Inhibitor GANT61 Research Articles

Non-canonical GLI1/2 activation by PI3K/AKT signaling in renal cell carcinoma: A novel potential therapeutic target

Renal cell carcinoma (RCC) is the most lethal urologic malignancy; however, the molecular events supporting RCC carcinogenesis and progression remain poorly understood. In this study, based on the analysis of gene expression profile data from human clear cell RCC (ccRCC) and the corresponding normal tissues, we discovered that Hedgehog (HH) pathway component genes GLI1 and GLI2 were significantly elevated in ccRCC. Survival analysis of a large cohort of ccRCC samples demonstrated that the expression of GLI1 and GLI2 was negatively correlated with patient overall survival. Clinical sample-based VHL mutation and cell model-based VHL manipulation studies all indicated that the activation of GLI1 and GLI2 was not affected by VHL status. Further signaling pathway dissections demonstrated that GLI1 and GLI2 were activated by the phosphoinositide 3-kinase (PI3K)/AKT pathway, but not mediated by the canonical HH/SMO/GLI signaling. Up-regulation of GLI1 and GLI2 promoted RCC proliferation and clonogenic ability, whereas, a combination of GLIs inhibitor Gant61 and AKT inhibitor Perifosine synergistically suppressed RCC growth and induced apoptosis in vitro and in vivo. Therefore, this study identifies that GLI1 and GLI2 are critical for RCC carcinogenesis, and also provides an alternative therapeutic strategy for RCC.

Research progress of hedgehog-Gli signal pathway and cytarabine resistance in acute myeloid leukemia

Drug resistance and relapse are bottlenecks of acute myeloid leukemia(AML) treatment, and conquering the drug resistance of AML cells is the key to improving treatment and helping patients fully recovered. As an indispensably crucial chemotherapeutic agent of AML treatment, drug resistance of cytarabine is getting increasing attention. Recently, several studies had demonstrated that the incorrectly triggered hedgehog (Hh)-glioma-associated oncogene (Gli) signal pathway plays a significant role in the drug resistance of cytarabine. Furthermore, Gli gene over-expression is the marker of AML poor prognosis. The inhibitor of Hh-Gli signal pathway, especially Gli specific small molecular inhibitor GANT61 is capable of inhibiting growth and accelerating apoptosis of AML cells. Therefore, this aricle reviews literatures on the relationship between the signaling pathway of Hh-Gli and drug resistance of cytarabine in AML. Key words: Leukemia, myeloid, acute; Cytarabine; Drug resistance; Hedgehog-Gli signal pathway

GLI1 orchestrates CXCR4/CXCR7 signaling to enhance migration and metastasis of breast cancer cells.

The up-regulation of chemokine receptors CXCR4 and CXCR7 impacts on the distant metastasis and prognosis of breast cancer, though knowledge about the regulatory mechanism of their expressions is limited. Meanwhile, the GLI transcription factors of Hedgehog signaling have been reported to play a pivotal role in the development and progression of many types of human cancer. In breast cancer, the increased expression of GLI1 correlated with metastasis and unfavorable overall prognosis, though its molecular mechanism is also not fully understood. Based on our findings that GLI1 enhanced the lung metastasis of breast cancer cells in a mouse model system, we comprehensively screened for genes up-regulated by GLI1 in breast cancer cells, and as such identified CXCR4, CXCR7/ACKR3, and actin-binding protein LCP1/L-PLASTIN, all of which have been reported to be involved in CXCL12-stimulating signaling. In breast cancer cells, we found that GLI1 and GLI2 up-regulated these expressions, while treatment with GLI-specific inhibitor GANT61 reduced the expressions. As for CXCR4, we confirmed it as a direct target of GLI1 through the reporter assay and the chromatin immunoprecipitation assay. We also found that GLI1 enhanced CXCL12-induced ERK phosphorylation and cell migration, both of which were blocked by either CXCR4-specific inhibitor or knockdown of CXCR7 or LCP1. These evidences suggest an indispensable role of GLI1 in the migration and metastasis of breast cancer cells through CXCL12/CXCR4 signaling enhancement.

Abstract 2135: Survivin is a novel target of the Hedgehog/GLI signaling pathway in human tumor cell lines

Abstract Survivin, a pivotal antiapoptotic protein is expressed in tumors whereas its expression in normal tissues is extremely low. It is believed that transcription of the survivin gene is driven mainly by the transcription factor Sp1. We have identified 11 potential consensus sites for GLI trancription factors in the survivin promoter, albeit non of them has been a full consensus. Interestingly, the distal part of the promoter (-1765 to -941, ending at +39) containing 4 GLI sites showed about 2-fold higher activity than the proximal promoter (-941 to +39). The survivin promoter-reporter has been found to be upregulated mainly by plasmids encoding GLI2 and deltaN-GLI2, a more active GLI2 truncated mutant. Its activity was even higher than the Sp1 activity. The promoter-reporter plasmid is also inhibited by the GLI1/2 inhibitor GANT61. Moreover, by analyzing 40 human tumor cell lines of various origin, we have found that the endogenous survivin protein level substantially decreases after the incubation with GANT61 in most cell lines. Real-time PCR for the survivin mRNA in GANT61-sensitive cell lines showed a decrease by up to about 50 per cent. These data suggest that survivin may be a novel important target regulated by the Hedgehog/GLI signaling pathway, which has been reported previously to be deregulated in the majority of tumor types. The work was supported by the grant NT/14005 from IGA, Ministry of Health, Czech Republic. Citation Format: Kateřina Vlčková, Lubica Ondrušová, Jiri Réda, Jiri Vachtenheim, Petra Záková. Survivin is a novel target of the Hedgehog/GLI signaling pathway in human tumor cell lines. [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 2135. doi:10.1158/1538-7445.AM2015-2135

Abstract 3565: Gli2 induces melanoma tumor growth and progression: A critical role of c-Myc

Abstract Gli2 is one of the key transcription factors in sonic hedgehog (Shh) pathway. Aberrant activation of Gli2 has been reported in several malignancies. However, the role of Gli2 in melanoma progression is unclear. Our results showed that knock down of Gli2 in melanoma cells inhibited the proliferation as well as induced apoptosis in melanoma cells in vitro, which was associated with down-regulation of c-Myc. Melanoma cells with stable Gli2 knockdown demonstrated diminished tumor growth when injected subcutaneously in athymic nude mice. In addition, Gli2 inhibitors GANT58 and GANT61 significantly inhibited the growth of melanoma cell lines. Furthermore, oral administration of 5mg/kg piplartine (PL), an alkaloid inhibited the growth of melanoma tumor xenograft by 84% as compared to control. The tumor suppression by PL was associated with inhibition of Gli2 and c-Myc as observed by western blot and immunohistochemistry analysis. The concentration of PL achieved in plasma was 8.5μM whereas in tumor was 6μmol/gm as analyzed by LC-MS/MS. Interestingly, the IC50 of PL at 48 hours was around 3-4μM in a panel of 15 melanoma cells lines, correlating well with the concentrations achieved in vivo. Melanoma cells treated with 2.5, 5 and 7.5μM PL for 48 hours induced significant apoptosis, with concomitant inhibition of Gli2 as well as c-Myc. Gli2 overexpression and treatment with Shh ligand significantly blocked PL mediated inhibition of cell proliferation and induction of apoptosis, establishing the role of Gli2 in PL-mediated growth suppression of melanoma cells. PL inhibited nuclear translocation as well as DNA binding of Gli2 as observed by analysis of Gli2 in nuclear fraction and Chromatin Immunoprecipitation (ChIP) assay, indicating the importance of Gli2 transcriptional activity in melanoma cell proliferation. In conclusion, our results provide convincing evidence that Gli2 mediated induction of c-Myc plays an important in melanoma tumor growth and subsequently establishes that PL mediated inhibition of melanoma cells in vitro and in vivo is associated with inhibition of Gli2. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Neel Manoj Fofaria, Sanjay K. Srivastava. Gli2 induces melanoma tumor growth and progression: A critical role of c-Myc. [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 3565. doi:10.1158/1538-7445.AM2015-3565

Abstract 3245: Targeted inhibition of Hedgehog (HH) signal transduction in rhabdomyosarcoma (RMS) reduces cell survival in vitro and tumor growth in vivo

Abstract RMS is the most common sarcoma of childhood. Approximately 60% of embryonal RMS (ERMS) and more rarely alveolar RMS (ARMS) express HH pathway components. However, roles for the HH signal transduction pathway in RMS biology and as a therapeutic target in RMS have been incompletely characterized. Therefore, we determined if RMS cells lines have HH signaling pathways that are responsive to pathway activation or inhibition in vitro. We demonstrated expression of HH pathway genes (Indian HH, Desert HH, PTCH1, SMO, GLI1, GLI2, and GLI3) by qRT PCR in ERMS (Rh18, Ruch2, SMS-CTR, and RD) and ARMS (Rh 30 and Rh41) cells. We showed high GLI1 protein expression, the downstream mediator of HH signaling, by Western blot in Rh18, Rh30, and Rh41 cells but not in RD or Rh28 cells. Exposure of Rh41 cells to Sonic HH-containing conditioned media caused a 12-fold increase in PTCH1 expression and exposure of Rh28 cells to Sonic HH peptide increased GLI1 expression 1.5-fold, suggesting intact HH signal transduction in some RMS cell lines. Down-regulation of HH pathway activity with the GLI1 inhibitor GANT61 reduced Rh41 and RD cell viability by MTT assays in a dose dependent manner. Reduced cell viability appeared to result at least in part from down-regulation of the anti-apoptotic GLI1 target gene, BCL2. These results suggest a role for HH signaling in RMS cell survival. Next, we used subcutaneous xenografts (with either Rh30 cells, n = 4; Rh41cells, n = 3; or RD cells, n = 3) to assess whether the HH pathway represents a potentially important therapeutic target in RMS in vivo. Mice were treated with either cyclopamine (subcutaneous SMO inhibitor), GANT61 (subcutaneous GLI1 inhibitor), or Vismodegib (oral SMO inhibitor) for 16 days once the tumor volume was 250 mm3. The study endpoints were HH pathway inhibition (qRT PCR), tumor volume (mm3), proliferation (BrdU immunostain), and apoptosis (Caspase immunostain) on day 17 of treatment. Cyclopamine and GANT61 reduced GLI1, PTCH1, and BCL2 expression in 7/10 and 4/10 mice, respectively. We observed partial responses to cyclopamine and GANT61 in Rh41 xenograft mice; GANT61 caused significant regression in tumor volume from baseline (p = 0.03). We did not detect changes in proliferation or apoptosis post-therapy by immunohistochemistry. Our results suggest that targeted inhibition of the HH signaling pathway with small-molecule inhibitors sometimes inhibits RMS xenograft tumor growth in vivo and support further RMS xenograft studies, assessing HH pathway inhibitors in combination with chemotherapeutic agents. Citation Format: Joon Won Yoon, Christopher Chandler, Marilyn Lamm, King-Fu Leong, Stephen Iannaccone, Gregory Taborn, Philip Iannaccone, David Walterhouse. Targeted inhibition of Hedgehog (HH) signal transduction in rhabdomyosarcoma (RMS) reduces cell survival in vitro and tumor growth in vivo. [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 3245. doi:10.1158/1538-7445.AM2015-3245

Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis.

Chronic kidney disease is characterized by interstitial fibrosis and proliferation of scar-secreting myofibroblasts, ultimately leading to end-stage renal disease. The hedgehog (Hh) pathway transcriptional effectors GLI1 and GLI2 are expressed in myofibroblast progenitors; however, the role of these effectors during fibrogenesis is poorly understood. Here, we demonstrated that GLI2, but not GLI1, drives myofibroblast cell-cycle progression in cultured mesenchymal stem cell-like progenitors. In animals exposed to unilateral ureteral obstruction, Hh pathway suppression by expression of the GLI3 repressor in GLI1+ myofibroblast progenitors limited kidney fibrosis. Myofibroblast-specific deletion of Gli2, but not Gli1, also limited kidney fibrosis, and induction of myofibroblast-specific cell-cycle arrest mediated this inhibition. Pharmacologic targeting of this pathway with darinaparsin, an arsenical in clinical trials, reduced fibrosis through reduction of GLI2 protein levels and subsequent cell-cycle arrest in myofibroblasts. GLI2 overexpression rescued the cell-cycle effect of darinaparsin in vitro. While darinaparsin ameliorated fibrosis in WT and Gli1-KO mice, it was not effective in conditional Gli2-KO mice, supporting GLI2 as a direct darinaparsin target. The GLI inhibitor GANT61 also reduced fibrosis in mice. Finally, GLI1 and GLI2 were upregulated in the kidneys of patients with high-grade fibrosis. Together, these data indicate that GLI inhibition has potential as a therapeutic strategy to limit myofibroblast proliferation in kidney fibrosis.

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.

Expression of Hedgehog Pathway Mediator GLI Represents a Negative Prognostic Marker in Human Acute Myeloid Leukemia and Its Inhibition Exerts Antileukemic Effects.

The Hedgehog pathway plays an important role in stem-cell biology and malignant transformation. Therefore, we investigated the expression and prognostic impact of Hedgehog pathway members in acute myeloid leukemia (AML). Pretreatment samples from 104 newly diagnosed AML patients (AMLSG 07-04 trial) were analyzed by qPCR, and expression of Hedgehog family members was correlated with clinical outcome. Inhibition of GLI by GANT61 or shRNA was investigated in AML cells in vitro and in vivo. Expression of receptors Smoothened and Patched-1 and their downstream mediators, GLI1, GLI2, and GLI3, was found in AML patients in contrast to Hedgehog ligands. GLI2 expression had a significant negative influence on event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS; P = 0.037, 0.026, and 0.013, respectively) and was correlated with FLT3 mutational status (P < 0.001). Analysis of a second, independent patient cohort confirmed the negative impact of GLI2 on EFS and OS (P = 0.007 and 0.003, respectively; n = 290). Within this cohort, GLI1 had a negative prognostic impact (P < 0.001 for both EFS and OS). Although AML cells did not express Hedgehog ligands by qPCR, AML patients had significantly increased Desert Hedgehog (DHH) plasma levels compared with healthy subjects (P = 0.002), in whom DHH was presumably provided by bone marrow niche cells. Moreover, the GLI inhibitor GANT61 or knockdown of GLI1/2 by shRNA caused antileukemic effects, including induction of apoptosis, reduced proliferation, and colony formation in AML cells, and a survival benefit in mice. GLI expression is a negative prognostic factor and might represent a novel druggable target in AML.

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.

Mitochondria-derived reactive oxygen species drive GANT61-induced mesothelioma cell apoptosis

Gli transcription factors of the Hedgehog (Hh) pathway have been reported to be drivers of malignant mesothelioma (MMe) cell survival. The Gli inhibitor GANT61 induces apoptosis in various cancer cell models, and has been associated directly with Gli inhibition. However various chemotherapeutics can induce cell death through generation of reactive oxygen species (ROS) but whether ROS mediates GANT61-induced apoptosis is unknown. In this study human MMe cells were treated with GANT61 and the mechanisms regulating cell death investigated. Exposure of MMe cells to GANT61 led to G1 phase arrest and apoptosis, which involved ROS but not its purported targets, GLI1 or GLI2. GANT61 triggered ROS generation and quenching of ROS protected MMe cells from GANT61-induced apoptosis. Furthermore, we demonstrated that mitochondria are important in mediating GANT61 effects: (1) ROS production and apoptosis were blocked by mitochondrial inhibitor rotenone; (2) GANT61 promoted superoxide formation in mitochondria; and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide, and were more resistant to apoptosis induced by GANT61 than wild-type cells. Our data demonstrate for the first time that GANT61 induces apoptosis by promoting mitochondrial superoxide generation independent of Gli inhibition, and highlights the therapeutic potential of mitochondrial ROS-mediated anticancer drugs in MMe.

ErbB2 signaling activates the Hedgehog pathway via PI3K–Akt in human esophageal adenocarcinoma: Identification of novel targets for concerted therapy concepts

The Hedgehog pathway plays an important role in the pathogenesis of several tumor types, including esophageal cancer. In our study, we show an expression of the ligand Indian hedgehog (Ihh) and its downstream mediator Gli-1 in primary resected adenocarcinoma tissue by immunohistochemistry and quantitative PCR in fifty percent of the cases, while matching healthy esophagus mucosa was negative for both proteins. Moreover, a functionally important regulation of Gli-1 by ErbB2–PI3K–mTORC signaling as well as a Gli-1-dependent regulation of Ihh in the ErbB2 amplified esophageal adenocarcinoma cell line OE19 was observed. Treatment of OE19 cells with the Her2 antibody trastuzumab, the PI3K–mTORC1 inhibitor NVP BEZ235 (BEZ235) or the knockdown of Akt1 resulted in a downregulation of Gli-1 and Ihh as well as in a reduction of viable OE19 cells in vitro. Interestingly, the Hedgehog receptor Smo, which acts upstream of Gli-1, was not expressed in OE19 cells and in the majority of primary human esophageal adenocarcinoma, suggesting a non-canonical upregulation of Gli-1 expression by the ErbB2–PI3K axis. To translate our findings into a therapeutic concept, we targeted ErbB2–PI3K–mTORC1 by trastuzumab and BEZ235, combining both compounds with the Gli-1/2 inhibitor GANT61. The triple combination led to significantly stronger reduction of tumor cell viability than cisplatinum or each biological alone. Therefore, concomitant blockage of the ErbB2–PI3K pathway and the Hedgehog downstream mediator Gli-1 may provide a new therapeutic strategy for esophageal cancer.

GLI inhibitor GANT-61 diminishes embryonal and alveolar rhabdomyosarcoma growth by inhibiting Shh/AKT-mTOR axis.

Rhabdomyosarcoma (RMS) typically arises from skeletal muscle. Currently, RMS in patients with recurrent and metastatic disease have no successful treatment. The molecular pathogenesis of RMS varies based on cancer sub-types. Some embryonal RMS but not other sub-types are driven by sonic hedgehog (Shh) signaling pathway. However, Shh pathway inhibitors particularly smoothened inhibitors are not highly effective in animals. Here, we show that Shh pathway effectors GLI1 and/or GLI2 are over-expressed in the majority of RMS cells and that GANT-61, a specific GLI1/2 inhibitor dampens the proliferation of both embryonal and alveolar RMS cells-derived xenograft tumors thereby blocking their growth. As compared to vehicle-treated control, about 50% tumor growth inhibition occurs in mice receiving GANT-61 treatment. The proliferation inhibition was associated with slowing of cell cycle progression which was mediated by the reduced expression of cyclins D1/2/3 & E and the concomitant induction of p21. GANT-61 not only reduced expression of GLI1/2 in these RMS but also significantly diminished AKT/mTOR signaling. The therapeutic action of GANT-61 was significantly augmented when combined with chemotherapeutic agents employed for RMS therapy such as temsirolimus or vincristine. Finally, reduced expression of proteins driving epithelial mesenchymal transition (EMT) characterized the residual tumors.

Reciprocal Regulation of Hypoxia-Inducible Factor 2α and GLI1 Expression Associated With the Radioresistance of Renal Cell Carcinoma

Renal cell carcinoma (RCC) is often considered a radioresistant tumor, but the molecular mechanism underlying its radioresistance is poorly understood. This study explored the roles of hypoxia-inducible factor 2α (HIF2α) and sonic hedgehog (SHH)-GLI1 signaling in mediating the radioresistance of RCC cells and to unveil the interaction between these 2 signaling pathways. The activities of SHH-GLI1 signaling pathway under normoxia and hypoxia in RCC cells were examined by real-time polymerase chain reaction, Western blot, and luciferase reporter assay. The expression of HIF2α and GLI1 in RCC patients was examined by immunohistochemistry, and their correlation was analyzed. Furthermore, RCC cells were treated with HIF2α-specific shRNA (sh-HIF2α), GLI1 inhibitor GANT61, or a combination to determine the effect of ionizing radiation (IR) on RCC cells based on clonogenic assay and double-strand break repair assay. RCC cells exhibited elevated SHH-GLI1 activities under hypoxia, which was mediated by HIF2α. Hypoxia induced GLI1 activation through SMO-independent pathways that could be ablated by PI3K inhibitor or MEK inhibitor. Remarkably, the SHH-GLI1 pathway also upregulated HIF2α expression in normoxia. Apparently, there was a positive correlation between HIF2α and GLI1 expression in RCC patients. The combination of sh-HIF2α and GLI1 inhibitor significantly sensitized RCC cells to IR. Cross-talk between the HIF2α and SHH-GLI1 pathways was demonstrated in RCC. Cotargeting these 2 pathways, significantly sensitizing RCC cells to IR, provides a novel strategy for RCC treatment.

Inhibition of autophagy potentiates the efficacy of Gli inhibitor GANT-61 in MYCN-amplified neuroblastoma cells.

BackgroundAberrant Hedgehog (Hh) signaling is often associated with neuroblastoma (NB), a childhood malignancy with varying clinical outcomes due to different molecular characteristics. Inhibition of Hh signaling with small molecule inhibitors, particularly with GANT-61, significantly suppresses NB growth. However, NB with MYCN amplification is less sensitive to GANT-61 than those without MYCN amplification.MethodsAutophagic process was examined in two MYCN amplified and two MYCN non-amplified NB cells treated with GANT-61. Subsequently, chemical and genetic approaches were applied with GANT-61 together to evaluate the role of autophagy in GANT-61 induced cell death.ResultsHere we show that GANT-61 enhanced autophagy in MYCN amplified NB cells. Both an autophagic inhibitor 3-methyladenine (3-MA) and genetic disruption of ATG5 or ATG7 expression suppressed GANT-61 induced autophagy and significantly increased apoptotic cell death, whereas pre-treatment with an apoptotic inhibitor, Z-VAD-FMK, rescued GANT-61 induced cell death and had no effect on the autophagic process. In the other hand, GANT-61 barely induced autophagy in MYCN non-amplified NB cells, but overexpression of MYCN in MYCN non-amplified NB cells recapitulated GANT-61 induced autophagy seen in MYCN amplified NB cells, suggesting that the level of GANT-61 induced autophagy in NB cells is related to MYCN expression level in cells.ConclusionAberrant Hh signaling activation as an oncogenic driver in NB renders inhibition of Hh signaling an effective measure to suppress NB growth. However, our data suggest that enhanced autophagy concomitant with Hh signaling inhibition acts as a pro-survival factor to maintain cell viability, which reduces GANT-61 efficacy. Besides, MYCN amplification is likely involved in the induction of the pro-survival autophagy. Overall, simultaneous inhibition of both Hh signaling and autophagy could be a better way to treat MYCN amplified NB.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-768) contains supplementary material, which is available to authorized users.

Abstract 2945: Characterization of a novel protein-coding gene named TIHL (Translocated in Hodgkin's Lymphoma)

Abstract Previously, we described the CIITA-BX648577 gene fusion (Steidl C. et al., Nature 2011) was highly expressed in Hodgkin's Lymphoma cell line KM-H2. The Class II Transactivator (CIITA) has been described to function in the regulation of immune responses and its deregulation may serve as a mechanism through which tumor cells evade immunosurveillance. In contrast, the expression and function of the gene/protein encoded by the novel locus BX648577/FLJ27352/C15orf65 is unknown. In the present study, we report the endogenous expression and describe the biological function of the novel gene which we named TIHL (Translocated in Hodgkin's Lymphoma). Comparative analysis in various species revealed high evolutionary conservation (≥50%) between human, mouse, gorilla and Drosophila TIHL orthologs. Using quantitative real time-PCR, we detected expression of full-length TIHL in both healthy and malignant hematopoietic cells. By immunoblot, we detected robust expression of the 13-16 kDa protein in leukemia and lymphoma cell lines using a TIHL-specific antibody. Lentiviral expression of TIHL in human NB4 leukemia cells and murine hematopoietic progenitor HPC-7 cells led to enhanced clonogenicity and increased proliferative capacity with significant increases in the integration of EdU (5-ethynyl-2′-deoxyuridine) and the percentage of cells in S-phase. Likewise, TIHL knockdown using 2 independent lentiviral shRNAs, led to decreased proliferation of both KG1a and NB4 leukemia cells grown in either suspension culture or semi-solid media. Ingenuity Pathway Analysis (IPA) after microarray analysis of NB4 cells overexpressing TIHL highlights significantly altered functional categories including ‘cell growth and proliferation,’ ‘cell death and survival,' and ‘cell cycle,' in line with our previous observations. In silico studies, predicted transcriptional regulators of TIHL including the gene GLI1 (Glioma-Associated Oncogene Homolog 1). GLI1, a positive regulator of the Hedgehog signaling pathway, targets genes involved in cell cycle regulation, cell proliferation and cell death. Treatment of KG1a and NB4 leukemia cells with the small molecule GLI inhibitor GANT61 led to significant inhibition of cell proliferation. Additionally, TIHL mRNA expression was significantly downregulated at increasing concentrations of GANT61 by qRT-PCR. Preliminary data suggests that TIHL overexpression may partially rescue the leukemia inhibitory effects of GANT61 treatment. These data strongly suggest that the novel gene TIHL is involved in cell cycle regulation/modulation in hematopoietic cells. Further studies will involve analysis of the effects of TIHL overexpression or knockdown in healthy donor-derived bone marrow cells, chromatin immunoprecipitation (ChIP)-PCR to confirm its direct transcriptional regulation by GLI1 and co-IP coupled to mass spectrometry to identify direct protein partners of this novel gene. Citation Format: Ujunwa C. Okoye-Okafor, Laura Barreyro, Mario Pujato, Heng Rui Wang, Boris Bartholdy, Britta Will, Tihomira I. Todorova, Masahiro Kawahara, Bruce Woolcock, Andras Fiser, Randy Gascoyne, Christian Steidl, Ulrich Steidl. Characterization of a novel protein-coding gene named TIHL (Translocated in Hodgkin's Lymphoma). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2945. doi:10.1158/1538-7445.AM2014-2945

Abstract 2350: Inhibition of Gli1 as a novel therapeutic target for lung squamous cell carcinoma

Abstract Purpose: It is clear that Hedgehog (Hh) signaling pathway can promote small cell lung cancer development but is not sufficient to drive tumor formation. However, the functional role of Hh pathway in non-small cell lung cancer remains unclear. We found that Gli1, a transcription factor of Hh pathway, is highly expressed in lung squamous cell carcinoma (SSC). The purpose of our study is to identify the functional importance of Gli1 in lung SCC. Methods: We performed a genome-wide gene expression analysis to examine Hh pathway component expression in 275 lung cancer patient specimens. qRT-PCR and western blot were used to validate their expression in SCC lines. To further explore its biological function in lung SCC we manipulated Gli1 expression genetically or pharmacologically followed by colony formation and tumor formation assays. Results: Microarray analysis revealed that Gli1, Gli2, Gli3, and Smoothened are significantly upregulated in lung SCC compared to lung adenocarcinoma (AD). Consistently we found the increased expression of Glis in lung SCC lines relative to AD lines by real-time PCR and western blot. Knocking down Gli1 by siRNA decreased liquid colony formation ability and Gli1-luciferase reporter activity in SCC lines. A significant reduction of cell proliferation and liquid colony formation was observed in shGli1 expressing SCC cells compared to control cells. Gli1 specific inhibitor GANT61 strongly inhibited SCC cell proliferation through inducing cell apoptosis. However, Gli1 expression or transcriptional activity is not regulated by classical Hh pathway upstream regulators in lung SCC. We found that blocking PI3K or ERK pathways by pharmacological inhibitors significantly reduced Gli1 expression and cell survival. In addition, TGF-β stimulation promoted Gli1 expression and transcriptional activity in SCC cells. Conclusions: Our data suggest that Hh pathway downstream factor Gli1 expression is essential for cell proliferation and colony formation in lung SCC. Gli1 is not regulated by canonical Hh pathway regulators, whereas PI3K, ERK, and TGF pathways are involved in its regulation. Blocking Gli1 function is a novel therapeutic strategy for lung SCC treatment. Citation Format: Chunli Shao. Inhibition of Gli1 as a novel therapeutic target for lung squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2350. doi:10.1158/1538-7445.AM2014-2350

Activated hedgehog pathway is a potential target for pharmacological intervention in biliary tract cancer.

Hedgehog (Hh) signalling contributes to carcinogenesis and represents a valid druggable target in human cancers, possibly also in biliary tract cancer (BTC). We analysed the expression of Hh components in BTC using eight heterogeneously differentiated cell lines, xenograft tumours and a human tissue microarray. The dose-, time- and cell line-dependent effects of two Hh inhibitors (cyclopamine and Gant-61) were analysed in vitro for survival, apoptosis, cell cycle distribution and possible synergism with conventional chemotherapeutic agents. In human BTC samples, the sonic Hh ligand and the Gli1 transcription factor showed increased expression in tumours compared to normal adjacent tissue and were significantly associated with high tumour grade and positive lymph node status. In BTC cell lines, we could confirm the Hh component expression at varying extent within the employed cell lines in vitro and in vivo indicating non-canonical signalling. Both Hh inhibitors showed dose-dependent cytotoxicity above 5 µM with a stronger effect for Gant-61 inducing apoptosis whereas cyclopamine rather inhibited proliferation. Cytotoxicity was associated with low cytokeratin expression and higher mesenchymal marker expression such as vimentin. Additionally, drug combinations of Gant-61 with conventional chemotherapy (cisplatin) exerted synergistic effects. In conclusion, Hh pathway is significantly activated in human BTC tissue compared to normal adjacent tissue. The current data demonstrate for the first time an effective anticancer activity of especially Gant-61 in BTC and suggest second generation Hh pathway inhibitors as a potential novel treatment strategy in BTC.

Mode and specificity of binding of the small molecule GANT61 to GLI determines inhibition of GLI-DNA binding.

The GLI genes, GLI1 and GLI2, are transcription factors that regulate target genes at the distal end of the canonical Hedgehog (HH) signaling pathway (SHH->PTCH->SMO->GLI), tightly regulated in embryonic development, tissue patterning and differentiation. Both GLI1 and GLI2 are oncogenes, constitutively activated in many types of human cancers. In colon cancer cells oncogenic KRAS-GLI signaling circumvents the HH-SMO-GLI axis to channel through and activate GLI in the transcriptional regulation of target genes. We have observed extensive cell death in a panel of 7 human colon carcinoma cell lines using the small molecule GLI inhibitor GANT61. Using computational docking and experimental confirmation by Surface Plasmon Resonance, GANT61 binds to the 5-zinc finger GLI1 protein between zinc fingers 2 and 3 at sites E119 and E167, independent of the GLI-DNA binding region, and conserved between GLI1 and GLI2. GANT61 does not bind to other zinc finger transcription factors (KLF4, TFIIβ). Mutating the predicted GANT61 binding sites in GLI1 significantly inhibits GANT61-GLI binding and GLI-luciferase activity. Data establish the specificity of GANT61 for targeting GLI, and substantiate the critical role of GLI in cancer cell survival. Thus, targeting GLI in cancer therapeutics may be of high impact.

Hedgehog–GLI Signaling Inhibition Suppresses Tumor Growth in Squamous Lung Cancer

Lung squamous cell carcinoma (LSCC) currently lacks effective targeted therapies. Previous studies reported overexpression of Hedgehog (HH)-GLI signaling components in LSCC. However, they addressed neither the tumor heterogeneity nor the requirement for HH-GLI signaling. Here, we investigated the role of HH-GLI signaling in LSCC, and studied the therapeutic potential of HH-GLI suppression. Gene expression datasets of two independent LSCC patient cohorts were analyzed to study the activation of HH-GLI signaling. Four human LSCC cell lines were examined for 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. Xenografts in immunodeficient mice were used to determine the in vivo efficacy of GLI inhibitor GANT61. In both cohorts, activation of HH-GLI signaling was significantly associated with the classical subtype of LSCC. In cell lines, genetic knockdown of Smoothened (SMO) produced minor effects on cell survival, whereas GLI2 knockdown significantly reduced proliferation and induced extensive apoptosis. Consistently, the SMO inhibitor GDC-0449 resulted in limited cytotoxicity in LSCC cells, whereas the GLI inhibitor GANT61 was very effective. Importantly, GANT61 demonstrated specific in vivo antitumor activity in xenograft models of GLI(+) cell lines. Our studies demonstrate an important role for GLI2 in LSCC, and suggest GLI inhibition as a novel and potent strategy to treat a subset of patients with LSCC.