Hh-Gli Signaling Research Articles

Molecular mechanisms of the tyrosine kinase inhibitor pralsetinib activity in in-vitro models of medullary thyroid carcinoma: Aberrant activation of the HH-Gli signaling pathway in acquired resistance

Medullary thyroid carcinoma (MTC) is a malignant tumor with challenging management. Multi-targeted kinase inhibitors (MKI) and tyrosine-kinase inhibitors (TKI) with high specificity for RET protein are approved for advanced MTC treatment. However, their efficacy is hindered by evasion mechanisms of tumor cells. Thus, the aim of this study was the identification of an escape mechanism in MTC cells exposed to a highly selective RET TKI. TT cells were treated with TKI, MKI, and/or the HH-Gli inhibitors, GANT61 and Arsenic Trioxide (ATO), in the presence or absence of hypoxia. RET modifications, oncogenic signaling activation, proliferation and apoptosis were assessed. Additionally, cell modifications and HH-Gli activation were also evaluated in pralsetinib-resistant TT cells. Pralsetinib inhibited RET autophosphorylation and RET downstream pathways activation in normoxic and hypoxic conditions. Additionally, pralsetinib impaired proliferation, induced the activation of apoptosis and, in hypoxic cells, downregulated HIF-1α. Focusing on escape molecular mechanisms associated with therapy, we observed increased Gli1 levels in a subset of cells. Indeed, pralsetinib stimulated the re-localization of Gli1 into the cell nuclei. Treatment of TT cells with both pralsetinib and ATO resulted in Gli1 down-regulation and impaired cell viability. Moreover, pralsetinib-resistant cells confirmed Gli1 activation and up-regulation of its transcriptionally regulated target genes. Altogether, we showed that pralsetinib impairs MTC cell growth and induces cell death, also in hypoxic conditions. The HH-Gli pathway is a new molecular mechanism of escape to pralsetinib therapy that can be overcome through combined therapy.

Signaling Switching from Hedgehog-GLI to MAPK Signaling Potentially Serves as a Compensatory Mechanism in Melanoma Cell Lines Resistant to GANT-61.

Melanoma represents the deadliest skin cancer due to its cell plasticity which results in high metastatic potential and chemoresistance. Melanomas frequently develop resistance to targeted therapy; therefore, new combination therapy strategies are required. Non-canonical signaling interactions between HH-GLI and RAS/RAF/ERK signaling were identified as one of the drivers of melanoma pathogenesis. Therefore, we decided to investigate the importance of these non-canonical interactions in chemoresistance, and examine the potential for HH-GLI and RAS/RAF/ERK combined therapy. We established two melanoma cell lines resistant to the GLI inhibitor, GANT-61, and characterized their response to other HH-GLI and RAS/RAF/ERK inhibitors. We successfully established two melanoma cell lines resistant to GANT-61. Both cell lines showed HH-GLI signaling downregulation and increased invasive cell properties like migration potential, colony forming capacity, and EMT. However, they differed in MAPK signaling activity, cell cycle regulation, and primary cilia formation, suggesting different potential mechanisms responsible for resistance occurrence. Our study provides the first ever insights into cell lines resistant to GANT-61 and shows potential mechanisms connected to HH-GLI and MAPK signaling which may represent new hot spots for noncanonical signaling interactions.

Hedgehog-GLI and Notch Pathways Sustain Chemoresistance and Invasiveness in Colorectal Cancer and Their Inhibition Restores Chemotherapy Efficacy

Colorectal cancer (CRC) is a leading cause of cancer-related mortality and chemoresistance is a major medical issue. The epithelial-to-mesenchymal transition (EMT) is the primary step in the emergence of the invasive phenotype and the Hedgehog-GLI (HH-GLI) and NOTCH signaling pathways are associated with poor prognosis and EMT in CRC. CRC cell lines harboring KRAS or BRAF mutations, grown as monolayers and organoids, were treated with the chemotherapeutic agent 5-Fluorouracil (5-FU) alone or combined with HH-GLI and NOTCH pathway inhibitors GANT61 and DAPT, or arsenic trioxide (ATO) to inhibit both pathways. Treatment with 5-FU led to the activation of HH-GLI and NOTCH pathways in both models. In KRAS mutant CRC, HH-GLI and NOTCH signaling activation co-operate to enhance chemoresistance and cell motility, while in BRAF mutant CRC, the HH-GLI pathway drives the chemoresistant and motile phenotype. We then showed that 5-FU promotes the mesenchymal and thus invasive phenotype in KRAS and BRAF mutant organoids and that chemosensitivity could be restored by targeting the HH-GLI pathway in BRAF mutant CRC or both HH-GLI and NOTCH pathways in KRAS mutant CRC. We suggest that in KRAS-driven CRC, the FDA-approved ATO acts as a chemotherapeutic sensitizer, whereas GANT61 is a promising chemotherapeutic sensitizer in BRAF-driven CRC.

Higher Expressions of SHH and AR Are Associated with a Positive Receptor Status and Have Impact on Survival in a Cohort of Croatian Breast Cancer Patients.

Breast cancers (BC) are usually classified into four molecular subtypes according to the expression of estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors and proliferation marker Ki-67. Despite available anti-hormonal therapies and due to the inherent propensity of some subtypes to develop metastasis, there is a permanent need to discover new prognostic and predictive biomarkers, as well as therapeutic targets for BC. In this study, we used immunohistochemical staining to determine the expression of androgen receptor (AR) and sonic hedgehog protein (SHH), the main ligand of the Hedgehog-GLI (HH-GLI) signaling pathway, in 185 archival primary BC tissue samples and correlated it with clinicopathological characteristics, molecular subtypes, receptors statuses, and survival in a cohort of Croatian BC patients. Results showed that higher SHH and AR expressions were associated with positive receptor status, but increased SHH expression had a negative impact on survival in receptor-negative BCs. On the contrary, higher AR expression was mostly protective. However, multivariate analysis showed that only higher AR expression could be considered as an independent prognostic biomarker for poorer overall survival in triple-negative breast cancer patients (TNBC) (HR 10.9, 95% CI 1.43–83.67; p = 0.021), what could be Croatian population-related. SHH could be a potential target for treating TNBCs and HER2-enriched BCs, in cases where HH-GLI signaling is canonical (SHH-dependent).

Disruption of Gprasp2 down-regulates Hedgehog signaling and leads to apoptosis in auditory cells

GPRASP2 is implicated in nervous system diseases, tumors and immune inflammation. In our previous study, G protein-coupled receptor associated sorting protein 2 (GPRASP2) was identified as a novel causal gene for X-linked recessive syndromic hearing loss (SHL). However, the role of GPRASP2 in auditory function has not been elucidated. The Gprasp2-knockout (KO) mouse HEI-OC1 auditory cells were constructed using CRISPR/Cas9-mediated gene editing. RNA-sequencing (RNA-seq) was used to investigate the differentially expressed genes (DEGs) and DEGs-enriched signaling pathways, which was verified by Western blot. Flow cytometry assay was used to examine cell apoptosis. The cytological pathology was evaluated by laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). Mitochondrial damage was observed in Gprasp2-KO HEI-OC1 cells. RNA-seq analysis suggested that Gprasp2-KO was implicated in the apoptosis process, which could be mediated by Hedgehog (Hh) signaling pathway. The key molecules in Hh signaling pathway (Smo, Gli1, Gli2) were detected to be down-regulated in Gprasp2-KO HEI-OC1 cells. The differential expression of apoptosis molecules (Bcl2, Bax, Caspase-3/cleaved-Caspase-3) indicated that Gprasp2-KO induced apoptosis in HEI-OC1 cells. The treatment of smoothened agonist (Purmorphamine, PUR) activated the Hh-Gli signaling pathway and reduced apoptosis in Gprasp2-KO HEI-OC1 cells. This study revealed that Gprasp2-disruption inhibited Hh signaling pathway and led to cell apoptosis in HEI-OC1 cells, which might provide the potential molecular mechanism of GPRASP2 mutation associated with human SHL.

SHH-N non-canonically sustains androgen receptor activity in androgen-independent prostate cancer cells

Prostate cancer is the second most frequent cancer diagnosed in men worldwide. Localized disease can be successfully treated, but advanced cases are more problematic. After initial effectiveness of androgen deprivation therapy, resistance quickly occurs. Therefore, we aimed to investigate the role of Hedgehog-GLI (HH-GLI) signaling in sustaining androgen-independent growth of prostate cancer cells. We found various modes of HH-GLI signaling activation in prostate cancer cells depending on androgen availability. When androgen was not deprived, we found evidence of non-canonical SMO signaling through the SRC kinase. After short-term androgen deprivation canonical HH-GLI signaling was activated, but we found little evidence of canonical HH-GLI signaling activity in androgen-independent prostate cancer cells. We show that in androgen-independent cells the pathway ligand, SHH-N, non-canonically binds to the androgen receptor through its cholesterol modification. Inhibition of this interaction leads to androgen receptor signaling downregulation. This implies that SHH-N activates the androgen receptor and sustains androgen-independence. Targeting this interaction might prove to be a valuable strategy for advanced prostate cancer treatment. Also, other non-canonical aspects of this signaling pathway should be investigated in more detail and considered when developing potential therapies.

Exosomes from cervical cancer cells facilitate pro-angiogenic endothelial reconditioning through transfer of Hedgehog\u2013GLI signaling components

BackgroundAngiogenic switch is a hallmark feature of transition from low-grade to high-grade cervical intraepithelial neoplasia (CIN) in cervical cancer progression. Therefore, early events leading to locally-advanced cervical metastatic lesions demand a greater understanding of the underlying mechanisms. Recent leads indicate the role of tumor-derived exosomes in altering the functions of endothelial cells in cervical cancer, which needs further investigation.MethodsExosomes isolated from cervical cancer cell lines were assessed for their angiogenic effect on the human umbilical vein endothelial cells (HUVEC) using tube formation and wound healing assay. The exosomal uptake by HUVEC cells was monitored using PKH-67 labelling followed by fluorescence microscopy. Alterations in Hh-GLI signaling components, PTCH1 and GLI1, in HUVEC were measured by immunoblotting. Changes in angiogenesis-related transcripts of vascular endothelial growth factor VEGF-A, VEGF-B, VEGFR2 and angiopoietin-1, angiopoietin-2, osteopontin were measured in exosome-treated HUVEC and in the exosomal RNA by RT-PCR.ResultsEnhanced tube formation, with an increased number of nodes and branching was observed in HUVEC’s treated with exosomes derived from different cervical cancer cell lines. HPV-positive (SiHa and HeLa) cells’ exosomes were more angiogenic. Exosome-treated HUVEC showed increased migration rate. PKH-67 labelled exosomes were found internalized in HUVEC. A high level of PTCH1 protein was detected in the exosome—treated endothelial cells. Subsequent RT-PCR analysis showed increased transcripts of Hh-GLI downstream target genes VEGF-A, VEGFR2, angiopoietin-2, and decreased expression of VEGF-B, and angiopoietin-1, suggestive of active Hh-GLI signaling. These effects were more pronounced in HUVEC’s treated with exosomes of HPV-positive cells. However, these effects were independent of tumor-derived VEGF-A as exosomal cargo lacked VEGF-A transcripts or proteins.ConclusionOverall, the data showed cervical cancer exosomes promote pro-angiogenic response in endothelial cells via upregulation of Hh-GLI signaling and modulate downstream angiogenesis-related target genes. The study provides a novel exosome-mediated mechanism potentially favoring cervical angiogenesis and thus identifies the exosomes as potential pharmacological targets against locally-advanced metastatic cervical lesions.Graphic abstract

Internal Oligoguanidinium Transporter: Mercury-Free Scalable Synthesis, Improvement of Cellular Localization, Endosomal Escape, Mitochondrial Localization, and Conjugation with Antisense Morpholino for NANOG Inhibition to Induce Chemosensitization of Taxol in MCF-7 Cells.

A nontoxic delivery vehicle is essential for the therapeutic applications of antisense phosphorodiamidate morpholino oligonucleotides (PMOs). Though guanidinium-rich or arginine-rich cellular transporter conjugated Vivo-PMO or PPMO has been developed for in vivo application, however, either their toxicity or stability has become an issue. Previously, we reported nonpeptidic internal guanidinium transporter (IGT) mediated delivery of PMO for gene silencing and got encouraging results. In this paper, we report the synthesis of IGT using a Hg-free method for scale up and N-terminal modification of IGT with a suitable hydrophobic or lipophilic group to improve the cell permeability, endosomal escape, and mitochondrial localization and to reduce toxicity in the MTT assay. For the delivery of PMO, IGT-PMO conjugate was synthesized to target NANOG in cells, a transcription factor required for cancer stem cell proliferation and embryonic development and is involved in many cancers. Our data shows IGT-PMO-facilitated NANOG inhibition, and thereby the prevention of EpCAM-N-Cadherin-Vimentin axis mediated epithelial to mesenchymal transition (EMT) in MCF-7 cells. Moreover, unlike taxol, NANOG inhibition influences the expression of stemness factor c-Myc, Hh-Gli signaling proteins, other cancer related factors, and their respective phenotypes in cancer cells. To the best of our knowledge, this is the first report to illustrate that the IGT-PMO-mediated NANOG inhibition increases the therapeutic potential of taxol and induces G0-G1 arrest in cancer cells to prevent cancer progression. However, it warrants further investigation in other cancer cells and preclinical platforms.

Non-canonical Hedgehog Signaling Pathway in Cancer: Activation of GLI Transcription Factors Beyond Smoothened.

The Hedgehog-GLI (HH-GLI) pathway is a highly conserved signaling that plays a critical role in controlling cell specification, cell–cell interaction and tissue patterning during embryonic development. Canonical activation of HH-GLI signaling occurs through binding of HH ligands to the twelve-pass transmembrane receptor Patched 1 (PTCH1), which derepresses the seven-pass transmembrane G protein-coupled receptor Smoothened (SMO). Thus, active SMO initiates a complex intracellular cascade that leads to the activation of the three GLI transcription factors, the final effectors of the HH-GLI pathway. Aberrant activation of this signaling has been implicated in a wide variety of tumors, such as those of the brain, skin, breast, gastrointestinal, lung, pancreas, prostate and ovary. In several of these cases, activation of HH-GLI signaling is mediated by overproduction of HH ligands (e.g., prostate cancer), loss-of-function mutations in PTCH1 or gain-of-function mutations in SMO, which occur in the majority of basal cell carcinoma (BCC), SHH-subtype medulloblastoma and rhabdomyosarcoma. Besides the classical canonical ligand-PTCH1-SMO route, mounting evidence points toward additional, non-canonical ways of GLI activation in cancer. By non-canonical we refer to all those mechanisms of activation of the GLI transcription factors occurring independently of SMO. Often, in a given cancer type canonical and non-canonical activation of HH-GLI signaling co-exist, and in some cancer types, more than one mechanism of non-canonical activation may occur. Tumors harboring non-canonical HH-GLI signaling are less sensitive to SMO inhibition, posing a threat for therapeutic efficacy of these antagonists. Here we will review the most recent findings on the involvement of alternative signaling pathways in inducing GLI activity in cancer and stem cells. We will also discuss the rationale of targeting these oncogenic pathways in combination with HH-GLI inhibitors as a promising anti-cancer therapies.

Role of GLI Transcription Factors in Pathogenesis and Their Potential as New Therapeutic Targets.

GLI transcription factors have important roles in intracellular signaling cascade, acting as the main mediators of the HH-GLI signaling pathway. This is one of the major developmental pathways, regulated both canonically and non-canonically. Deregulation of the pathway during development leads to a number of developmental malformations, depending on the deregulated pathway component. The HH-GLI pathway is mostly inactive in the adult organism but retains its function in stem cells. Aberrant activation in adult cells leads to carcinogenesis through overactivation of several tightly regulated cellular processes such as proliferation, angiogenesis, EMT. Targeting GLI transcription factors has recently become a major focus of potential therapeutic protocols.

PTTG1 cooperated with GLI1 leads to epithelial-mesenchymal transition in esophageal squamous cell cancer.

Pituitary tumor-transforming gene-1 (PTTG1) could acquire its metastasis-promoting effects via inducing epithelial-mesenchymal transition (EMT). However, its role and mechanism in EMT in esophageal squamous cell cancer (ESCC) had not been clearly elucidated. Here, we demonstrated that PTTG1 was overexpressed in ESCC cell lines and tissues especially those with lymph node metastasis. Down regulation of PTTG1 levels dampened the ESCC cells invasion, migration, proliferation ability and colony formation in vitro and inhibited the growth of mouse xenograft model of ESCC cells in vivo. In addition, our in vitro and in vivo experiments consistently showed that decreased PTTG1 led to the inhibition of EMT process. Glioma-associated oncogene homolog1 (GLI1), a key factor in HH-GLI signaling pathway, was also overexpressed in ESCC cells and tissues. Mechanistic studies demonstrated that decreased PTTG1 mitigated the expression levels of GLI1 in vitro and in vivo and ChIP assay also indicated that PTTG1 cooperated with GLI1 by binding to its promoter. Furthermore, overexpression of GLI1 rescued the EMT inhibited by down regulation of PTTG1 in vitro. Together, these data suggested that PTTG1 promoted the invasion ability of ESCC cells via EMT, more important, PTTG1 participated in EMT via activating the expression of GLI1 in ESCC. PTTG1 could be a candidate biomarker for defining ESCC metastasis and useful target for therapy.

Abstract 2442: EWS/FLI1 activates GLI1 indirectly through GLI2 in Ewing Sarcoma

Abstract GLI1 expression in Ewing tumors has been shown to be enhanced by EWS/FLI1 expression in both model transformation systems and in Ewing cell lines. In Ewing cells, GLI1 has been shown to produce significant phenotypic effects consistent with a role in Ewing tumor formation and propagation. Also consistent with an important role is the demonstrated correspondence between the transcriptional target sets of EWS/FLI1 and of GLI1 in Ewing tumor cells. The principal physiologic developmental regulator of GLI1 is the diffusible Sonic Hedgehog ligand which binds the cell surface tumor suppressor Patched with consequent activation of the transmembrane protein Smoothened. In Ewing tumors, the enhancement of GLI1 expression has been shown to be Hedgehog independent and to occur downstream of Smoothened. Indeed, evidence has been published that EWS/FLI1 directly activates the GLI1 promoter. While there is experimental evidence that GLI1 inhibition may be clinically useful in Ewing tumors, preclinical evidence for one inhibitory approach has been disappointing. Interestingly, direct transcriptional activation of GLI1 has not been demonstrated to be of significance in other most other physiologic or pathologic contexts. We speculated that direct transcriptional activation of GLI1 by EWS/FLI1 might be only part of the story in Ewing tumors. Since GLI2 is the most potent physiologic transcriptional activator of GLI1, we undertook to investigate the role of GLI2 in Hedgehog/GLI (HH-GLI) signaling in Ewing Tumors. We found that GLI2 is broadly expressed in Ewing tumors. Based on GLI2 overexpression and on shRNA knockdown of GLI2, we found that GLI2 expression increases GLI1 expression in Ewing cell lines. Via GLI2 knockdown, we have found that GLI2 has biologic effects similar to or greater than GLI1 in Ewing cells. Finally, we demonstrate that EWS/FLI1 enhances nuclear localization of GLI2, suggesting an additional indirect mechanism by which EWS/FLI1 activates HH-GLI signaling and GLI1 expression in Ewing tumors. These data indicate that GLI2 is an active player in HH-GLI pathway activation in Ewing tumors. They also suggest that measures which target GLI2 may have equal or greater efficacy to those aimed at GLI1. Citation Format: Laura Christensen, William A. May. EWS/FLI1 activates GLI1 indirectly through GLI2 in Ewing Sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2442.

GSK3β and Gli3 play a role in activation of Hedgehog-Gli pathway in human colon cancer — Targeting GSK3β downregulates the signaling pathway and reduces cell proliferation

The role of Hedgehog-Gli (Hh-Gli) signaling in colon cancer tumorigenesis has not yet been completely elucidated. Here we provide strong evidence of Hh-Gli signaling involvement in survival of colon cancer cells, with the main trigger of activation being deregulated GSK3β.Our clinical data reveals high expression levels of GSK3β and Gli3 in human colon cancer tissue samples, with positive correlation between GSK3β expression and DUKES' stage. Further experiments on colon cancer cell lines have shown that a deregulated GSK3β upregulates Hh-Gli signaling and positively affects colon cancer cell survival. We show that inhibition of GSK3β with lithium chloride enhances Gli3 processing into its repressor form, consequently downregulating Hh-Gli signaling, reducing cell proliferation and inducing cell death. Analysis of the molecular mechanisms revealed that lithium chloride enhances Gli3–SuFu–GSK3β complex formation leading to more efficient Gli3 cleavage and Hh-Gli signaling downregulation. This work proposes that activation of the Hh-Gli signaling pathway in colon cancer cells occurs non-canonically via deregulated GSK3β. Gli3 seems to be the main pathway effector, highlighting the activator potential of this transcription factor, which is highly dependent on GSK3β function and fine tuning of the Gli3–SuFu–GSK3β platform.

Combination of cyclopamine and tamoxifen promotes survival and migration of mcf-7 breast cancer cells--interaction of hedgehog-gli and estrogen receptor signaling pathways.

Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role in the development of hormone-dependent breast cancer. We aimed to investigate the effects of inhibiting both pathways simultaneously on breast cancer cell survival and the potential interactions between these two signaling pathways. ER-positive MCF-7 cells show decreased viability after treatment with cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor) treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other hand, causes short-term survival of cells, and increased migration. We found upregulated Hh-Gli signaling under these conditions and protein profiling revealed increased expression of proteins involved in cell proliferation and migration. Therefore, even though Hh-Gli signaling seems to be a good potential target for breast cancer therapy, caution must be advised, especially when combining therapies. In addition, we also show a potential direct interaction between the Shh protein and ERα in MCF-7 cells. Our data suggest that the Shh protein is able to activate ERα independently of the canonical Hh-Gli signaling pathway. Therefore, this may present an additional boost for ER-positive cells that express Shh, even in the absence of estrogen.

491: Interaction of Hedgehog-Gli and estrogen receptor signaling pathways in breast cancer cells

Introduction Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role in the development of hormone-dependent breast cancer. The aim of this study is to determine the possible combined effects of Hh-Gli pathway inhibitor cyclopamine with ER inhibitor tamoxifen in breast cancer therapy, and to establish a potential interactions between Hh-Gli and ER signaling in breast cancer. Materials and Methods ER-positive MCF-7 and ER-negative SkBr-3 breast cancer cell lines were used. Drug treatments and Competition experiments were with: cyclopamine 0.5-7.5 μM (Toronto Research Chemicals) and tamoxifen 1-10 μM (Toronto Research Chemicals). MTT and cell migration assays were used. Gene expression studies with : cyclopamine (2.5 μM), Shh protein (3 ng/ml), tamoxifen (1 μM for MCF-7 or 5 µM for SkBr-3), β-estradiol (5 nM, Sigma) or cyclopamine + tamoxifen and Transfection with GLI1 and PTCH1 silencing (Life Technologies) in MCF cells were performed, and QRT-PCR with primers for ERα, c-MYC, RPLP0, PTCH1, SMO, GLI1, SHH, andSUFU.Immunofluorescent staining, Immunoprecipitation and Western blot were performed with primary antibodies: anti-Hh (Santa Cruz, sc-9024), anti-ERα (Santa Cruz, sc-8002), anti-Ptch1 (Santa Cruz, sc-6147), anti-Gli1 (Santa Cruz, sc-20687) and Actin (Santa Cruz, sc-1616). Confocal images were examined using the Manders coefficient plugin of the ImageJ software (v 1.45e). Dynabeads Protein G (Life Technologies) were coated with anti-ERα antibody and cell lysates were immunoprecipitated as per manufacturer’s instructions. Results and Discussion ER-positive cells show decreased viability after treatment with cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor) treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other hand, causes short-term survival of cells, and only longer treatment decreases cell proliferation. The survival effect is strongest 48 hours after simultaneous treatment with both inhibitors, and we found upregulated Hh-Gli signaling under these conditions. In addition to promoting survival, the combination of these two drugs promotes cell migration and longer treatment decreases Hh-Gli signaling as well as ERα levels. We also show a direct interaction between Shh and ERα. Shh protein is able to bind and activate ERα independently of the canonical Hh-Gli signaling pathway. Conclusion The mechanism which is responsible for the increased viability of ER-positive cell line after combined treatment with cyclopamine and tamoxifen is not clear. Although Hh-Gli signaling seems to be a good potential target for breast cancer therapy, combined treatment of cyclopamine and tamoxifen may induce an opposite effect, providing cells with short-term survival.

Abstract A25: GLI inhibition as a new strategy to treat lung squamous cell carcinoma.

Abstract Purpose: Lung squamous cell carcinoma (LSCC) comprises ~30% of non-small cell lung cancers and currently lacks effective targeted therapies. Although the overexpression of HEDGEHOG (HH)-GLI signaling components has been reported in LSCC patient samples, the requirement for HH-GLI signaling in regulating LSCC cellular survival and proliferation remains unknown. In this study, we investigated the role of HH-GLI signaling in LSCC, and studied the therapeutic potential of HH-GLI signaling inhibition. Experimental Design: Two independent LSCC patient cohorts were analyzed for gene expression to study the activation pattern of HH-GLI signaling. Four representative human LSCC cell lines were examined for expression of HH-GLI signaling components. Cell proliferation and apoptosis were assayed in these cells after blocking the HH-GLI pathway by lentiviral-shRNA knockdown or small molecule inhibitors. A xenograft model in immunodeficient mice was used to determine the in vivo therapeutic efficacy of GLI inhibitors. Results: LSCC has been classified into four distinct mRNA expression subtypes. In both cohorts, the classical subtype (~36% of LSCC) showed a HH-GLI activation pattern with increased expression of positive regulators and decreased expression of negative regulators relative to the other three subtypes. Particularly, GLI2 was consistently highly expressed in the classical subtype, and strong positive correlations between GLI2 and the classical subtype markers (SOX2, TP63 and PIK3CA) were observed, suggesting an important role of GLI2 in this subtype. In three GLI2-positive LSCC cell lines, genetic deletion of GLI2 by lentiviral-shRNAs caused extensive apoptosis and significant growth inhibition, indicating a critical role of GLI2 in regulating cell survival and cell death. Pharmacological inhibition of HH-GLI signaling by GLI inhibitor GANT61 phenocopied the loss of GLI2 in GLI2-positive cells, but had little effect on a GLI-negative cell line. In the xenograft model, GANT61 significantly suppressed tumor progression and reduced final tumor weight for GLI2-positive cell lines in comparison to solvent control, but had no effects on the GLI-negative LSCC tumors, suggesting a specific anti-tumor activity of GANT61. The expression of HH downstream targets was significantly reduced by GANT61 both in vitro and in vivo, demonstrating a successful blockage of the HH-GLI pathway. In contrast to GANT61, GDC-0449, a clinically available SMO inhibitor, only produced limited cytotoxicity in vitro despite the universal expression of SMO, indicating the existence of SMO-independent activation of GLI signaling in these cells. Arsenic trioxide (ATO), an FDA-approved treatment of acute promyelocytic leukemia, has recently been shown to inhibit GLI2 ciliary accumulation and promote its degradation in other cell types. Therefore, we are also investigating the clinical relevance of ATO in treating a selected subset of LSCC that has GLI2 overexpression. Conclusions: Our studies reveal an important role of GLI2 in LSCC cell survival. Different from standard-of-care chemotherapy or inhibiting kinase signaling cascades, we present a potential strategy to treat a particular subset of lung squamous cell carcinoma by targeting the GLI transcriptional network. Citation Format: Lingling Huang, Vonn Walter, Neil Hayes, Mark Onaitis. GLI inhibition as a new strategy to treat lung squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A25.

Abstract B236: Inhibition of Hedgehog-GLI signaling suppresses tumor growth in squamous lung cancer.

Abstract Purpose: Lung squamous cell carcinoma (LSCC) comprises ∼30% of non-small cell lung cancers and currently lacks effective targeted therapies. Previous studies reported overexpression of Hedgehog (HH)-GLI signaling components in LSCC, but the requirement for HH-GLI signaling in regulation of cellular survival and proliferation in LSCC remains unknown. In this study, we investigated the role of HH-GLI signaling in LSCC, and studied the therapeutic potential of HH signaling inhibition. Experimental Design: Gene expression datasets of two independent LSCC patient cohorts were analyzed to study the activation pattern of HH-GLI signaling. Four representative human LSCC cell lines were examined for expression of HH-GLI signaling components. Cell proliferation and apoptosis were assayed in these cells after blocking the HH-GLI pathway by lentiviral-shRNA knockdown or small molecule inhibitors. A xenograft model in immunodeficient mice was used to determine the in vivo therapeutic efficacy of GLI inhibitor GANT61. Results: LSCC has been classified into four distinct mRNA expression subtypes. The classical subtype (∼36% of LSCC) displayed increased expression of positive regulators (PTCH1, SMO, GLI1, GLI2) and decreased expression of negative regulators (SUFU, GLI3) relative to the other three subtypes. GLI2 was consistently highly expressed in the classical subtype, and strong positive correlations between GLI2 and the classical subtype makers (SOX2, TP63 and PIK3CA) were observed, indicating a critical role of GLI2 in LSCC. In three cell lines (NCI-H520, NCI-H226 and SK-MES-1), high level of GLI2 was consistently expressed, whereas GLI1 was hardly detectable. Neither GLI1 nor GLI2 was detected in NCI-H2170. Knockdown of GLI2 by lentiviral-shRNAs caused extensive apoptosis and significant growth inhibition in GLI2-positive cells, suggest an indispensable role of GLI2 in regulating cell survival. Pharmacological inhibition of HH-GLI signaling by GLI inhibitor GANT61 phenocopied the loss of GLI2 in GLI-positive cells, but had little effect on the GLI-negative NCI-H2170. In the xenograft model, GANT61 suppressed tumor progression and led to a significant 40% reduction of tumor weight for GLI-positive cell lines in comparison to solvent control, but had no effects on the GLI-negative NCI-H2170 tumors, suggesting a specific anti-tumor efficacy of GANT61. The expression of HH downstream targets (GLI2, PTCH1, HHIP, and CCND1) was significantly reduced by GANT61, demonstrating a successful blockage of the HH-GLI pathway. In contrast to GANT61, GDC-0449, a clinically available SMO inhibitor, only produced limited cytotoxicity despite the universal expression of SMO. Conclusions: Our studies reveal an important role of GLI2 in LSCC cell survival, and suggest GLI inhibition as a novel and potent strategy to treat patients with classical LSCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B236. Citation Format: Lingling Huang, Vonn Walter, Neil Hayes, Mark Onaitis. Inhibition of Hedgehog-GLI signaling suppresses tumor growth in squamous lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B236.

Histone Acetyltransferase PCAF Is Required for Hedgehog–Gli-Dependent Transcription and Cancer Cell Proliferation

The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. In addition, we found that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally, we observed that PCAF silencing reduces the tumor-forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.

Nonclassical hedgehog‐gli signaling and its clinical implications

Hedgehog (Hh) signaling regulates embryonic patterning and organ morphogenesis. It is also involved in regeneration and repair of tissues. Aberrant Hh pathway activation is a feature of many human malignancies. Classical Hh signaling is activated by Hh ligands that can signal in an autocrine or paracrine manner generating a tumor-stromal crosstalk. In contrast to canonical Hh signaling that culminates in the activation of GLI transcription factors, "noncanonical" Hh signaling does not involve GLI transcriptional activity. Several Hh pathway inhibitors have progressed to clinical trials, where the outcomes have not been very encouraging in many solid tumors. Here we discuss the likely role of "nonclassical" Hh-GLI signaling that is activated by growth factors and cytokines from the tumor and/or its microenvironment; these uncouple Hh signaling from the vital regulatory protein Smoothened, and result in the activation of GLI. While efforts are being made to target tumor-intrinsic Hh targets, it is imperative to acknowledge the role of the complex molecular networks and crosstalk between different components of the tumor microenvironment that can result in the emergence of resistance to conventional Hh therapy. These considerations have an important bearing on appreciating the need to mitigate the effects of tumor microenvironment to combat resistance to Hh inhibitors.

Abstract A19: The histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and proliferation

Abstract The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of an aberrant Hh-Gli signaling has thus emerged as an attractive target for anticancer therapy, however the mechanisms that mediate Hh-Gli signaling in vertebrates remain still poorly understood. Here, we show that the histone acetyltransferase (HAT) PCAF/KAT2B is an important regulator of the Hh pathway. Specifically, we demonstrate that PCAF depletion leads to an impairment of Hh activity and reduced expression of Hh target genes. Consequently, downregulation of PCAF in medulloblastoma and glioblastoma cell lines leads to decreased cell proliferation and increased apoptosis. Furthermore, we show that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Our study identifies the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, and we propose PCAF as a potential interesting novel therapeutic target for the treatment of patients with medulloblastoma and glioblastoma. Citation Format: Martina Malatesta, Cornelia Steinhauer, Massimo Squatrito, Kristian Helin. The histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and proliferation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A19.