Hedgehog Target Gene Expression Research Articles

Dual and opposing roles for the kinesin-2 motor, KIF17, in Hedgehog-dependent cerebellar development.

While the kinesin-2 motors KIF3A and KIF3B have essential roles in ciliogenesis and Hedgehog (HH) signal transduction, potential role(s) for another kinesin-2 motor, KIF17, in HH signaling have yet to be explored. Here, we investigated the contribution of KIF17 to HH-dependent cerebellar development, where Kif17 is expressed in both HH-producing Purkinje cells and HH-responding cerebellar granule neuron progenitors (CGNPs). Germline Kif17 deletion in mice results in cerebellar hypoplasia due to reduced CGNP proliferation, a consequence of decreased HH pathway activity mediated through decreased Sonic HH (SHH) protein. Notably, Purkinje cell-specific Kif17 deletion partially phenocopies Kif17 germline mutants. Unexpectedly, CGNP-specific Kif17 deletion results in the opposite phenotype-increased CGNP proliferation and HH target gene expression due to altered GLI transcription factor processing. Together, these data identify KIF17 as a key regulator of HH-dependent cerebellar development, with dual and opposing roles in HH-producing Purkinje cells and HH-responding CGNPs.

Timing is everything: Transcriptional repression is not the default mode for regulating Hedgehog signaling.

Hedgehog (HH) signaling is a conserved pathway that drives developmental growth and is essential for the formation of most organs. The expression of HH target genes is regulated by a dual switch mechanism where GLI proteins function as bifunctional transcriptional activators (in the presence of HH signaling) and transcriptional repressors (in the absence of HH signaling). This results in a tight control of GLI target gene expression during rapidly changing levels of pathway activity. It has long been presumed that GLI proteins also repress target genes prior to the initial expression of HH in a given tissue. This idea forms the basis for the limb bud pre-patterning model for regulating digit number. Recent findings indicate that GLI repressor proteins are indeed present prior to HH signaling but contrary to this model, GLI proteins are inert as they do not regulate transcriptional responses or enhancer chromatin modifications at this time. These findings suggest that GLI transcriptional repressor activity is not a default state as assumed, but is itself regulated in an unknown fashion. We discuss these findings and their implications for understanding pre-patterning, digit regulation, and HH-driven disease.

Casein Kinase 1D Encodes a Novel Drug Target in Hedgehog-GLI-Driven Cancers and Tumor-Initiating Cells Resistant to SMO Inhibition.

Simple SummaryUncontrolled activation of hedgehog (HH)—GLI signaling contributes to the development of several human malignancies. Targeted inhibition of the HH—GLI signaling cascade with small-molecule inhibitors can reduce cancer growth, but patient relapse is very common due to the development of drug resistance. Therefore, a high unmet medical need exists for new drug targets and inhibitors to achieve efficient and durable responses. In the current study, we identified CSNK1D as a novel drug target in the HH—GLI signaling pathway. Genetic and pharmacological inhibition of CSNK1D activity leads to suppression of oncogenic HH—GLI signaling, even in cancer cells in which already approved HH inhibitors are no longer effective due to resistance mechanisms. Inhibition of CSNK1D function reduces the malignant properties of so-called tumor-initiating cells, thereby limiting cancer growth and presumably metastasis. The results of this study form the basis for the development of efficient CSNK1D inhibitors for the therapy of HH—GLI-associated cancers.(1) Background: Aberrant activation of the hedgehog (HH)—GLI pathway in stem-like tumor-initiating cells (TIC) is a frequent oncogenic driver signal in various human malignancies. Remarkable efficacy of anti-HH therapeutics led to the approval of HH inhibitors targeting the key pathway effector smoothened (SMO) in basal cell carcinoma and acute myeloid leukemia. However, frequent development of drug resistance and severe adverse effects of SMO inhibitors pose major challenges that require alternative treatment strategies targeting HH—GLI in TIC downstream of SMO. We therefore investigated members of the casein kinase 1 (CSNK1) family as novel drug targets in HH—GLI-driven malignancies. (2) Methods: We genetically and pharmacologically inhibited CSNK1D in HH-dependent cancer cells displaying either sensitivity or resistance to SMO inhibitors. To address the role of CSNK1D in oncogenic HH signaling and tumor growth and initiation, we quantitatively analyzed HH target gene expression, performed genetic and chemical perturbations of CSNK1D activity, and monitored the oncogenic transformation of TIC in vitro and in vivo using 3D clonogenic tumor spheroid assays and xenograft models. (3) Results: We show that CSNK1D plays a critical role in controlling oncogenic GLI activity downstream of SMO. We provide evidence that inhibition of CSNK1D interferes with oncogenic HH signaling in both SMO inhibitor-sensitive and -resistant tumor settings. Furthermore, genetic and pharmacologic perturbation of CSNK1D decreases the clonogenic growth of GLI-dependent TIC in vitro and in vivo. (4) Conclusions: Pharmacologic targeting of CSNK1D represents a novel therapeutic approach for the treatment of both SMO inhibitor-sensitive and -resistant tumors.

Inflammation-induced JMJD2D promotes colitis recovery and colon tumorigenesis by activating Hedgehog signaling

Histone demethylase JMJD2D can promote gene expression by specifically demethylating H3K9me2/3. The role of JMJD2D in colitis and colitis-associated colorectal cancer (CRC) progression remains unclear. Here, we show that colonic JMJD2D is induced by TNFα during dextran sulfate sodium-induced colitis. JMJD2D-deficient mice exhibit more severe colon damage and defective colon regeneration due to impaired Hedgehog signaling activation after colitis. JMJD2D knockdown in CRC cells suppresses Hedgehog signaling, resulting in reduced CRC growth and metastasis. Mechanistically, JMJD2D promotes Hedgehog target gene expression through interacting with Gli2 to reduce H3K9me3 levels at the promoter. Clinically, JMJD2D expression is upregulated and positively correlated with Gli2 expression in human inflammatory bowel disease specimens and CRC specimens. The JMJD2D inhibitor 5-c-8HQ or aspirin synergizes with Hedgehog inhibitor vismodegib to inhibit CRC cell proliferation and tumorigenesis. Collectively, our findings unveil an essential role of JMJD2D in activating the processes of colonic protection, regeneration, and tumorigenesis.

The hedgehog co-receptor BOC differentially regulates SHH signaling during craniofacial development.

The Hedgehog (HH) pathway controls multiple aspects of craniofacial development. HH ligands signal through the canonical receptor PTCH1, and three co-receptors: GAS1, CDON and BOC. Together, these co-receptors are required during embryogenesis to mediate proper HH signaling. Here, we investigated the individual and combined contributions of GAS1, CDON and BOC to HH-dependent mammalian craniofacial development. Notably, individual deletion of either Gas1 or Cdon results in variable holoprosencephaly phenotypes in mice, even on a congenic background. In contrast, we find that Boc deletion results in facial widening that correlates with increased HH target gene expression. In addition, Boc deletion in a Gas1 null background partially ameliorates the craniofacial defects observed in Gas1 single mutants; a phenotype that persists over developmental time, resulting in significant improvements to a subset of craniofacial structures. This contrasts with HH-dependent phenotypes in other tissues that significantly worsen following combined deletion of Gas1 and Boc Together, these data indicate that BOC acts as a multi-functional regulator of HH signaling during craniofacial development, alternately promoting or restraining HH pathway activity in a tissue-specific fashion.

PS1452 GLI‐1, EFFECTOR OF CANONICAL HEDGEHOG SIGNALING PATHWAY, DOES NOT SEEM TO BE DYSREGULATED IN PATIENTS WITH MYELOFIBROSIS

Background:Although activation of Hedgehog signaling pathway was reported to be present in murine bone marrow transplant model of primary myelofibrosis (PMF) and increased expression of Hedgehog target genes was reported in granulocytes isolated from unselected patients with myeloproliferative neoplasms, therapeutic Hedgehog inhibition showed only limited results in myelofibrosis so far. Also, inconsistent effects of such therapy on Gli1 (effector of hedgehog signaling) were observed.Aims:We aimed to investigate Gli‐1 expression in bone‐marrow tissues of patients with primary and secondary myelofibrosis (SMF) and to assess its clinical correlations.Methods:We retrospectively investigated Gli‐1 mRNA expression in 32 diseased (28 PMF, 4 SMF) and 16 healthy (limited stage agressive Non‐Hodgkin lymphoma) bone marrow samples using real‐time‐polymerase‐chain‐reaction (GLI1 Hs01110766_m1 Thermo Fischer Scientific TaqMan assay). Expression was normalized to Abl and expressed as a ΔCT value. Correlations with clinical parameters were made. Study was approved by the institutional review board. All procedures were in accordance with the Helsinki Declaration. All patients provided written informed consent.Results:Median age of our patients was 70 years, there were 21 (66%) males and 11 (34%) females. Gli‐1 expression did not significantly differ between PMF, SMF and controls (median ΔCT 7.2, 7.3 and 6.9 respectively; P = 0.864).Higher Gli‐1 bone marrow expression was not significantly associated with neither age, gender, JAK2, CALR, MPL mutational status nor degree of bone marrow fibrosis (P>0.05 for all analyses). Only statistically significant associations were present between higher Gli‐1 expression and higher transferrin saturation (P = 0.039; Rho = 0.4) and higher absolute lymphocyte count (P = 0.032; Rho = 0.4).Gli‐1 expression separated at median (ΔCT 7.25) had neutral effect on overall survival in our cohort of patients (P = 0.651) as shown in a Figure.Summary/Conclusion:Our findings indicate Gli‐1 mRNA expression might not be dysregulated in patients with myelofibrosis and has neutral impact on prognosis of these patients. Although limited by small number of patients, retrospective study design and single center experience, our results are in line with previous observations of modest clinical effects of Hedgehog inhibition in patients with myelofibrosis.image

Gli Proteins: Regulation in Development and Cancer.

Gli proteins are transcriptional effectors of the Hedgehog signaling pathway. They play key roles in the development of many organs and tissues, and are deregulated in birth defects and cancer. We review the molecular mechanisms of Gli protein regulation in mammals, with special emphasis on posttranslational modifications and intracellular transport. We also discuss how Gli proteins interact with co-activators and co-repressors to fine-tune the expression of Hedgehog target genes. Finally, we provide an overview of the regulation of developmental processes and tissue regeneration by Gli proteins and discuss how these proteins are involved in cancer progression, both through canonical regulation via the Hedgehog pathway and through cross-talk with other signaling pathways.

Analysis of the transcriptional logic governing differential spatial expression in Hh target genes.

This work provides theoretical tools to analyse the transcriptional effects of certain biochemical mechanisms (i.e. affinity and cooperativity) that have been proposed in previous literature to explain the proper spatial expression of Hedgehog target genes involved in Drosophila development. Specifically we have focused on the expression of decapentaplegic, wingless, stripe and patched. The transcription of these genes is believed to be controlled by enhancer modules able to interpret opposing gradients of the activator and repressor forms of the transcription factor Cubitus interruptus (Ci). This study is based on a thermodynamic approach, which provides expression rates for these genes. These expression rates are controlled by transcription factors which are competing and cooperating for common binding sites. We have made mathematical representations of the different expression rates which depend on multiple factors and variables. The expressions obtained with the model have been refined to produce simpler equivalent formulae which allow for their mathematical analysis. Thanks to this, we can evaluate the correlation between the different interactions involved in transcription and the biological features observed at tissular level. These mathematical models can be applied to other morphogenes to help understand the complex transcriptional logic of opposing activator and repressor gradients.

RITA downregulates Hedgehog-GLI in medulloblastoma and rhabdomyosarcoma via JNK-dependent but p53-independent mechanism

Overactivation of the Hedgehog (HH) signaling pathway is implicated in many cancers. In this study, we demonstrate that the small molecule RITA, a p53 activator, effectively downregulates HH signaling in human medulloblastoma and rhabdomyosarcoma cells irrespective of p53. This is mediated by a ROS-independent activation of the MAP kinase JNK. We also show that in vitro RITA sensitized cells to the GLI antagonist GANT61, as co-administration of the two drugs had more pronounced effects on cell proliferation and apoptosis. In vivo administration of RITA or GANT61 suppressed rhabdomyosarcoma xenograft growth in nude mice; however, co-administration did not further enhance tumor suppression, even though cell proliferation was decreased. RITA was more potent than GANT61 in downregulating HH target gene expression; surprisingly, this suppressive effect was almost completely eliminated when the two drugs were administered together. Notably, RNA-seq demonstrated a broader response of pathways involved in cancer cell growth in the combination treatment, providing a plausible interpretation for tumor reduction in the absence of HH signaling downregulation.

Different Response of Ptch Mutant and Ptch Wildtype Rhabdomyosarcoma Toward SMO and PI3K Inhibitors

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. RMS frequently show Hedgehog (HH) pathway activity, which is predominantly seen in the embryonal subtype (ERMS). They also show activation of Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. Here we compared the therapeutic effectiveness and the impact on HH target gene expression of Smoothened (SMO) antagonists with those of the PI3K inhibitor pictilisib in ERMS with and without mutations in the HH receptor Patched1 (PTCH). Our data demonstrate that growth of ERMS showing canonical Hh signaling activity due to Ptch germline mutations is efficiently reduced by SMO antagonists. This goes along with strong downregulation of the Hh target Gli1. Likewise Ptch mutant tumors are highly responsive toward the PI3K inhibitor pictilisib, which involves modulation of AKT and caspase activity. Pictilisib also modulates Hh target gene expression, which, however, is rather not correlated with its antitumoral effects. In contrast, sporadic ERMS, which usually express HH target genes without having PTCH mutation, apparently lack canonical HH signaling activity. Thus, stimulation by Sonic HE (SHH) or SAG (Smoothened agonist) or inhibition by SMO antagonists do not modulate HH target gene expression. In addition, SMO antagonists do not provoke efficient anticancer effects and rather exert off-target effects. In contrast, pictilisib and other PI3K/AKT/mTOR inhibitors potently inhibit cellular growth. They also efficiently inhibit HH target gene expression. However, of whether this is correlated with their antitumoral effects it is not clear. Together, these data suggest that PI3K inhibitors are a good and reliable therapeutic option for all ERMS, whereas SMO inhibitors might only be beneficial for ERMS driven by PTCH mutations.

SMO Inhibition Modulates Cellular Plasticity and Invasiveness in Colorectal Cancer.

HIGHLIGHTS Preliminary results of this work were presented at the 2016 Academic Surgical Congress, Jacksonville (FL), February 2–4 2016 (Original title: Selective Smo-Inhibition Interferes With Cellular Energetic Metabolism In Colorectal Cancer)This study was funded by “Sapienza—University of Rome” (Funds for young researchers) and “AIRC” (Italian Association for Cancer Research)Hedgehog inhibitor was kindly provided by Genentech, Inc.®.Colon Cancer (CC) is the fourth most frequently diagnosed tumor and the second leading cause of death in the USA. Abnormalities of Hedgehog pathway have been demonstrated in several types of human cancers, however the role of Hedgehog (Hh) in CC remain controversial. In this study, we analyzed the association between increased mRNA expression of GLI1 and GLI2, two Hh target genes, and CC survival and recurrence by gene expression microarray from a cohort of 382 CC patients. We found that patients with increased expression of GLI1 showed a statistically significant reduction in survival. In order to demonstrate a causal role of Hh pathway activation in the pathogenesis of CC, we treated HCT 116, SW480 and SW620 CC cells lines with GDC-0449, a pharmacological inhibitor of Smoothened (SMO). Treatment with GDC-0449 markedly reduced expression of Hh target genes GLI1, PTCH1, HIP1, MUC5AC, thus indicating that this pathway is constitutively active in CC cell lines. Moreover, GDC-0449 partially reduced cell proliferation, which was associated with upregulation of p21 and downregulation of CycD1. Finally, treatment with the same drug reduced migration and three-dimensional invasion, which were associated with downregulation of Snail1, the EMT master gene, and with induction of the epithelial markers Cytokeratin-18 and E-cadherin. These results were confirmed by SMO genetic silencing. Notably, treatment with 5E1, a Sonic Hedgehog-specific mAb, markedly reduced the expression of Hedgehog target genes, as well as inhibited cell proliferation and mediated reversion toward an epithelial phenotype. This suggests the existence of a Hedgehog autocrine signaling loop affecting cell plasticity and fostering cell proliferation and migration/invasion in CC cell lines. These discoveries encourage future investigations to better characterize the role of Hedgehog in cellular plasticity and invasion during the different steps of CC pathogenesis.

Targeting class I histone deacetylases by the novel small molecule inhibitor 4SC-202 blocks oncogenic hedgehog-GLI signaling and overcomes smoothened inhibitor resistance.

Aberrant activation of Hedgehog (HH)/GLI signaling is causally involved in numerous human malignancies, including basal cell carcinoma (BCC) and medulloblastoma. HH pathway antagonists targeting smoothened (SMO), an essential effector of canonical HH/GLI signaling, show significant clinical success in BCC patients and have recently been approved for the treatment of advanced and metastatic BCC. However, rapid and frequent development of drug resistance to SMO inhibitors (SMOi) together with severe side effects caused by prolonged SMOi treatment call for alternative treatment strategies targeting HH/GLI signaling downstream of SMO. In this study, we report that 4SC‐202, a novel clinically validated inhibitor of class I histone deacetylases (HDACs), efficiently blocks HH/GLI signaling. Notably, 4SC‐202 treatment abrogates GLI activation and HH target gene expression in both SMOi‐sensitive and ‐resistant cells. Mechanistically, we propose that the inhibition of HDACs 1/2/3 is crucial for targeting oncogenic HH/GLI signaling, and that class I HDAC inhibitors either in combination with SMOi or as second‐line therapy may improve the treatment options for HH‐associated malignancies with SMOi resistance.

Inhibition of Ciliogenesis Promotes Hedgehog Signaling, Tumorigenesis, and Metastasis in Breast Cancer.

Primary cilia are chemosensors that play a dual role to either activate or repress Hedgehog signaling, depending on presence or absence of ligand, respectively. While inhibition of ciliogenesis has been shown to be characteristic of breast cancers, the functional consequence is unknown. Here, for the first time, inhibition of ciliogenesis led to earlier tumor formation, faster tumor growth rate, higher grade tumor formation, and increased metastasis in the polyoma middle T (PyMT) mouse model of breast cancer. In in vitro model systems, inhibition of ciliogenesis resulted in increased expression of Hedgehog-target genes through a mechanism involving loss of the repressor form of the GLI transcription factor (GLIR) and activation of Hedgehog target gene expression through cross-talk with TGF-alpha (TGFA) signaling. Bioinformatics analysis revealed that increased Hedgehog signaling is frequently associated with increased TGFA; signaling in patients with triple-negative breast cancers (TNBC), a particularly aggressive breast cancer subtype. These results identify a previously unrecognized role for inhibition of ciliogenesis in breast cancer progression. This study identifies inhibition of ciliogenesis as an important event for activation of Hedgehog signaling and progression of breast cancer to a more aggressive, metastatic disease.Implications: These findings change the way we understand how cancer cells turn on a critical signaling pathways and a provide rationale for developing novel therapeutic approaches to target noncanonical Hedgehog signaling for the treatment of breast cancer. Mol Cancer Res; 15(10); 1421-30. ©2017 AACR.

Highlights of This Issue

Epigenetic cancer drivers represent a widely unexplored territory with a high potential for finding theranostic targets. Horie and colleagues combined the powers of promoter resolution Cap Analysis of Gene Expression (CAGE) data from the FANTOM5 consortium with RNA-seq and DNA-methylation data from The Cancer Genome Atlas (TCGA) project and reported a number of epigenetically regulated genes in non-small cell lung cancer (NSCLC); such genes may complement the known mutational drivers. The data also demonstrate that multiple DNA repeats of the REP522 family are epigenetically activated in NSCLC cells and act as promoters for cancer-specific lncRNAs such as RP1-90G24.10, AL022344.4, and PCAT7.Synonymous mutations do not change a protein sequence but can have wide-ranging effects on protein expression, and evidence indicates that synonymous mutations are enriched in tumors. However, previous studies have generally failed to determine whether these mutations have functional effects. In the current Rapid Impact, Bhagavatula and colleagues develop a massively parallel fluorescence assay to measure the effects of synonymous mutations in exon 6 of the tumor suppressor gene TP53, and identify several mutations that cause decreased protein expression due to splicing defects. This methodology suggests a general framework to assay synonymous mutations for their functional role in oncogenesis.Interferon signaling has been shown to be critical in immune-mediated elimination of early malignancies. Here, Walter and colleagues have identified a novel subset of genes, normally produced during active interferon signaling (interferon-stimulated genes, ISG), to be transcriptionally repressed by the progesterone receptor (PR) in both breast cancer cells and patient specimens. PR modulation of interferon transcriptional machinery and subsequent down-regulation of ISG expression represents a mechanism by which early malignant breast lesions evade immunosurveillance processes and progress to clinically relevant tumors.Inhibition of ciliogenesis is common in breast cancers. In this study, it is demonstrated that inhibition of ciliogenesis leads to earlier tumor formation, faster tumor growth, higher grade tumors, and increased metastasis. Furthermore, it is revealed that inhibition of ciliogenesis results in increased expression of Hedgehog-target genes through cross-talk with the TGF-alpha signaling pathway. Activation of the Hedgehog pathway is most frequently observed in the triple-negative breast cancer (TNBC) subtype and is associated with decreased overall survival. These mechanistic insights into how Hedgehog signaling is activated open the door to novel strategies to treat this aggressive form of breast cancer.

Chondrocyte expansion is associated with loss of primary cilia and disrupted hedgehog signalling.

Tissue engineering-based therapies targeting cartilage diseases, such as osteoarthritis, require in vitro expansion of articular chondrocytes. A major obstacle for these therapies is the dedifferentiation and loss of phenotype accompanying chondrocyte expansion. Recent studies suggest that manipulation of hedgehog signalling may be used to promote chondrocyte re-differentiation. Hedgehog signalling requires the primary cilium, a microtubule-based signalling compartment, the integrity of which is linked to the cytoskeleton. We tested the hypothesis that alterations in cilia expression occurred as consequence of chondrocyte dedifferentiation and influenced hedgehog responsiveness. In vitro chondrocyte expansion to passage 5 (P5) was associated with increased actin stress fibre formation, dedifferentiation and progressive loss of primary cilia, compared to primary (P0) cells. P5 chondrocytes exhibited ~50 % fewer cilia with a reduced mean length. Cilia loss was associated with disruption of ligand-induced hedgehog signalling, such that P5 chondrocytes did not significantly regulate the expression of hedgehog target genes (GLI1 and PTCH1). This phenomenon could be recapitulated by applying 24 h cyclic tensile strain, which reduced cilia prevalence and length in P0 cells. LiCl treatment rescued cilia loss in P5 cells, partially restoring hedgehog signalling, so that GLI1 expression was significantly increased by Indian hedgehog. This study demonstrated that monolayer expansion disrupted primary cilia structure and hedgehog signalling associated with chondrocyte dedifferentiation. This excluded the possibility to use hedgehog ligands to stimulate re-differentiation without first restoring cilia expression. Furthermore, primary cilia loss during chondrocyte expansion would likely impact other cilia pathways important for cartilage health and tissue engineering, including transforming growth factor (TGF), Wnt and mechanosignalling.

Inhibition of the hedgehog pathway in patients with basal-cell nevus syndrome: final results from the multicentre, randomised, double-blind, placebo-controlled, phase 2 trial

Aberrant hedgehog signalling underlies the development of basal-cell carcinomas. We previously reported the interim analysis of a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial in patients with the basal-cell nevus (Gorlin) syndrome indicating that the smoothened inhibitor vismodegib reduces basal-cell carcinoma tumour burden and prevents new basal-cell carcinoma growth in patients with basal-cell nevus syndrome. We report the final results of this 36 month trial. In our multicentre, randomised, double-blind, placebo-controlled, phase 2 trial we enrolled patients aged 35-75 years with basal-cell nevus syndrome with at least ten surgically eligible basal-cell carcinomas at the Children's Hospital Oakland, Columbia University outpatient dermatology clinic (NY, USA) and a private practice outpatient dermatology office in Newport Beach (CA, USA). Patients were assigned to vismodegib or placebo (2:1) according to a randomisation sequence generated by computer code. The primary endpoint of the trial of 41 patients was to compare the effect of oral vismodegib (150 mg/day) versus placebo on the incidence of new surgically eligible basal-cell carcinomas after 3 months of treatment. In the subsequent, open-label phase (n=37) patients continued vismodegib at two sites for as long as month 36 (n=25) and at the third site were monitored up to month 36 (n=12). Additional endpoints for this phase were: whether continuous versus interrupted dosing differentially affected tumour burden; time to reach various levels of reduction in tumour burden; reduction in tumour size in patients who took less than 50% of the expected number of vismodegib tablets; reduction in the number of surgical excisions required per year before, during, and after treatment; and the effect of vismodegib on hedgehog target gene expression. We monitored patients at visits every 3 months for up to 36 months. The primary endpoint was analysed on a modified intention-to-treat basis. This trial is registered with ClinicalTrials.gov, number NCT00957229. Between Sept 22, 2009, and Jan 24, 2011, 41 patients were monitored for a median of 36 months (IQR 36-36). Patients treated with vismodegib (n=26) had a mean reduced rate of new surgically eligible basal-cell carcinomas compared with patients randomly assigned to placebo (n=15; two [SD 0·12] new surgically eligible basal-cell carcinomas per patient per year vs 34 [1·32] new surgically eligible basal-cell carcinomas per patient per year, p<0·0001). In the 11 patients initially assigned to placebo, mean cross over to vismodegib reduced the development of new surgically eligible basal-cell carcinomas compared with placebo (0·4 [SD 0·2] new surgically eligible basal-cell carcinomas per patient per year vs 30·0 [7·8] new surgically eligible basal-cell carcinomas per patient per year, p<0·0001). Only three (17%) of 18 patients tolerated vismodegib continuously for the full 36 months. Fewer new surgically eligible basal-cell carcinomas developed in patients receiving vismodegib continuously than in those who interrupted dosing (mean 0·6 [0·72] new surgically eligible basal-cell carcinomas per patient per year vs 1·7 [1·8] new surgically eligible basal-cell carcinomas per patient per year, p<0·0001). Treatment-related grade 3-4 adverse events included weight loss of 20% or more (n=6) and muscle cramps (n=2). Two patients died during the course of the trial, one each from laryngeal and metastatic prostate cancer, deemed probably unrelated to drug. Vismodegib reduces basal-cell carcinoma tumour burden in patients with basal-cell nevus syndrome. Adverse events associated with vismodegib frequently led to interruption of treatment, which is followed by basal-cell carcinoma recurrence. Genentech investigator-initiated trial funding, Clinical and Translational Science Award from the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Cancer Institute, Damon Runyon Cancer Research Foundation Clinical Investigator Award, Swim across America Foundation, and Michael J Rainen Family Foundation.

BCC or not: Sufu keeps it in check.

BCC or not: Sufu keeps it in check.

The BET Bromodomain Inhibitor I-BET151 Acts Downstream of Smoothened Protein to Abrogate the Growth of Hedgehog Protein-driven Cancers

Epigenetic enzymes modulate signal transduction pathways in different biological contexts. We reasoned that epigenetic regulators might modulate the Hedgehog (HH) signaling pathway, a main driver of cell proliferation in various cancers including medulloblastoma. To test this hypothesis, we performed an unbiased small-molecule screen utilizing an HH-dependent reporter cell line (Light2 cells). We incubated Light2 cells with small molecules targeting different epigenetic modulators and identified four histone deacetylase inhibitors and a bromodomain and extra terminal domain (BET) protein inhibitor (I-BET151) that attenuate HH activity. I-BET151 was also able to inhibit the expression of HH target genes in Sufu(-/-) mouse embryonic fibroblasts, in which constitutive Gli activity is activated in a Smoothened (Smo)-independent fashion, consistent with it acting downstream of Smo. Knockdown of Brd4 (which encodes one of the BET proteins) phenocopies I-BET151 treatment, suggesting that Brd4 is a regulator of the HH signaling pathway. Consistent with this suggestion, Brd4 associates with the proximal promoter region of the Gli1 locus, and does so in a manner that can be reversed by I-BET151. Importantly, I-BET151 also suppressed the HH activity-dependent growth of medulloblastoma cells, in vitro and in vivo. These studies suggest that BET protein modulation may be an attractive therapeutic strategy for attenuating the growth of HH-dependent cancers, such as medulloblastoma.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Arsenic Trioxide Prevents Osteosarcoma Growth by Inhibition of GLI Transcription via DNA Damage Accumulation

The Hedgehog pathway is activated in various types of malignancies. We previously reported that inhibition of SMO or GLI prevents osteosarcoma growth in vitro and in vivo. Recently, it has been reported that arsenic trioxide (ATO) inhibits cancer growth by blocking GLI transcription. In this study, we analyzed the function of ATO in the pathogenesis of osteosarcoma. Real-time PCR showed that ATO decreased the expression of Hedgehog target genes, including PTCH1, GLI1, and GLI2, in human osteosarcoma cell lines. WST-1 assay and colony formation assay revealed that ATO prevented osteosarcoma growth. These findings show that ATO prevents GLI transcription and osteosarcoma growth in vitro. Flow cytometric analysis showed that ATO promoted apoptotic cell death. Comet assay showed that ATO treatment increased accumulation of DNA damage. Western blot analysis showed that ATO treatment increased the expression of γH2AX, cleaved PARP, and cleaved caspase-3. In addition, ATO treatment decreased the expression of Bcl-2 and Bcl-xL. These findings suggest that ATO treatment promoted apoptotic cell death caused by accumulation of DNA damage. In contrast, Sonic Hedgehog treatment decreased the expression of γH2AX induced by cisplatin treatment. ATO re-induced the accumulation of DNA damage attenuated by Sonic Hedgehog treatment. These findings suggest that ATO inhibits the activation of Hedgehog signaling and promotes apoptotic cell death in osteosarcoma cells by accumulation of DNA damage. Finally, examination of mouse xenograft models showed that ATO administration prevented the growth of osteosarcoma in nude mice. Because ATO is an FDA-approved drug for treatment of leukemia, our findings suggest that ATO is a new therapeutic option for treatment of patients with osteosarcoma.