Gulay Bulut

Arsenic trioxide inhibits human cancer cell growth and tumor development in mice by blocking Hedgehog/GLI pathway

The Hedgehog (Hh) pathway is activated in some human cancers, including medulloblastoma. The glioma-associated oncogene homolog (GLI) transcription factors are critical mediators of the activated Hh pathway, and their expression may be elevated in some tumors independent of upstream Hh signaling. Thus, therapies targeting GLI transcription factors may benefit a wide spectrum of patients with mutations at different nodal points of the Hh pathway. In this study, we present evidence that arsenic trioxide (ATO) suppresses human cancer cell growth and tumor development in mice by inhibiting GLI1. Mechanistically, ATO directly bound to GLI1 protein, inhibited its transcriptional activity, and decreased expression of endogenous GLI target genes. Consistent with this, ATO inhibited the growth of human cancer cell lines that depended on upregulated GLI expression in vitro and in vivo in a xenograft model of Ewing sarcoma. Furthermore, ATO improved survival of a clinically relevant spontaneous mouse model of medulloblastoma with activated Hh pathway signaling. Our results establish ATO as a Hh pathway inhibitor acting at the level of GLI1 both in vitro and in vivo. These results warrant the clinical investigation of ATO for tumors with activated Hh/GLI signaling, in particular patients who develop resistance to current therapies targeting the Hh pathway upstream of GLI.

GLI1 Is a Direct Transcriptional Target of EWS-FLI1 Oncoprotein

Ewing sarcoma family of tumors (ESFT) is an undifferentiated neoplasm of the bone and soft tissue. ESFT is characterized by a specific chromosomal translocation occurring between chromosome 22 and (in most cases) chromosome 11, which generates an aberrant transcription factor, EWS-FLI1. The function of EWS-FLI1 is essential for the maintenance of ESFT cell survival and tumorigenesis. The Hedgehog pathway is activated in several cancers. Oncogenic potential of the Hedgehog pathway is mediated by increasing the activity of the GLI family of transcription factors. Recent evidence suggests that EWS-FLI1 increases expression of GLI1 by an unknown mechanism. Our data from chromatin immunoprecipitation and promoter reporter studies indicated GLI1 as a direct transcriptional target of EWS-FLI1. Expression of EWS-FLI1 in non-ESFT cells increased GLI1 expression and GLI-dependent transcription. We also detected high levels of GLI1 protein in ESFT cell lines. Pharmacological inhibition of GLI1 protein function decreased proliferation and soft agar colony formation of ESFT cells. Our results establish GLI1 as a direct transcriptional target of EWS-FLI1 and suggest a potential role for GLI1 in ESFT tumorigenesis.

Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells.

Ezrin is a multifunctional protein that connects the actin cytoskeleton to the extracellular matrix through transmembrane proteins. High ezrin expression is associated with lung metastasis and poor survival in cancer. We screened small molecule libraries for compounds that directly interact with ezrin protein using surface plasmon resonance to identify lead compounds. The secondary functional assays used for lead compound selection included ezrin phosphorylation as measured by immunoprecipitation and in vitro kinase assays, actin binding, chemotaxis, invasion into an endothelial cell monolayer, zebrafish and Xenopus embryonic development, mouse lung organ culture and an in vivo lung metastasis model. Two molecules, NSC305787 and NSC668394, that directly bind to ezrin with low micromolar affinity were selected based on inhibition of ezrin function in multiple assays. They inhibited ezrin phosphorylation, ezrin–actin interaction and ezrin-mediated motility of osteosarcoma (OS) cells in culture. NSC305787 mimicked the ezrin morpholino phenotype, and NSC668394 caused a unique developmental defect consistent with reduced cell motility in zebrafish. Following tail vein injection of OS cells into mice, both molecules inhibited lung metastasis of ezrin-sensitive cells, but not ezrin-resistant cells. The small molecule inhibitors NSC305787 and NSC668394 demonstrate a novel targeted therapy that directly inhibits ezrin protein as an approach to prevent tumor metastasis.

Beta-Catenin Accelerates Human Papilloma Virus Type-16 Mediated Cervical Carcinogenesis in Transgenic Mice

Human papilloma virus (HPV) is the principal etiological agent of cervical cancer in women, and its DNA is present in virtually all of these tumors. However, exposure to the high-risk HPV types alone is insufficient for tumor development. Identifying specific collaborating factors that will lead to cervical cancer remains an unanswered question, especially because millions of women are exposed to HPV. Our earlier work using an in vitro model indicated that activation of the canonical Wnt pathway in HPV-positive epithelial cells was sufficient to induce anchorage independent growth. We therefore hypothesized that constitutive activation of this pathway might function as the “second hit.” To address this possibility, we developed two double-transgenic (DT) mouse models, K14-E7/ΔN87βcat and K14-HPV16/ΔN87βcat that express either the proteins encoded by the E7 oncogene or the HPV16 early region along with constitutively active β-catenin, which was expressed by linking it to the keratin-14 (K14) promoter. We initiated tumor formation by treating all groups with estrogen for six months. Invasive cervical cancer was observed in 11% of the K14-ΔN87βcat mice, expressing activated β-catenin and in 50% of the animals expressing the HPV16 E7 oncogene. In double-transgenic mice, coexpression of β-catenin and HPV16 E7 induced invasive cervical cancer at about 7 months in 94% of the cases. We did not observe cervical cancer in any group unless the mice were treated with estrogen. In the second model, K14-HPV16 mice suffered cervical dysplasias, but this phenotype was not augmented in HPV16/ΔN87βcat mice. In summary, the phenotypes of the K14-E7/ΔN87βcat mice support the hypothesis that activation of the Wnt/β-catenin pathway in HPV-associated premalignant lesions plays a functional role in accelerating cervical carcinogenesis.

HGF/c-Met Overexpressions, but not Met Mutation, Correlates with Progression of Non-small Cell Lung Cancer

Hepatocyte Growth Factor (HGF) and its receptor c-Met are suggested to play an important role in progression of solid organ tumors by mediating cell motility, invasion and metastasis. Overexpression of HGF and c-Met have been shown in non-small-cell lung cancer (NSCLC). However, their role in tumor progression is not clearly defined. The aim of this study is to determine the role of HGF/c-Met pathway and its association with invasion related markers and clinicopathologic parameters in NSCLC. Immunohistochemical analysis was performed on 63 paraffin-embedded NSCLC tumor sections. The expressions of invasion related markers such as Matrix Metalloproteinases (MMPs) 2 and 9, Tissue Inhibitor Metalloproteinase (TIMP) 1 and 3 and RhoA were also examined. Co-expression of HGF/c-Met was significantly associated with lymph node invasion and TIMP-3 and RhoA overexpressions. There were positive correlation between TIMP-3 overexpression and advanced stage and negative correlation between RhoA overexpression and survival. DNA sequencing for Met mutations in both nonkinase and tyrosine kinase (TK) domain was established. A single nucleotide polymorphism (SNP) in sema domain and two SNPs in TK domain of c-Met were found. There was no statistically significant correlation between the presence of c-Met alterations and clinicopathologic parameters except shorter survival time in cases with two SNPs in TK domain. These results suggest that HGF/c-Met might exert their effects in tumor progression in association with RhoA and probably with TIMP-3. The blockade of the HGF/c-Met pathway with RhoA and/or TIMP-3 inhibitors may be an effective therapeutic target for NSCLC treatment.

The E6 Oncoprotein from HPV16 Enhances the Canonical Wnt/β-catenin Pathway in Skin Epidermis in vivo.

The contribution of the Wnt signaling pathway to human papilloma virus (HPV)-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk HPVs (HR-HPV), β-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the PDZ-binding domain of E6 protein. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of β-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of β-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2+/LacZ mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of β-catenin, the accumulation of cellular β-catenin–responsive genes, and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice) or in combination with Axin2+/LacZ. Conversely, cotransfection with either E6 or E6ΔPDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that used a luciferase Wnt/β-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only. Mol Cancer Res; 10(2); 250–8. ©2011 AACR.

Ezrin Inhibition Up-regulates Stress Response Gene Expression

Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes.

Abstract 3906: Design, synthesis and biological evaluation of 2nd generation ezrin inhibitors for metastatic osteosarcoma

Though advancements in chemotherapy and surgical techniques have ameliorated treatment of primary osteosarcoma (OS), the metastatic phenotype remains a clinical challenge. Overall five-year survival rates for patients with localized OS have improved to 60-70%, yet survival rates of patients with metastasis remains at 20-30% with mortality linked to metastatis-induced respiratory failure. Therefore, targeting fundamental molecular events that lead to metastasis may yield significant benefit to patients with OS. Accumulating evidence from clinical samples and pre-clinical animal models suggests that ezrin is a key regulator in the metastasis of OS cells. Ezrin is a multifunctional protein that connects the actin cytoskeleton to extracellular matrix through transmembrane proteins and a critical component for cell motility, adhesion and shape. We have recently identified that a small molecule, NSC 668394 acts as a potent and selective inhibitor of ezrin function and inhibits migration in both in-vitro and in-vivo models. Moreover, suppression of ezrin phosphorylation by NSC 668394 significantly reduced the metastatic behavior in cellular and animal models and has thus emerged as an important lead inhibitor. Consequently, we conducted a series of structure-activity-relationship (SAR) studies that monitored direct binding; OS cell migration and HUVEC monolayer invasion in ‘real-time’ with surface-plasmon resonance and electrical impedance technology. From our 2nd generation library, we have designed novel candidate inhibitors which feature enhanced ezrin-binding affinity, and improved anti-migration and anti-invasion activities. In addition to their potency against the different stages of OS metastasis, these compounds possess other desirable attributes for development including good druggable physical-chemical characteristics and are currently undergoing further preclinical evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3906. doi:1538-7445.AM2012-3906

Generation of K14-E7/∆N87βcat double transgenic mice as a model of cervical cancer.

Nearly all cervical cancers are initiated by a subset of high-risk human papilloma viruses (HPVs). However, cervical cancers develop only in a small fraction of women who are infected with these viruses. HPV is required, but not sufficient for developing cervical cancer. Activation of complementary signaling pathways appears to be necessary for malignant transformation of cervical epithelial cells that are immortalized by HPV. Here, we describe the creation and maintenance of a double transgenic mouse model that is based on constitutively active Wnt/β-catenin signaling in cervical epithelial cells expressing the HPV oncoprotein E7. These mice develop invasive cervical squamous carcinomas within 6 months with an average penetrance of 94 %.

Mutation Analysis of the Vangl2 Coding Region Revealed No Common Cause for Tetralogy of Fallot

Vangl2 (Van Gogh-like 2) protein acts via non-canonical Wnt signalling to regulate polarized cell movements during development of the proximal outflow tract in vertebrate embryos. Recently, it has been shown that mutations of the Vangl2 gene cause aortic arch defects that are characteristic of the loop-tail (Lp) mouse and they have also became a strong candidate for causing congenital outflow tract defects in humans. Thus, in this study Tetralogy of Fallot (ToF), which comprises a group of syndromes that constitutes the most frequent cause of congenital cardiac outflow abnormalities in humans, was analysed for mutations within all coding regions of the Vangl2 gene. Based on direct sequencing data from a combination of 20 patients with ToF and 22 healthy people, three polymorphisms have been identified in exon 6 and exon 7 which do not change the amino acid sequence. It was concluded, therefore, that there is no specific mutation responsible for the ToF phenotype in the Vangl2 gene.