Hope M. Amm

Combined modality therapy with TRAIL or agonistic death receptor antibodies

Molecularly targeted therapies, such as antibodies and small molecule inhibitors have emerged as an important breakthrough in the treatment of many human cancers. One targeted therapy under development is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) due to its ability to induce apoptosis in a variety of human cancer cell lines and xenografts, while lacking toxicity in most normal cells. TRAIL and apoptosis-inducing agonistic antibodies to the TRAIL death receptors have been the subject of many preclinical and clinical studies in the past decade. However, the sensitivity of individual cancer cell lines of a particular tumor type to these agents varies from highly sensitive to resistant. Various chemotherapy agents have been shown to enhance the apoptosis-inducing capacity of TRAIL receptor-targeted therapies and induce sensitization of TRAIL-resistant cells. This review provides an overview of the mechanisms associated with chemotherapy enhancement of TRAIL receptor-targeted therapies including modulation of the apoptotic (death receptor expression, FLIP, and Bcl-2 or inhibitors of apoptosis (IAP) families) as well as cell signaling (NFκB, Akt, p53) pathways. These mechanisms will be important in establishing effective combinations to pursue clinically and in determining relevant targets for future cancer therapies.

Mechanisms of drug sensitization to TRA-8, an agonistic death receptor 5 antibody, involve modulation of the intrinsic apoptotic pathway in human breast cancer cells

TRA-8, a monoclonal antibody to death receptor 5 induces apoptosis in various cancer cells; however, the degree of sensitivity varies from highly sensitive to resistant. We have previously shown that resistance to TRA-8 can be reversed by using chemotherapeutic agents, but the mechanism underlying this sensitization was not fully understood. Here, we examined the combination of TRA-8 with doxorubicin or bortezomib in breast cancer cells. In TRA-8–resistant BT-474 and T47D cells, both chemotherapy agents synergistically sensitized cells to TRA-8 cytotoxicity with enhanced activation of apoptosis shown by cleavage of caspases and PARP, reduced Bid, increased proapoptotic Bcl-2 proteins, and increased mitochondrial membrane depolarization. Doxorubicin or bortezomib combined with TRA-8 also reduced Bcl-XL and X-linked inhibitors of apoptosis (XIAP) in treated cells. Furthermore, targeting these proteins with pharmacologic modulators, AT-101, BH3I-2′ and AT-406, produced sensitization to TRA-8. TRA-8 combined with AT-101 or BH3I-2′, inhibitors of antiapoptotic Bcl-2 proteins, produced synergistic cytotoxicity against ZR-75-1, BT-474, and T47D cells. The IAP-targeting compound, AT-406, was synergistic with TRA-8 in BT-474 cells, and to a lesser extent T47D cells. Activation of the intrinsic apoptotic pathway was a common mechanism associated with sensitization of TRA-8–resistant breast cancer cell lines. Collectively, these studies show that the Bcl-2 and IAP families of proteins are involved in TRA-8 and chemotherapy resistance via their modulation of the intrinsic apoptotic pathway. Targeting these proteins with novel agents sensitized TRA-8–resistant breast cancer cells, suggesting this approach may represent a potent therapeutic strategy in the treatment of breast cancer. Mol Cancer Res; 9(4); 403–17. ©2011 AACR.

Targeting the sonic hedgehog pathway in keratocystic odontogenic tumor

Background: Keratocystic odontogenic tumors (KCOT) have a high rate of recurrence and very limited treatment options beyond surgery. Results: Cyclopamine, a smoothened antagonist, reduced KCOT cell viability and signaling components of the hedgehog and NOTCH signaling pathways. Conclusion: Hedgehog signaling mediates KCOT cell survival. Significance: Inhibitors of hedgehog signaling may be valuable in the treatment of KCOT. Keratocystic odontogenic tumors (KCOT) may occur sporadically or associated with the nevoid basal cell carcinoma syndrome. It is a benign aggressive tumor of odontogenic epithelial origin with a high rate of recurrence. A primary human keratocystic odontogenic tumor cell population, KCOT-1, has been established from a tumor explant culture. The KCOT-1 cells were characterized by growth rate, gene expression profiles of major tooth enamel matrix proteins (EMPs), amelogenin (AMELX), enamelin (ENAM), ameloblastin (AMBN), amelotin (AMTN), tumor-related proteins enamelysin (MMP-20), kallikrein-4 (KLK-4), and odontogenic ameloblast-associated protein (ODAM) using quantitative real-time reverse transcription-polymerase chain reaction. Cytokeratin 14 (CK14) was examined by immunohistochemistry. In addition, expression of the members of the sonic hedgehog (SHH) pathway, SHH, patched (PTCH-1), smoothened (SMO), GLI-1, and GLI-2 and of the NOTCH signaling pathway, NOTCH-1, NOTCH-2, NOTCH-3, JAG-2 (Jagged-2), and Delta-like-1 (DLL-1) were evaluated. KCOT-1 cells were treated with SMO antagonist cyclopamine. We found that cyclopamine significantly arrested the growth of KCOT-1 cells in a dose-dependent manner and that the effects of cyclopamine were abolished by adding SHH protein. The protein expression of the SHH pathway was down-regulated by cyclopamine, further confirming that cyclopamine inhibits the SHH signaling pathway; SHH down-regulation correlated with the down-regulation of the NOTCH signaling pathway as well. In conclusion, using an established KCOT-1 cell population, we characterized the gene expression profiles related to the EMPs, SHH, and NOTCH signaling pathway and confirmed that cyclopamine significantly arrested the growth of KCOT-1 cells and may be a viable agent as a novel therapeutic.

Anti-tumor activity of an anti-DR5 monoclonal antibody, TRA-8, in combination with taxane/platinum-based chemotherapy in an ovarian cancer model

OBJECTIVE Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis via binding to death receptors and enhances the anti-tumor effect of conventional cancer therapies. We evaluated the efficacy of TRA-8, an agonistic antibody to DR5, combined with docetaxel and carboplatin in vitro in an intraperitoneal (IP) ovarian cancer model. METHODS Luciferase positive ES2 cells (ES2H) were treated in 96 well plates with TRA-8, carboplatin, docetaxel, and combination therapy. Cell viability was assessed using ATP-lite assay. Apoptosis was confirmed via Western blot analysis. ES2H cells were injected IP into female athymic nude mice. Animals were sorted based on bioluminescent signal with the following treatments: 1) untreated; 2) TRA-8 alone; 3) docetaxel+carboplatin; and 4) docetaxel+carboplatin+TRA-8. Animals receiving TRA-8 antibody were injected IP with 200 μg of TRA-8 twice weekly until death. Animals receiving docetaxel+carboplatin were injected IP with 5mg/kg and 15 mg/kg respectively every 3 weeks until death. Animals were assessed for tumor burden using bioluminescence imaging and overall survival. RESULTS Combination therapy reduced viability of ES2H cells in vitro over single agent therapy. Tumor burden was lowest in the chemotherapy+TRA-8 group at days 23 (p<0.001) and 30 (p = 0.04). Mean survival was greatest in the chemotherapy+TRA-8 group (41 days) compared to the chemotherapy only group (34 days) and control group (27 days) as determined by Kaplan-Meier analysis (p<0.001). CONCLUSION Conventional chemotherapy combined with TRA-8 reduced cell-viability via activation of apoptotic pathways, reduced tumor burden and improved survival in this ovarian cancer model.

Differential Enamel and Osteogenic Gene Expression Profiles in Odontogenic Tumors

Odontogenic tumors occur within the jaw bones and may be derived from odontogenic epithelium or ectomesenchyme or contain active components of both tissue types. We investigated the gene expression profile of enamel matrix proteins (EMPs), genes related to osteogenesis, and the mineralization process in odontogenic tumor cell populations focusing on an ameloblastoma (AB-1), a keratocystic odontogenic tumor (KCOT-1), and a calcifying epithelial odontogenic tumor (CEOT-1). All cell populations were shown to be epithelial in origin by CK14 expression. All tested EMPs were expressed by all odontogenic tumor cell types, with higher transcript levels seen in the AB-1 population especially for AMEL, AMBN, and ODAM. CEOT-1 cell populations showed a greater content of ALP-positive cells as well as higher ALP mRNA levels. Using qRT-PCR, we found a higher expression of 8 genes in the CEOT-1 compared to the AB-1 and KCOT-1. In this study we demonstrated the establishment of AB-1, KCOT-1 and CEOT-1 cell populations. The unique gene expression profiles of AB-1, KCOT-1, and CEOT-1 cells and their interactions with the surrounding microenvironment may support their unique tumor development, progression, and survival.

Relationship between galectin-3 expression and TRAIL sensitivity in breast cancer

Evaluation of: Mazurek N, Byrd JC, Sun Y, Ueno S, Bresalier RS. A galectin-3 sequence polymorphism confers TRAIL sensitivity to human breast cancer cells. Cancer DOI: 10.1002/cncr.26078 (2011) (Epub ahead of print). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors expressed on cancer cells and induces apoptosis. Triple-negative breast cancer cell lines are more sensitive to TRAIL or TRAIL-death receptor agonistic monoclonal antibody-induced apoptosis compared with HER-2/neu-overexpressing or luminal cell lines. The paper under evaluation sought to determine whether the His64/Pro64 polymorphism of galectin-3, which is associated with breast cancer incidence, affects sensitivity to TRAIL. None of five breast cancer cell lines homozygous for Pro64 galectin-3 were sensitive to TRAIL, but two out of two homozygous His64 cell lines and one out of two heterozygous His64 cell lines were sensitive. Transfection of galectin-3 null BT549 breast cancer cells with His64 galectin-3 rendered them sensitive to TRAIL, while Pro64 galectin-3-transfected cells remained resistant to TRAIL. This article highlights that galectin-3 receptor expression and genotype may be useful markers in predicting TRAIL or agonistic antibody sensitivity of breast cancer patients.

Establishment and characterization of a primary calcifying epithelial odontogenic tumor cell population

UNLABELLED Calcifying epithelial odontogenic tumors (CEOTs) are rare neoplasms derived from dental tissue with the unique characteristic of calcifying amyloid-like material. OBJECTIVES To establish primary CEOT epithelial-derived cell populations, investigate the expression of enamel matrix proteins (EMPs), and identify potential ameloblastin (AMBN) and patched 1 (PTCH1) gene alterations. MATERIALS AND METHODS A 28-year-old patient with a lesion of the posterior maxilla, radiographically characterized by a radiolucency with well-defined borders containing mixed radiopacities, agreed to participate with informed consent. The patients biopsy confirmed the diagnosis of CEOT, and a small representative tumor fragment was ascertained for cell culture. Explant cultures were established and used to establish primary cell populations. These were analyzed for morphology, cell proliferation, mineralization activity, expression of epithelial-associated markers (qRT-PCR and immunocytochemistry), and gene mutations of AMBN or PTCH1. DNA was extracted from tumor cells and gene coding and exon-intron boundaries overlapping fragments amplified. PCR products were bidirectional DNA sequenced and compared against reference sequence. RESULTS A CEOT cell population was established and proliferated in culture and could be maintained for several passages. Expression of EMPs, cytokeratin 14 and 17, and patched (PTCH1), as well as ALP activity, was detected. These cells also had the ability to mineralize, similar to the primary tumor. Two AMBN alterations were identified in the sample: c.1323G>A/A441A (rs7680880) and c.1344*+111delA. Two single-nucleotide polymorphisms were identified in the PTCH1 gene. CONCLUSIONS Our data support the establishment of a CEOT-derived cell population, which expresses known epithelial-associated proteins.

Establishment of Singleton-Merten Syndrome pulp cells: evidence of mineralization dysregulation

Abstract Singleton-Merten syndrome (SMS) is a rare disease with a phenotype of dental dysplasia. Currently, the underlying mechanism of this disease is unknown. In order to investigate the functional mechanism of the SMS tooth phenotypes, we isolated dental pulp tissue and established SMS primary pulp cells. These cells exhibited normal morphology and could be maintained in culture. Their ability to express alkaline phosphatase and mineralize was confirmed by in vitro staining. A comparative osteogenesis polymerase chain reaction array analysis was performed revealing 22 genes up-regulated and 8 genes down-regulated greater than 2-fold in SMS versus unaffected pulp cells. Down-regulated genes included ALP, IGF2, TGFBR2 and COL1A1. Collagen type I was reduced in SMS cells as shown by Western blot analysis. Furthermore, matrix metallopeptidase 13 was found to be dramatically increased in SMS pulp cells. Our findings suggest that dentin mineralization is dysregulated in SMS and may contribute to the root phenotype found in this disease.

Matrix metalloproteinase expression in keratocystic odontogenic tumors and primary cells

Abstract Keratocystic odontogenic tumors (KCOTs) are locally invasive, rapidly proliferating cystic lesions of the jaw. The bone-invasive nature of these tumors has been previously associated with the expression of matrix metalloproteinases (MMPs), which degrade the extracellular matrix. The purpose of this study was to assess the expression and activity of MMPs in primary KCOT cells and tumor tissue. Methods: Four independently established KCOT primary cell populations were grown in Dulbecco’s modified Eagle medium supplemented with 10% FBS and antibiotics. Primary cells were analyzed by qRT-PCR and immunohistochemistry (IHC), and for secretion of active MMPs. Primary tumor sections were analyzed by IHC. Results: Of the 18 human MMPs examined, 9 were consistently expressed in primary KCOT cells. MMP-2 and MMP-14 were highly expressed in all KCOT populations, while MMP-1, 3, 11, 12, 16, 17, and 19 were moderately expressed. MMP-3, 11, 12, 16, 17 and 19 were shown to be expressed in KCOTs for the first time. No significant differences in MMPS profiles were found between syndromic (KCOT-3) and non-syndromic cell populations (KCOT-1/2/4). Protein expression of MMP-1, 11, 12, 14 and 16 was confirmed in each KCOT cell populations by IHC. KCOT-3 cells secreted active MMP-2 as determined by a gel zymography assay. Expression of MMP-1, 2, 3, 11, 12, 14, and 16 was confirmed in matching primary KCOT tumor sections representing syndromic and non-syndromic KCOTs. Conclusion: KCOT primary cell populations and tumors express a wide range of MMPs, which likely play a role in the bone-invasive nature of these tumors.

Molecular Signaling in Benign Odontogenic Neoplasia Pathogenesis

Several molecular pathways have been shown to play critical roles in the pathogenesis of odontogenic tumors. These neoplasms arise from the epithelial or mesenchymal cells of the dental apparatus in the jaw or oral mucosa. Next-generation genomic sequencing has identified gene mutations or single nucleotide polymorphisms associated with many of these tumors. In this review, we focus on two of the most common odontogenic tumor subtypes: ameloblastoma and keratocystic odontogenic tumors. We highlight gene expression and protein immunohistological findings and known genetic alterations in the hedgehog, BRAF/Ras/MAPK, epidermal growth factor receptor, Wnt, and Akt signaling pathways relevant to these tumors. These various pathways are explored to potentially target odontogenic tumor cells and prevent growth and recurrence of disease. Through an understanding of these signaling pathways and their crosstalk, molecular diagnostics may emerge as well as the ability to exploit identified molecular differences to develop novel molecular therapeutics for the treatment of odontogenic tumors.