Overcoming Resistance to Standard-of-Care Therapies for Head and Neck Squamous Cell Carcinomas.

Although there have been some advances during in recent decades, the treatment of head and neck squamous cell carcinoma (HNSCC) remains challenging. Resistance is a major issue for various treatments that are used, including both the conventional standards of care (radiotherapy and platinum-based chemotherapy) and the newer EGFR and checkpoint inhibitors. In fact, all the non-surgical treatments currently used for HNSCC are associated with intrinsic and/or acquired resistance. Herein, we explore the cellular mechanisms of resistance reported in HNSCC, including those related to epigenetic factors, DNA repair defects, and several signaling pathways. This article discusses these mechanisms and possible approaches that can be used to target different pathways to sensitize HNSCC to the existing treatments, obtain better responses to new agents, and ultimately improve the patient outcomes.

A Retrospective Analysis of 129 Ameloblastoma Cases: Clinical and Demographical Trends from a Single Institution.

Ameloblastomas are benign neoplasms of the jaw, but frequently require extensive surgery. The aim of the study was to analyze the demographic and clinicopathological features of ameloblastoma cases at a single Oral and Maxillofacial Surgery group in the United States. STUDY DESIGN: A retrospective chart review of patients evaluated for ameloblastoma between 2010 and 2020 at a single tertiary care center. Age, race, sex, tumor size, tumor location, and histological subtypes were recorded. RESULTS: A total of 129 cases of ameloblastoma were recorded with a mean patient age of 42 ± 18.6 years (range 9-91 years old), male to female ratio 1.08:1. Ameloblastoma presenting in the mandible outnumbered maxilla in primary (118 to 8, respectively) and recurrent cases (8 to 1, respectively). There was a higher prevalence of ameloblastoma in Black patients (61.3%) with mean age of Black patients occurring at 40.5 years and the mean age of White patients occurring at 47.8 years and mean tumor size trended larger in the Black patients (15.7 cm2) compared to White patients (11.8 cm2). CONCLUSION: Data suggests a strong influence of racial factors on the incidence of ameloblastoma, with regards to size, Black patients with ameloblastoma trended higher and more data is needed to clearly elucidate any relationship between the tumor size and race, as other factors may influence the size (such as time to discovery).

Recurrent central odontogenic fibroma in a patient with nevoid basal cell carcinoma syndrome: case report and in vitro analysis.

OBJECTIVE: Central odontogenic fibromas (COF) are rare, benign tumors derived from dental mesenchymal tissue that may occur in the maxilla or mandible. This report describes primary and recurrent COF in the mandible of a patient with nevoid basal cell carcinoma syndrome (NBCCS). STUDY DESIGN: A 36-year-old African American male presented with a COF and its recurrence 17 months later. Tissue pieces were obtained from both occurrences with IRB-approved signed consent. Collected tissue pieces were dissected; one portion was formalin-fixed and paraffin-embedded, and the other was cultured for the isolation of cell populations from the primary (COdF-1) and recurrent (COdF-1a) tumors. Quantification real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, and DNA sequencing were used for gene and protein analysis of the primary tumor and cell populations. RESULTS: Histopathologic analysis of the tumor showed sparse odontogenic epithelial cords in fibrous connective tissue, and qRT-PCR analysis of tumor and cell populations (COdF-1 and COdF-1a) detected VIM, CK14, CD34, CD99 and ALPL mRNA expression. Protein expression was confirmed by immunohistochemistry. CD34 expression in primary tissues was higher than in tumor cells due to tumor vascularization. DNA sequencing indicated the patient had PTCH1 mutations. CONCLUSIONS: Histopathology, mRNA, and protein expression indicate the rare occurrence of COF in a patient with mutated PTCH1 gene and NBCCS.

89Zr-panitumumab PET imaging for preoperative assessment of ameloblastoma in a PDX model.

Accurate assessment of tumor margins with specific, non-invasive imaging would result in the preservation of healthy tissue and improve long-term local tumor control, thereby reducing the risk of recurrence. Overexpression of epidermal growth factor receptor (EGFR) has been used in other cancers as an imaging biomarker to identify cancerous tissue. We hypothesize that expression of EGFR in ameloblastomas may be used to specifically visualize tumors. The aims of this study are to measure the specificity of radiolabeled 89Zr-panitumumab (an EGFR antibody) in vivo using patient-derived xenograft (PDX) models of ameloblastoma and positron emission tomography/computed tomography (PET/CT) scans. In PDX of ameloblastomas from four patients (AB-36, AB-37, AB-39 AB-53), the biodistribution of 89Zr-panitumumab was measured 120 h post-injection and was reported as the injected dose per gram of tissue (%ID/g; AB-36, 40%; AB-37, 62%; AB-39 18%; AB-53, 65%). The radiolabeled %ID/g was significantly greater in tumors of 89Zr-panitumumab-treated mice that did not receive unlabeled panitumumab as a blocking control for AB-36, AB-37, and AB-53. Radiolabeled anti-EGFR demonstrates specificity for ameloblastoma PDX tumor xenografts, we believe 89Zr-panitumumab is an attractive target for pre-surgical imaging of ameloblastomas. With this technology, we could more accurately assess tumor margins for the surgical removal of ameloblastomas.

The use of hedgehog antagonists in cancer therapy: a comparison of clinical outcomes and gene expression analyses.

Hedgehog (HH) signaling, a critical developmental pathway, has been implicated in cancer initiation and progression. With vismodegib and sonidegib having been approved for clinical use, increasing numbers of HH inhibitors alone and in combination with chemotherapies are in clinical trials. Here we highlight the clinical research on HH antagonists and the genetics of response to these compounds in human cancers. Selectivity of HH inhibitors, determined by decreased pathway transcriptional activity, has been demonstrated in many clinical trials. Patients with advanced/metastatic basal cell carcinoma have benefited the most, whereas HH antagonists did little to improve survival rates in other cancers. Correlation between clinical response and HH gene expression vary among different cancer types. Predicting response and resistance to HH inhibitors presents a challenge and continues to remain an important area of research. New approaches combine standard of care chemotherapies and molecularly targeted therapies to increase the clinical utility of HH inhibitors.

Mandibular undifferentiated pleomorphic sarcoma: Molecular analysis of a primary cell population.

BACKGROUND: Undifferentiated pleomorphic sarcomas are one of the most common subtypes of soft tissue sarcomas. These are aggressive mesenchymal tumors and are devoid of the major known biomarkers except vimentin. Our objective was to establish and characterize a primary cell population from a mandibular UPS specimen. METHODS: The tumor was surgically removed from the right mandible of a 24-year-old male with IRB approved signed consent. Tumor was dissected, cultured ex vivo, and a cell population, MUPS-1, were isolated from outgrowths. Gene and protein expression profiles of both the primary tumor and the derived there from cells were obtained by quantitative RT-PCR and immunohistochemistry and included markers of epithelial, endothelial, and mesenchymal differentiation. To better define potential biomarkers, MUPS-1 cells were additionally characterized by RNA sequencing analysis. RESULTS: Pathological analysis of primary tumor tissue revealed a sarcoma demonstrating multiple pathways of differentiation simultaneously with myxoid, fibrous, and osseous tissue. The isolated cells had a spindle cell-like morphology, were maintained in culture for greater than 20 passages, and formed colonies in soft agar indicating tumorigenicity. The cells, similar to the primary tumor, were strongly positive for vimentin and moderately expressed alkaline phosphatase. RNA-seq analysis revealed the tumor over-expressed several genes compared to normal tissue, including components of the Notch signaling pathway, NOTCH3 and JAG1. CONCLUSIONS: We have successfully established an undifferentiated pleomorphic sarcoma cell population, which will provide a valuable resource for studying fundamental processes and potentially serving as a platform for exploring therapeutic strategies for sarcomas.

Comparison of Panitumumab-IRDye800CW and 5-Aminolevulinic Acid to Provide Optical Contrast in a Model of Glioblastoma Multiforme.

Maximal safe resection of malignant tissue is associated with improved progression-free survival and better response to radiation and chemotherapy for patients with glioblastoma (GBM). 5-Aminolevulinic acid (5-ALA) is the current FDA-approved standard for intraoperative brain tumor visualization. Unfortunately, autofluorescence in diffuse areas and high fluorescence in dense tissues significantly limit discrimination at tumor margins. This study is the first to compare 5-ALA to an investigational new drug, panitumumab-IRDye800CW, in the same animal model. A patient-derived GBM xenograft model was established in 16 nude mice, which later received injections of 5-ALA, panitumumab-IRDye800CW, IRDye800CW, 5-ALA and IRDye800CW, or 5-ALA and panitumumab-IRDye800CW. Brains were prepared for multi-instrument fluorescence imaging, IHC, and quantitative analysis of tumor-to-background ratio (TBR) and tumor margin accuracy. Statistical analysis was compared with Wilcoxon rank-sum or paired t test. Panitumumab-IRDye800CW had a 30% higher comprehensive TBR compared with 5-ALA (P = 0.0079). SDs for core and margin regions of interest in 5-ALA-treated tissues were significantly higher than those found in panitumumab-IRDye800CW-treated tissues (P = 0.0240 and P = 0.0284, respectively). Panitumumab-IRDye800CW specificities for tumor core and margin were more than 10% higher than those of 5-ALA. Higher AUC for panitumumab-IRDye800CW indicated strong capability to discriminate between normal and malignant brain tissue when compared with 5-ALA. This work demonstrates that panitumumab-IRDye800CW shows potential as a targeting agent for fluorescence intraoperative detection of GBM. Improved margin definition and surgical resection using panitumumab-IRDye800 has the potential to improve surgical outcomes and survival in patients with GBM compared with 5-ALA.

Fluorescently Labeled Cetuximab-IRDye800 for Guided Surgical Excision of Ameloblastoma: A Proof of Principle Study.

PURPOSE: Fluorescently labeled epidermal growth factor receptor (EGFR) antibodies have successfully identified microscopic tumors in multiple in vivo models of human cancers with limited toxicity. The present study sought to demonstrate the ability of fluorescently labeled anti-EGFR, cetuximab-IRDye800, to localize to ameloblastoma (AB) tumor cells in vitro and in vivo. MATERIAL AND METHODS: EGFR expression in AB cells was confirmed by quantitative real-time polymerase chain reaction and immunohistochemistry. Primary AB cells were labeled in vitro with cetuximab-IRDye800 or nonspecific IgG-IRDye800. An in vivo patient-derived xenograft (PDX) model of AB was developed. The tumor tissue from 3 patients was implanted subcutaneously into immunocompromised mice. The mice received an intravenous injection of cetuximab-IRDye800 or IgG-IRDye800 and underwent imaging to detect infrared fluorescence using a Pearl imaging system (LI-COR Biosciences, Lincoln, NE). After resection of the overlying skin, the tumor/background ratios (TBRs) were calculated and statistically analyzed using a paired t test. RESULTS: EGFR expression was seen in all AB samples. Tumor-specific labeling was achieved, as evidenced by a positive fluorescence signal from cetuximab-IRDye800 binding to AB cells, with little staining seen in the negative controls treated with IgG-IRDye800. In the animal PDX model, imaging revealed that the TBRs produced by cetuximab were significantly greater than those produced by IgG on days 7 to 14 for AB-20 tumors. After skin flap removal to simulate a preresection state, the TBRs increased with cetuximab and were significantly greater than the TBRs with the IgG control for PDX tumors derived from the 3 patients with AB. The excised tissues were embedded in paraffin and examined to confirm the presence of tumor. CONCLUSIONS: Fluorescently labeled anti-EGFR demonstrated specificity for AB cells and PDX tumors. The present study is the first report of tumor-specific, antibody-based imaging of odontogenic tumors, of which AB is one of the most clinically aggressive. We expect this technology will ultimately assist surgeons treating AB by helping to accurately assess the tumor margins during surgery, leading to improved long-term local tumor control and less surgical morbidity.

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 tumors 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.

Proteasome inhibition reverses hedgehog inhibitor and taxane resistance in ovarian cancer.

The goal of this study was to determine whether combined targeted therapies, specifically those against the Notch, hedgehog and ubiquitin-proteasome pathways, could overcome ovarian cancer chemoresistance. Chemoresistant ovarian cancer cells were exposed to gamma-secretase inhibitors (GSI-I, Compound E) or the proteasome inhibitor bortezomib, alone and in combination with the hedgehog antagonist, LDE225. Bortezomib, alone and in combination with LDE225, was evaluated for effects on paclitaxel efficacy. Cell viability and cell cycle analysis were assessed by MTT assay and propidium iodide staining, respectively. Proteasome activity and gene expression were determined by luminescence assay and qPCR, respectively. Studies demonstrated that GSI-I, but not Compound E, inhibited proteasome activity, similar to bortezomib. Proteasome inhibition decreased hedgehog target genes (PTCH1, GLI1 and GLI2) and increased LDE225 sensitivity in vitro. Bortezomib, alone and in combination with LDE225, increased paclitaxel sensitivity through apoptosis and G2/M arrest. Expression of the multi-drug resistance gene ABCB1/MDR1 was decreased and acetylation of α-tubulin, a marker of microtubule stabilization, was increased following bortezomib treatment. HDAC6 inhibitor tubastatin-a demonstrated that microtubule effects are associated with hedgehog inhibition and sensitization to paclitaxel and LDE225. These results suggest that proteasome inhibition, through alteration of microtubule dynamics and hedgehog signaling, can reverse taxane-mediated chemoresistance.

Matrix metalloproteinase expression in keratocystic odontogenic tumors and primary cells.

UNLABELLED: 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.

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 patient's 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.

Lung resistance-related protein (LRP) expression in malignant ascitic cells as a prognostic marker for advanced ovarian serous carcinoma.

PURPOSE: Ovarian serous carcinoma is an aggressive cancer that often presents with metastatic disease. Although primary tumor and established metastatic foci in the omentum are generally compared to identify proteins involved in drug resistance, we investigated a potential bridge, the malignant cells from ascites, as facilitator of drug resistance and recurrence. METHODS: We evaluated the expression of drug resistance markers P-glycoprotein (P-gp), canalicular multispecific organic anion transporter (MRP2), and lung resistance-related protein (LRP) in malignant cells from ascites and matched omental metastasis from 25 patients with advanced-stage ovarian serous carcinoma who were chemotherapeutic naïve and undergoing initial cytoreductive surgery. Cell viability in vitro, patient response to chemotherapy, and patient survival were correlated with these biomarkers. RESULTS: Of the 25 patients evaluated for a correlation of LRP to 1-year recurrence, we correctly predicted the 1-year recurrence of 24 patients based solely on the presence of LRP in ascitic tumor cells (p=0.01). P-gp and MRP2 were not expressed in malignant cells of ascites or omental metastases. Malignant cells from ascites had higher expression of LRP and were found to be more resistant to carboplatin treatment than cells from omental metastasis (p=0.00375) by in vitro assay. LRP expression in the malignant cells of ascites correlated with carboplatin resistance (p=0.001) by in vitro assay and recurrence at 1 year (p=0.0125). CONCLUSIONS: LRP expression in malignant cells of ascites is a promising marker to predict response to first-line chemotherapy in patients with advanced ovarian serous carcinoma.

Targeting the sonic hedgehog pathway in keratocystic odontogenic tumor.

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.

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

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.

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.

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.

Gli3 mediates cell survival and sensitivity to cyclopamine in pancreatic cancer.

Activation of the hedgehog (HH) pathway plays a critical role in the development and continued growth of pancreatic adenocarcinoma (PAC). Cyclopamine, a HH pathway inhibitor, has been shown to suppress PAC cell proliferation in vitro and in vivo. However, the molecular basis of response to cyclopamine has not been fully elucidated nor have genes that predict sensitivity to this compound been identified. To better understand these features of HH pathway inhibition, we evaluated the biological and molecular effects of cyclopamine in vitro. The viability of 9 human PAC cell lines following cyclopamine exposure was determined using MTS assay. Proliferation and induction of apoptosis in treated cells were examined by bromo-deoxyuridine incorporation, caspase activation, and mitochondrial membrane potential. Gene expression before and after cyclopamine treatment was determined using Taqman real-time quantitative polymerase chain reaction (RTQ-PCR) and Taqman low-density array (TLDA). Among the cell lines examined, cyclopamine IC50 values ranged from 8.79 to >30 µM. Response to cyclopamine included reduced cell proliferation and induction of apoptosis with and without mitochondrial membrane depolarization. Regression analysis revealed that GLI3 expression significantly correlated with cyclopamine resistance (r = 0.80; p = 0.0102). Knockdown of GLI3 using siRNAs increased sensitivity to cyclopamine. In addition, GLI3 siRNAs decreased PAC cell viability and reduced expression of genes involved in HH signaling (Patched 1 and GLI1) and cell proliferation, similar to cyclopamine. These effects were not observed in PAC cells with undetectable GLI3 expression. These data suggest that Gli3 mediates cell survival and sensitivity to cyclopamine in pancreatic cancer.