survival assays. The therapeutic efficacy was assessed by calculating combination index (CI) values using CalcuSyn software. Simultaneously, we have also assessed the status of Hh signaling and several DNA damage response and repair networks that respond to platinum drugs. Interestingly, both the inhibitors of Hh signaling attenuated OVCA cells’ growth and their ability to form colonies. Consistent with this, inhibition of Hh signaling alone induced replication stress associated DNA damage responses (as evidenced by γH2AX foci) and compromised ATR-mediated cell cycle checkpoint responses. Similarly, combination treatment of Hh inhibitor (either SMO inhibitor or GLI inhibitor) with carboplatin potentiated the DNA damage induced by carboplatin and its cytotoxic effects in several ovarian cancer cell lines. Additionally, evaluation of combination treatments efficacies was confirmed in isogenic platinum sensitive (A2780) and resistant (A2780/CP70) OVCA cell lines. Moreover, analysis of combination therapeutic indexes revealed synergistic effects by demonstrating the CI values in the range of 0.3 to 0.49 (synergy defined as CI <1) at multiple dosage combinations. Collectively, our studies suggest the combination of Hh signaling inhibitors with carboplatin could be an effective therapeutic modality in synergistic killing of ovarian cancer cells and overcoming platinum resistance. Citation Format: Sebastian M. Spencer, Kaushlendra Tripathi, Erhong Meng, Jennifer Scalici, Rodney P. Rocconi, Komaraiah Palle. A novel combination of Hedgehog inhibitors with carboplatin exhibits synergy in ovarian cancer treatment by altered regulation of DNA repair networks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3631. doi:10.1158/1538-7445.AM2015-3631

Abstract 5084: A multifaceted role of the Hedgehog pathway in modulating pancreatic ductal adenocarcinoma biology

Abstract The Hedgehog (Hh) developmental pathway is reactivated in pancreatic ductal adenocarcinoma (PDA) and forms a paracrine axis from the tumor cells to the supporting stromal cells. Previous work from this laboratory found that, in established tumors, inhibition of the Hedgehog (Hh) pathway caused a loss of tumor stroma implicating this axis in the development of the profuse desmoplastic stroma observed in PDA. However, the mechanisms by which Hh signaling modulates the stroma in PDA remain unknown. In this study we have attempted to identify the target genes of the Hh pathway in the stroma of pancreatic tumors. Using a novel tumor biopsy procedure coupled with RNA-SEQ analysis we have identified genes acutely sensitive to Hh pathway inhibition in the KPC mouse model of PDA. The gene signature of down-regulated genes following Hh inhibition implicated the Hh pathway in the proliferation of its target stromal cells as well as a possible indirect effect on the proliferation of tumor cells. Interestingly, a significant number of genes were also up-regulated following inhibition of the Hh pathway. This gene signature was consistent with the migration and infiltration of leukocytes. Taken together these data suggest that the Hh pathway can drive proliferation of both the stromal and epithelial compartments as well as actively suppress leukocyte infiltration into pancreatic tumors. In addition we have utilized a Gli1-GFP reporter mouse to study the Hh target cells in the stroma of KPC mice. Previous work has suggested that Hh target cells in pancreatic tumors are myofibroblasts and thus α-Smooth muscle actin (α-SMA) positive. However, we observed that while some Gli1 positive stromal cells are α-SMA positive a significant proportion are α-SMA negative. In addition, costaining KPC-Gli1-GFP tumors for other stromal markers, including Fibroblast specific protein-1 (FSP-1), Glial Fibrillary Acidic Protein (GFAP), Desmin, Vimentin and Platelet-derived growth factor receptor β (PDGFRβ) reveals subsets of Hh target cells in the tumor stroma. These data suggest the Hh pathway targets a complex set of stromal fibroblasts to mediate its effects. Taken together, these data suggest that the Hh pathway regulates multiple facets of pancreatic tumor biology both in negative and positive manner and does so through targeting a complex array of stromal cells. Citation Format: Dafydd Huw Thomas, Albert Lee, Carmine Palermo, Stephen Sastra, Raul Rabadan, Kenneth Olive. A multifaceted role of the Hedgehog pathway in modulating pancreatic ductal adenocarcinoma biology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5084. doi:10.1158/1538-7445.AM2015-5084

Abstract 3665: Reconstructing the itraconazole scaffold towards improved specificity for hedeghog signaling inhibition

Abstract Inappropriate regulation of the hedgehog (Hh) signaling pathway contributes to the development and progression of a number of human cancers; most notably, basal cell carcinoma (BCC) and medulloblastoma (MB). Clinical validation of small molecule mediated silencing of the Hh pathway was recently demonstrated by the FDA approval of vismodegib for the treatment of locally advanced BCC, representing a first-in-class inhibitor of the Hh signaling pathway. Vismodegib antagonizes Hh signaling by directly binding smoothened (SMO), a critical regulator of the pathway and target of numerous small molecule preclinical and clinical drug candidates. Vismodegib treatments in MB patients revealed novel SMO mutants that demonstrated resistance to vismodegib therapy, which underscores the need for continued development of novel Hh pathway inhibitors. Recently, itraconazole (ITZ) was identified as an inhibitor of Hh signaling in vitro and in vivo. Characterization indicated ITZ, a clinically used antifungal agent, maintains Hh inhibition against vismodegib-resistant SMO mutants. Ongoing structure-activity analysis for the ITZ scaffold is currently being investigated to identify analogues with improved potency and reduced non-specific toxicity. Results from these studies to identify candidates with improved pharmacological profiles and selectivity for Hh signaling modulation will be described herein. Citation Format: Albert M. DeBerardinis, M. Kyle Hadden. Reconstructing the itraconazole scaffold towards improved specificity for hedeghog signaling inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2015-3665

Hedgehog inhibitors from Artocarpus communis and Hyptis suaveolens

The hedgehog (Hh) signaling pathway plays crucial roles in cell maintenance and proliferation during embryonic development. Naturally occurring Hh inhibitors were isolated from Artocarpus communis and Hyptis suaveolens using our previously constructed cell-based assay system. Bioactivity guided fractionation led to the isolation of 15 compounds, including seven new compounds (4, 5, 6, 7, and 9–11). The isolated compounds showed cytotoxicity against a cancer cell line (PANC1) in which Hh signaling was abnormally activated. Several compounds (12–14; GLI1 transcriptional inhibition IC50=7.6, 4.7, and 4.0μM, respectively) inhibited Hh related protein (BCL2) expression. Moreover, compounds 1, 12, and 13 disrupted GLI1 and DNA complex formation.

Abstract A51: Novel Hedgehog co-receptors in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies, with a dismal six month median survival. The Hedgehog (Hh) signaling pathway is upregulated in PDAC. 75% of PDAC cases display increased expression of Hh ligands. In fact, Hh signaling is active in early PanIN lesions and persists as the cancer progresses. Interestingly, Hh ligands are secreted by the tumor cells and function in a paracrine manner, signaling to the surrounding tumor stroma, which in turn promotes tumor growth. PDAC is characterized by hypovascularity and a highly desmoplastic stroma. The downstream effects of Hh activation in the stroma are currently not well understood, and represent potential therapeutic targets. Hh pathway activation involves the binding of Hh ligands to the canonical receptor, Patched1 (Ptch1). This terminates Ptch1-mediated repression of Smoothened (Smo) and furthers downstream effects through activation of the Gli family of transcription factors. Recently, new co-receptors were identified that play an essential role in Hh pathway function. CAMrelated/down-regulated by oncogenes (Cdon), Brother of Cdo (Boc), and Growth arrest-specific 1 (Gas1) all cooperate with Ptch1 to promote Hh signaling during development. A central question is to what degree these receptors act to mediate Hh pathway function in adult tissues, especially in Hh-driven diseases, such as PDAC. Our data reveals that in the healthy pancreas, Gas1, Boc, and Cdon are expressed in a perivascular and periductal manner in both fibroblasts and stellate cells as shown by co-stains with αSMA and vimentin. During cancer progression, expression of all three co-receptors is prevalent throughout the desmoplastic stroma. Functional studies on Gas1-/-;Boc-/- mouse embryonic fibroblasts (MEFs) treated with Hh ligand showed a drastic reduction in Hh response compared to wildtype MEFs, indicating that these co-receptors are important for Hh signal transduction. Despite a reduced Hh-response, we found that Gas1-/-;Boc-/- MEFs promoted the growth of significantly larger, more vascularized tumors than their wildtype counterparts. A more physiologically relevant experiment with pancreatic fibroblasts and primary pancreatic tumor cells yielded the same counterintuitive result: removal of Gas1 and Boc from fibroblasts decreases Hh-response, and yet tumor-promoting potential was significantly increased. Moreover, chick chorioallantoic membrane (CAM) assays with tumor cells and fibroblasts revealed that Gas1-/-;Boc-/- MEFs stimulated blood vessel growth. We repeated these experiments with MEFs in which all three co-receptors were removed. Gas1-/-;Boc-/-;Cdon-/- MEFs displayed a more significantly abrogated Hh-response than both their Gas1-/-;Boc-/- and wildtype counterparts. However subcutaneous co-injection and CAM experiments revealed that these MEFs did not promote tumor growth, behaving similarly to Smo-/- MEFs in previously published findings. These results indicate the importance of Hh dosage in pancreatic cancer, which has important clinical implications. In particular, we found that intermediate levels of Hh signaling, such as that resulting from pharmaceutical inhibition, resulted in a marked angiogenesis response. This finding may partially account for the recent failure of Hh inhibition in a pancreatic cancer clinical trial. Citation Format: Esha Mathew, Yaqing Zhang, Alexander M. Holtz, Kevin T. Kane, Jane Song, Benjamin L. Allen, Marina Pasca di Magliano. Novel Hedgehog co-receptors in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A51.

Synthesis and antiproliferative activity of novel 4-substituted-phenoxy-benzamide derivatives

A series of novel 4-substituted-phenoxy-benzamide derivatives bearing an aryl cycloaliphatic amine moiety were synthesized and evaluated for antiproliferative activity against SW620, HT29 and MGC803 cancer cell lines in vitro. The pharmacological data demonstrated that the majority of target compounds exhibited moderate efficacy in HT29 and MGC803 cell lines. Compound 10c showed promising inhibition of hedgehog (Hh) signaling pathway in an Hh-related assay. In addition, the superposition pattern of 10c showed a good fit for a pharmacophoric model generated by Hh inhibitors and provided a basis for further structural optimization.

Hedgehog Inhibitors in Rhabdomyosarcoma: A Comparison of Four Compounds and Responsiveness of Four Cell Lines.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and is divided into two major histological subgroups, i.e., embryonal (ERMS) and alveolar RMS (ARMS). RMS can show HEDGEHOG/SMOOTHENED (HH/SMO) signaling activity and several clinical trials using HH inhibitors for therapy of RMS have been launched. We here compared the antitumoral effects of the SMO inhibitors GDC-0449, LDE225, HhA, and cyclopamine in two ERMS (RD, RUCH-2) and two ARMS (RMS-13, Rh41) cell lines. Our data show that the antitumoral effects of these SMO inhibitors are highly divers and do not necessarily correlate with inhibition of HH signaling. In addition, the responsiveness of the RMS cell lines to the drugs is highly heterogeneous. Whereas some SMO inhibitors (i.e., LDE225 and HhA) induce strong proapoptotic and antiproliferative effects in some RMS cell lines, others paradoxically induce cellular proliferation at certain concentrations (e.g., 10 μM GDC-0449 or 5 μM cyclopamine in RUCH-2 and Rh41 cells) or can increase HH signaling activity as judged by GLI1 expression (i.e., LDE225, HhA, and cyclopamine). Similarly, some drugs (e.g., HhA) inhibit PI3K/AKT signaling or induce autophagy (e.g., LDE225) in some cell lines, whereas others cannot (e.g., GDC-0449). In addition, the effects of SMO inhibitors are concentration-dependent (e.g., 1 and 10 μM GDC-0449 decrease GLI1 expression in RD cells whereas 30 μM GDC-0449 does not). Together these data show that some SMO inhibitors can induce strong antitumoral effects in some, but not all, RMS cell lines. Due to the highly heterogeneous response, we propose to conduct thorough pretesting of SMO inhibitors in patient-derived short-term RMS cultures or patient-derived xenograft mouse models before applying these drugs to RMS patients.

Inhibition of hedgehog signalling attenuates UVB-induced skin photoageing.

The hedgehog (Hh) signalling pathway regulates normal development and cell proliferation in metazoan organisms, but its aberrant activation can promote tumorigenesis and progression of a variety of aggressive human cancers including skin cancer. Despite its importance, little is known about its role in photoageing, a type of UV-induced skin lesions. In this study, we investigated the involvement of Hh signalling in the photoageing process as well as the use of an Hh-regulating alkaloid compound as a novel therapeutic drug to regulate photoageing in keratinocytes. We found that UVB induced Hh signalling by the expression of Hh ligands and Hh-mediated transcription factors, respectively. Moreover, UVB-induced Hh activation relied on mitogen-activated protein kinase (p38, ERK and JNK) activity and inflammatory responses (upregulation of COX-2, IL-1β, IL-6 and TNF-α), resulting in premature senescence and photoageing in vitro and in vivo. Notably, a selected Hh inhibitor, evodiamine, mediated photoageing blockade in a mouse skin model. Taken together, our findings demonstrated that Hh signalling is associated with UVB-induced photoageing, while pharmacological inhibition of Hh signalling significantly reduced experimental photoageing, indicating its potential for use as a therapeutic target for this disease.

Targeting the Hedgehog signaling pathway in cancer: beyond Smoothened.

An essential role for Hedgehog (Hh) signaling in human cancer has been established beyond doubt. At present, targeting Hh signaling has mainly been investigated with SMO inhibitors. Unfortunately, resistance against currently used SMO inhibitors has already been observed in basal cell carcinoma (BCC) patients. Therefore, the use of Hh inhibitors targeting the signaling cascade more downstream of SMO could represent a more promising strategy. Furthermore, besides the classical canonical way of Hh signaling activation, non-canonical activation of the GLI transcription factors by multiple important signaling pathways (e.g. MAPK, PI3K, TGFβ) has also been described, pinpointing the importance of targeting the transcription factors GLI1/2. The most promising agent in this context is probably the GLI1/2 inhibitor GANT61 which has been investigated preclinically in numerous tumor types in the last few years. In this review, the emerging role of Hh signaling in cancer is critically evaluated focusing on the potential of targeting Hh signaling more downstream of SMO, i.e. at the level of the GLI transcription factors. Furthermore, the working mechanism and therapeutic potential of the most extensively studied GLI inhibitor in human cancer, i.e. GANT61, is discussed in detail. In conclusion, GANT61 appears to be highly effective against human cancer cells and in xenograft mouse models, targeting almost all of the classical hallmarks of cancer and could hence represent a promising treatment option for human cancer.

An in vivo chemical genetic screen identifies phosphodiesterase 4 as a pharmacological target for hedgehog signaling inhibition.

Hedgehog (Hh) signaling plays an integral role in vertebrate development, and its dysregulation has been accepted widely as a driver of numerous malignancies. While a variety of small molecules target Smoothened (Smo) as a strategy for Hh inhibition, Smo gain-of-function mutations have limited their clinical implementation. Modulation of targets downstream of Smo could define a paradigm for treatment of Hh-dependent cancers. Here, we describe eggmanone, a small molecule identified from a chemical genetic zebrafish screen, which induced an Hh-null phenotype. Eggmanone exerts its Hh-inhibitory effects through selective antagonism of phosphodiesterase 4 (PDE4), leading to protein kinase A activation and subsequent Hh blockade. Our study implicates PDE4 as a target for Hh inhibition, suggests an improved strategy for Hh-dependent cancer therapy, and identifies a unique probe of downstream-of-Smo Hh modulation.

Inhibition of Hedgehog signaling pathway impedes cancer cell proliferation by promotion of autophagy

Multiple lines of evidence implicate that aberrant activation of Hedgehog (Hh) signaling is involved in a variety of human cancers. However, the molecular mechanisms underlying how cancer cells respond to Hh inhibition remain to be elucidated. In this study, we found that blockade of Hh signaling suppresses cell proliferation in human cancer cells. Microarray analysis revealed that differentially expressed genes (DEGs) in human cancer cells are enriched in autophagy pathway in response to the inhibition of Hh signaling. Interestingly, inhibition of Hh signaling induced autophagy, whereas activation of Hh signaling by ligand treatments prevented the induction of autophagy. In addition, inhibition of autophagy by 3-methyladenine (3-MA) partially suppressed cytotoxicity induced by inhibition of Hh signaling. Finally, in autophagy deficient cells, cytotoxic effect triggered by inhibition of Hh signaling was partially reversed, indicating the modulation of autophagy by Hh signaling is autophagy-specific. These results suggest that inhibition of Hh signaling impedes cancer cell proliferation in part through induction of autophagy.

Hedgehog signaling and urological cancers.

Hedgehog (Hh) signaling is aberrantly activated in several hematological and solid cancers. Therapeutic options are sometimes lacking for urological cancers because their mechanisms of progression are imperfectly understood. Studies establishing the anti-tumor effects and safety of inhibitors of Hh pathways are needed for tumors in which the Hh pathways are activated. At present vismodegib is clinically available for basal cell carcinoma, and is expected to be extended to treat other cancers. Cholecalciferol, the precursor of active vitamin D3, is a strong inhibitor of Shh-Gli signaling and may have growth inhibitory effects in renal cancer. As a supplementary therapy it may promote tumor regression. Preclinical data in prostate cancer suggest that while suppressing Hh signaling could reduce invasion and metastasis, it may also result in acquired drug resistance after long-term use. Combining Hh inhibitors with ionizing radiation and/or chemotherapy could improve treatment while lessening the risk of acquired drug resistance. Expression of Shh-related ligand gene and Shh-Gli-inducible target genes like FOXM1 or IGF2 is characteristic of urothelial tumor samples. Overexpression of Shh is observed in 96% of non-muscle invasive bladder cancer and 52% of muscle invasive bladder cancer samples. This review summarizes recently reported trends in Hh signaling activation studies in urological cancer, especially focusing on possible clinical applications.

Hedgehog pathway inhibitors promote adaptive immune responses in basal cell carcinoma.

Basal cell carcinomas (BCCs) are tumors ignored by immune surveillance. Activated Hedgehog (Hh) signaling within primary cilia is a key driver in the pathogenesis of BCCs. We examined immune alterations during treatment with systemic Hh inhibitors. We investigated biopsies from patients with BCC before (23 patients) and after 4 weeks of treatment (5 patients) with Hh signaling inhibitor. Ber-Ep4, BCL-2, Ki-67, CD4, CD8, MHC class I, HLA-DR-class II, and SOX9 were analyzed by immunohistochemistry. Primary cilia were analyzed by double immunofluorescence of acetylated tubulin and SOX9. Differential gene expression for 84 cytokines and chemokines was analyzed in 3 patients. After 4 weeks of treatment, we found reduction of Ki-67, SOX9, Ber-EP4, and BCL-2 expression in tumors associated with morphologic signs of squamous differentiation. In addition, the number of cilia-positive BCC cells was significantly decreased. An upregulation of MHC I expression on the cell membranes of residual tumor cells and an influx of CD4(+), HLA-DR-class II(+), and CD8(+) cells with invasion into the tumor cell nests were found. Finally, qPCR arrays showed the differential expression of genes involved in modulating immune responses. We show that Hh pathway inhibitor-induced tumor regression is accompanied by a dynamic change of the microenvironment with a disruption of immune privilege involving an influx of cytotoxic T cells, activation of the adaptive immune functions, and a profound alteration of the local chemokine/cytokine network.

Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma.

We previously reported that aberrant HH pathway activation confers a poor prognosis in rhabdomyosarcoma (RMS). Searching for new treatment strategies we therefore targeted HH signaling. Here, we identify a novel synthetic lethality of concomitant inhibition of HH and PI3K/AKT/mTOR pathways in RMS by GLI1/2 inhibitor GANT61 and PI3K/mTOR inhibitor PI103. Synergistic drug interaction is confirmed by calculation of combination index (CI < 0.2). Similarly, genetic silencing of GLI1/2 significantly increases PI103-induced apoptosis. GANT61 and PI103 also synergize to induce apoptosis in cultured primary RMS cells emphasizing the clinical relevance of this combination. Importantly, GANT61/PI103 cotreatment suppresses clonogenic survival, three-dimensional sphere formation and tumor growth in an in vivo model of RMS. Mechanistic studies reveal that GANT61 and PI103 cooperate to trigger caspase-dependent apoptosis via the mitochondrial pathway, as demonstrated by several lines of evidence. First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis. Second, GANT61/PI103 cotreatment triggers BAK/BAX activation, which contributes to GANT61/PI103-mediated apoptosis, since knockdown of BAK provides protection. Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis. Fourth, GANT61/PI103 cotreatment initiate activation of the caspase cascade via apoptosome-mediated cleavage of the initiator caspase-9, as indicated by changes in the cleavage pattern of caspases (e.g. accumulation of the caspase-9 p35 cleavage fragment) upon addition of the caspase inhibitor zVAD.fmk. Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS.

Efficacy of Hedgehog pathway inhibitors in Basal cell carcinoma.

Basal cell carcinoma (BCC) is the most commonly diagnosed cancer. While most BCCs are amenable to surgery, some tumors can reach a more advanced stage or metastasize, and become ineligible for surgical resection or radiotherapy. Abnormal activation of the Hedgehog (Hh) pathway is a key driver in BCC pathophysiology. Consequently, inhibitors of the Hh pathway have been developed. Molecules that inhibit the receptor protein Smoothened (SMO) are the most advanced in clinical development. Vismodegib is the first-in-class SMO inhibitor and has been approved in a number of countries for the treatment of metastatic or locally advanced BCC. Several molecules have demonstrated antitumoral activity, but treatment may be limited in duration by a number of side effects, and it is not yet established whether these agents are truly curative or whether continued treatment will be required. Resistance to SMO inhibition has been reported in the clinic for which incidence and mechanisms must be elucidated to inform future therapeutic strategies. Intermittent dosing regimens to improve tolerability, as well as neoadjuvant use of Hh pathway inhibitors, are currently under investigation. Here, we review the most recent outcomes obtained with Hh inhibitors under clinical investigation in BCC.

Hedgehog Inhibition Results in the Upregulation of Pro-Tumourigenic αvβ6 Integrin Expression and Function in Cancer

OBJECTIVE: Aberrant Hedgehog (Hh) signalling has been reported in a number of cancers, and Hh inhibitors are in clinical trials. We have previously reported αvβ6 integrin upregulation in cancer. The objective of this study was to examine the relationship between Hedgehog signalling and αvβ6 integrin. STUDY DESIGN:To examine a link between Hh and αvβ6, we overexpressed Gli1 in immortalised keratinocytes. Three-dimensional organotypic assays were used in vitro; and archival human clinical samples were used for in vivo studies.

Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis.