Search

Introduction: To assess the technical feasibility and safety of repeated percutaneous computed tomography (CT)-guided transthoracic biopsies and intratumoral injections of gene-modified dendritic cells in metastatic NSCLC., Methods: A total of 15 patients with 15 NSCLC lesions measuring greater than 1.0 cm underwent two cycles of intratumoral biopsies and CCL21 dendritic cell injections separated by 7 days. All needle placements and injections were done under CT guidance. Clinical and imaging follow-up was done approximately 4 weeks after the first procedure. Safety and feasibility were determined as: (1) safety and feasibility similar to that of single-needle biopsy, and (2) an absence of serious adverse events defined as grade greater than or equal to three according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0., Results: A total of 30 percutaneous, transthoracic intratumoral biopsies and injections into the lung cancer were performed, two cycles (at d 0 and 7) received by each patient (311 biopsies and 96 intratumoral injections). All percutaneous cases achieved technical success with respect to needle placement for both biopsy and injection of CCL21 dendritic cells. Only minor complications were observed (grade <3), including pneumothorax (n = 10, 33%) and small postbiopsy hemorrhage (n = 2, 7%). Pneumothorax was moderate (n = 1) or trace (n = 9), with resolution of the moderate pneumothorax after manual aspiration without chest tube placement. No patient required chest tube placement. No other complications or serious adverse effects related to the biopsy or dendritic cell injection were noted. All patients were in stable condition after up to 4 hours in the recovery unit and were discharged home on the same day. No procedure-related complications were observed on imaging or clinical follow-up at 4 weeks., Conclusions: Repeated percutaneous, transthoracic CT-guided biopsies and intratumoral gene-modified cell-based immunotherapy injections into lung cancers are technically feasible, safe, and reproducible. There were no procedure-related serious (defined as grade ≥3) adverse events., (© 2021 THE AUTHORS.)

Liver is the most common metastatic site for colorectal cancer (CRC), there is no satisfied approach to treat CRC liver metastasis (CRCLM). Here, we investigated the role of a polycomb protein BMI-1 in CRCLM. Immunohistochemical analysis showed that BMI-1 expression in liver metastases was upregulated and associated with T4 stage, invasion depth and right-sided primary tumor. Knockdown BMI-1 in high metastatic HCT116 and LOVO cells repressed the migratory/invasive phenotype and reversed epithelial-mesenchymal transition (EMT), while BMI-1 overexpression in low metastatic Ls174T and DLD1 cells enhanced invasiveness and EMT. The effects of BMI-1 in CRC cells were related to upregulating snail via AKT/GSK-3 β pathway. Furthermore, knockdown BMI-1 in HCT116 and LOVO cells reduced CRCLM using experimental liver metastasis mice model. Meanwhile, BMI-1 overexpression in Ls174T and DLD1 significantly increased CRCLM. Moreover, sodium butyrate, a histone deacetylase and BMI-1 inhibitor, reduced HCT116 and LOVO liver metastasis in immunodeficient mice. Our results suggest that BMI-1 is a major regulator of CRCLM and provide a potent molecular target for CRCLM treatment., Competing Interests: The authors declare no conflicts of interest., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Background: Immune checkpoint blockade that downregulates T cell evasion for effective immunity has provided a renewed interest in therapeutic cancer vaccines., Methods: Utilizing murine lung cancer models, we determined: tumor burden, TIL cytolysis, immunohistochemistry, flow cytometry, RNA Sequencing, CD4 T cells, CD8 T cells, CXCL9 chemokine, and CXCL10 chemokine neutralization to evaluate the efficacy of Programmed cell death protein 1 (PD-1) blockade combined with chemokine (C-C motif) ligand 21-dendritic cell tumor antigen (CCL21-DC tumor Ag) vaccine., Results: Anti-PD1 combined with CCL21-DC tumor Ag vaccine eradicated 75% of 12-day established tumors (150 mm 3 ) that was enhanced to 90% by administering CCL21-DC tumor Ag vaccine prior to combined therapy. The effect of combined therapy was blocked by CD4, CD8, CXCL9, and CXCL10 neutralizing antibodies., Conclusion: PD-1 blockade therapy plus CCL21-DC tumor Ag vaccine could be beneficial to lung cancer patients.

Background : Targeting inhibitory immune checkpoint molecules has highlighted the need to find approaches enabling the activation of immune responses against cancer. Therapeutic vaccination, which induces specific immune responses against tumor antigens (Ags), is an attractive option. Methods : Utilizing a K-RasG12Dp53null murine lung cancer model we determined tumor burden, tumor-infiltrating T cell (TIL) cytolysis, immunohistochemistry, flow cytometry, and CD4 and CD8 depletion to evaluate the efficacy of PD-1 blockade combined with CCL21-DC tumor lysate vaccine. Results : Anti-PD-1 plus CCL21-DC tumor lysate vaccine administered to mice bearing established tumors (150 mm 3 ) increased expression of perforin and granzyme B in the tumor microenvironment (TME), increased tumor-infiltrating T cell (TIL) activity, and caused 80% tumor eradication. Mice with treatment-induced tumor eradication developed immunological memory, enabling tumor rejection upon challenge and cancer-recurrence-free survival. The depletion of CD4 or CD8 abrogated the antitumor activity of combined therapy. PD-1 blockade or CCL21-DC tumor lysate vaccine monotherapy reduced tumor burden without tumor eradication. Conclusion : Immune checkpoint blockade promotes the activity of the therapeutic cancer vaccine. PD-1 blockade plus CCL21-DC tumor lysate vaccine therapy could benefit lung cancer patients.

CCL21 promotes immune activity in the tumor microenvironment (TME) by colocalizing dendritic cells (DC) and T cells programing ectopic lymph node architectural structures that correlate with cancer prognosis. Innovative strategies to deliver CCL21 in cancer patients will reactivate the downregulated immune activity in the TME. Immune escape mechanisms are upregulated in the TME that promote tumor immune evasion. CCL21 combined with inhibition of dominant pathways of immune evasion will aid in the development of effective immunotherapy for cancer.

Epithelial cells in the field of lung injury can give rise to distinct premalignant lesions that may bear unique genetic aberrations. A subset of these lesions may escape immune surveillance and progress to invasive cancer; however, the mutational landscape that may predict progression has not been determined. Knowledge of premalignant lesion composition and the associated microenvironment is critical for understanding tumorigenesis and the development of effective preventive and interception strategies. To identify somatic mutations and the extent of immune cell infiltration in adenomatous premalignancy and associated lung adenocarcinomas, we sequenced exomes from 41 lung cancer resection specimens, including 89 premalignant atypical adenomatous hyperplasia lesions, 15 adenocarcinomas in situ , and 55 invasive adenocarcinomas and their adjacent normal lung tissues. We defined nonsynonymous somatic mutations occurring in both premalignancy and the associated tumor as progression-associated mutations whose predicted neoantigens were highly correlated with infiltration of CD8 + and CD4 + T cells as well as upregulation of PD-L1 in premalignant lesions, suggesting the presence of an adaptive immune response to these neoantigens. Each patient had a unique repertoire of somatic mutations and associated neoantigens. Collectively, these results provide evidence for mutational heterogeneity, pathway dysregulation, and immune recognition in pulmonary premalignancy. Significance: These findings identify progression-associated somatic mutations, oncogenic pathways, and association between the mutational landscape and adaptive immune responses in adenomatous premalignancy. See related commentary by Merrick, p. 4811 ., (©2019 American Association for Cancer Research.)

Radiation-induced oral mucositis (RIOM) is one of the most common side effects of radiotherapy in cancer patients, especially in almost all head and neck cancer patients. It presents as severe pain and ulceration. The development of RIOM is composed of five stages: initiation, primary damage response, signal amplification, ulceration, and healing. However, the key regulators involved in the RIOM pathogenesis remain largely unknown. In this study, we reveal a novel role of miR-200c, a member of the miR-200 family, in modulating RIOM pathogenesis. Using a mouse model mimicking RIOM, we found that the miR-200 family numbers (miR-141, miR-200a, miR-200b, and miR-200c) except miR-429 were significantly induced during the RIOM formation. Besides, in RIOM mice, miR-200c expression level was also increased dramatically in the normal human keratinocytes (NHKs) after irradiation. Knockdown of miR-200c expression with miR-200c-3p-shRNA significantly reduced senescence phenotype and enhanced cell proliferation in NHKs after irradiation. The generation of reactive oxygen species (ROS) and p47 enzyme involved in ROS production was increased after irradiation but both were markedly reduced in NHKs by miR-200c inhibition. Knockdown of miR-200c expression in NHKs increased DNA double-strand break repair after irradiation compared with control NHKs. Furthermore, miR-200c inhibition repressed the production of proinflammatory cytokines (TGF- β , TNF- α , and IL-1 α ) via inhibiting NF- κ B and Smad2 activation in NHKs exposed to IR. Additionally, miR-200c inhibition promoted NHK migration and increased the expression of molecules that regulate epithelial to mesenchymal transition, including Snail, Vimentin, Zeb1, and Bmi-1. These results not only identify the key role of miR-200c in the pathogenesis of RIOM but also provide a novel therapeutic target to treat RIOM.

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated, including in non-small cell lung cancer (NSCLC). Here, we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo Snail-mediated transformation relied upon silencing of the tumor-suppressive RNA splicing regulatory protein ESRP1. In clinical specimens of NSCLC, ESRP1 loss was documented in Snail-expressing premalignant pulmonary lesions. Mechanistic investigations showed that Snail drives malignant progression in an ALDH + CD44 + CD24 - pulmonary stem cell subset in which ESRP1 and stemness-repressing microRNAs are inhibited. Collectively, our results show how ESRP1 loss is a critical event in lung carcinogenesis, and they identify new candidate directions for targeted therapy of NSCLC. Significance: This study defines a Snail-ESRP1 cancer axis that is crucial for human lung carcinogenesis, with implications for new intervention strategies and translational opportunities. Cancer Res; 78(8); 1986-99. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Scope: Short-chain fatty acid sodium butyrate (NaB) is the byproduct of bacterial anaerobic fermentation of dietary fiber in the colon, and has been shown to have an antitumor effect on colorectal cancer (CRC). The miR-200 family is a key regulator of the epithelial-mesenchymal transition (EMT). We investigate the role of miR-200s expression on cell migration in NaB-treated CRC cells., Methods and Results: HCT116 and LOVO CRC cells treated with NaB depicted reduced cell proliferation, enhanced apoptosis, and cell cycle arrest. NaB inhibited cell migration in the wound healing and transwell assays, and in spheriod cultures while regulating EMT-related protein expression. NaB reciprocally increased miR-200s but reduced expression of their target genes (Bmi-1, Zeb1, EZH2). Cells transfected with miR-200c shRNA displayed a significant blockade of NaB-induced anti-invasive activity. Upregulation of Bmi-1 expression partially reversed the effect of NaB. In addition to inhibition of tumor growth in vivo, qRT-PCR results showed that NaB increased miR-200c/200b/492 expression in the tumor tissues. Immunohistochemistry and Western blotting results demonstrated that NaB decreased Bmi-1 expression in vivo., Conclusion: NaB inhibits CRC cell migration by enhancing miR-200c expression-mediated downregulation of Bmi-1. These findings support the utility of NaB in colorectal cancer therapy., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective To evaluate the adverse effects and therapeutic efficacy of our biocompatible polymer platform delivering targeted local therapy of cytokine CCL21 and cisplatin in a partially resected xenograft animal model of head and neck squamous cell carcinoma. In addition, to evaluate the efficacy of cotreatment with radiotherapy and assess the biocompatibility of the cisplatin-eluting polymer in the murine neck. Study Design Experimental animal study. Setting Academic research laboratory. Subjects and Methods SCCVII/SF cell injection established head and neck squamous cell carcinoma tumors in C3H/HeJ mice. Subjects underwent surgery, and a chemokine-eluting polymer was implanted into the resected site. Subjects treated with cisplatin received radiation or no radiation, and tissue was harvested after 8 weeks to assess polymer biocompatibility. Results Our results with the polymer platform significantly ( P < .05) reduced SCCVII/SF tumor size in C3H/HeJ mice with cisplatin (49% ± 8.7%, Δ3.4 ± 0.6 cm 3 [95% CI]), CCL21 (42% ± 4.8%, Δ3.5 ± 0.4 cm 3 ), and cisplatin/CCL21 dual-agent polymer (82% ± 4.4%, Δ8.0 ± 0.4 cm 3 ) as compared with controls. Cisplatin polymer with high-dose (16 Gy) and low-dose (4 Gy) radiation reduced tumor mass (respectively, 92% ± 7.2%, Δ6.1 ± 0.5 cm 3 ; 85% ± 7.4%, Δ5.7 ± 0.5 cm 3 ) as compared with the reduction from high-dose radiotherapy alone (70% ± 7.9%, Δ4.7 ± 0.5 cm 3 ). No significant toxicity or inflammation was noted on histopathology after radiotherapy and cisplatin-eluting polymer treatment. Conclusion Cisplatin, CCL21, and cisplatin/CCL21 dual-agent polymer all exhibit significant antitumor effects and decrease tumor burden. Moreover, combination cisplatin polymer with radiotherapy may permit a decrease in intensity of radiation therapy in patients having received the cisplatin polymer. Histopathologic analysis suggests that the polymer is free from significant adverse effects in this model and warrants clinical trial.

Background: MicroRNAs (miRNAs) exhibit essential regulatory functions related to cell growth, apoptosis, development and differentiation. Dysregulated expression of miRNAs is associated with a wide variety of human diseases. As such miRNA signatures are valuable as biomarkers for disease and for making treatment decisions. Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC). Here we screened for miRNAs in chronic HBV associated HCC., Methods: To determine the miRNAs in HCC occurrence associated with HBV infection, we analyzed global miRNA expression profiles in 12 pairs of HCC and adjacent matched non-HCC tissues from HBV-positive and HBV-negative patients using microarray analyses. The microarray result was validated by real-time PCR in 32 HBV-positive and 24 HBV-negative patient HCC samples. The potential candidate target genes of the miRNAs were predicted by miRWalk software. Genes simultaneously predicted as targets by two or more miRNAs were subjected to GO and KEGG pathway analysis. The miRNA regulatory network analysis was performed using the Ingenuity Pathway Analysis (IPA) software., Results: Eight miRNAs (miR-223, miR-98, miR-15b, miR-199a-5p, miR-19b, miR-22, miR-451, and miR-101) were involved in HBV-unrelated HCC, 5 miRNAs (miR-98, miR-375, miR-335, miR-199a-5p, and miR-22) were involved in HBV infection, and 7 miRNAs (miR-150, miR-342-3p, miR-663, miR-20b, miR-92a-3p, miR-376c-3p and miR-92b) were specifically altered in HBV-related HCC. Gene Ontology and KEGG analyses predict that these HBV-related HCC miRNAs are involved in the regulation of: transcription, RNA polymerase II promoter, phosphorylation of proteins through MAPK signaling pathway, focal adhesion, and actin cytoskeleton. IPA analysis also suggest that these miRNAs act on AGO2, TP53, CCND1, and 11 other genes that significantly influence HCC occurrence and HBV infection., Conclusion: Our data indicates that the unique 7 miRNAs expression signature could be involved in the development HBV- related HCC.

Casitas B-lineage lymphoma (CBL) is an E3 ubiquitin ligase and a molecule of adaptor that we have shown is important for non-small-cell lung cancer (NSCLC). We investigated if MET is a target of CBL and if enhanced in CBL-altered NSCLC. We showed that CBL wildtype cells have lower MET expression than CBL mutant cells. Ubiquitination of MET was also decreased in CBL mutant cells compared to wildtype cells. Mutant cells were also more sensitive to MET inhibitor SU11274 than wild-type cells. sh-RNA-mediated knockdown of CBL enhanced cell motility and colony formation in NSCLC cells, and these activities were inhibited by SU11274. Assessment of the phospho-kinome showed decreased phosphorylation of pathways involving MET, paxillin, EPHA2, and VEGFR. When CBL was knocked down in the mutant cell line H1975 (erlotinib-resistant), it became sensitive to MET inhibition. Our findings suggest that CBL status is a potential positive indicator for MET-targeted therapeutics in NSCLC.

Purpose: A phase I study was conducted to determine safety, clinical efficacy, and antitumor immune responses in patients with advanced non-small cell lung carcinoma (NSCLC) following intratumoral administration of autologous dendritic cells (DC) transduced with an adenoviral (Ad) vector expressing the CCL21 gene (Ad-CCL21-DC). We evaluated safety and tumor antigen-specific immune responses following in situ vaccination (ClinicalTrials.gov: NCT01574222). Experimental Design: Sixteen stage IIIB/IV NSCLC subjects received two vaccinations (1 × 10 6 , 5 × 10 6 , 1 × 10 7 , or 3 × 10 7 DCs/injection) by CT- or bronchoscopic-guided intratumoral injections (days 0 and 7). Immune responses were assessed by tumor antigen-specific peripheral blood lymphocyte induction of IFNγ in ELISPOT assays. Tumor biopsies were evaluated for CD8 + T cells by IHC and for PD-L1 expression by IHC and real-time PCR (RT-PCR). Results: Twenty-five percent (4/16) of patients had stable disease at day 56. Median survival was 3.9 months. ELISPOT assays revealed 6 of 16 patients had systemic responses against tumor-associated antigens (TAA). Tumor CD8 + T-cell infiltration was induced in 54% of subjects (7/13; 3.4-fold average increase in the number of CD8 + T cells per mm 2 ). Patients with increased CD8 + T cells following vaccination showed significantly increased PD-L1 mRNA expression. Conclusions: Intratumoral vaccination with Ad-CCL21-DC resulted in (i) induction of systemic tumor antigen-specific immune responses; (ii) enhanced tumor CD8 + T-cell infiltration; and (iii) increased tumor PD-L1 expression. Future studies will evaluate the role of combination therapies with PD-1/PD-L1 checkpoint inhibition combined with DC-CCL21 in situ vaccination. Clin Cancer Res; 23(16); 4556-68. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: In the present study, we investigated the role of p38-p38IP signaling in the inflammation-induced promotion of epithelial-to-mesenchymal transition (EMT) in Head and Neck Squamous Cell Carcinoma (HNSCC)., Methods: Quantitative RT-PCR, western blot analysis, spheroid modeling and immunohistochemical staining of human HNSCC tissue sections were used., Results: p38 inhibitor treated and p38 shRNA HNSCC cell lines demonstrate a significant upregulation in E-cadherin mRNA and a decrease in the mRNA expression of Snail. p38 binds to and stabilizes p38IP, a subunit of histone SPT3-TAF9-GCN5 acetyltransferase (STAGA), resulting in enhanced transcription of Snail. p38 shRNA HNSCC cell lines show a less invasive phenotype in a spheroid model. In clinical HNSCC samples, p38 interacting protein (p38IP) is significantly increased compared to adjacent normal tissue. An inverse relationship between p38, p38IP and E-cadherin is demonstrated., Conclusions: Herein we provide the first report that p38-p38IP is required for the Snail-induced E-cadherin down-regulation and cell invasion in HNSCC., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objectives: To investigate the role of p53 in NF-κB mediated epithelial-to-mesenchymal (EMT) in head and neck squamous cell carcinoma (HNSCC)., Materials and Methods: We utilized HNSCC and normal oral epithelial cell lines as our model system. We used a lentiviral shRNA system to silence the expression of p65 and p53 in these cell lines. Mutant and wild-type (WT) p53 background genotypes were analyzed. The expression of epithelial and mesenchymal markers was determined using western blotting and quantitative PCR assays. Cell morphology, growth, and invasion were determined using a 3-dimensional spheroid culture and anchorage independent growth (AIG) assays., Results: In HNSCC cells with mutant p53 we found that silencing p65 expression promoted EMT. In contrast, in the context of WT p53, ectopic p65 over-expression promoted EMT. Ablation of WT p53 in normal oral epithelial cells blocked EMT induced by p65 over-expression. We demonstrate that AIG and apoptosis induced by NF-κB activation is regulated by p53., Conclusion: Our data demonstrates that p53 mutational status is critical in determining the outcome of NF-κB activation in HNSCC. In the presence of WT p53, excess p65 signal can promote EMT. Conversely, ablation of p65 in the context of mutant p53 drives EMT. These results demonstrate that p53 mutational status alters the outcome of NF-κB signaling. These results, though preliminary, demonstrate the critical role of p53 mutational status in determining the outcome of NF-κB signaling and suggest that monitoring p53 status may inform the utility of NF-κB inhibitor treatment in HNSCC., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

An immune tolerant tumor microenvironment promotes immune evasion of lung cancer. Agents that antagonize immune tolerance will thus aid the fight against this devastating disease. Members of the tumor necrosis factor receptor (TNFR) family modulate the magnitude, duration and phenotype of immune responsiveness to antigens. Among these, GITR expressed on immune cells functions as a key regulator in inflammatory and immune responses. Here, we evaluate the GITR agonistic antibody (DTA-1) as a mono-therapy and in combination with therapeutic vaccination in murine lung cancer models. We found that DTA-1 treatment of tumor-bearing mice increased: (i) the frequency and activation of intratumoral natural killer (NK) cells and T lymphocytes, (ii) the antigen presenting cell (APC) activity in the tumor, and (iii) systemic T-cell specific tumor cell cytolysis. DTA-1 treatment enhanced tumor cell apoptosis as quantified by cleaved caspase-3 staining in the tumors. DTA-1 treatment increased expression of IFNγ, TNFα and IL-12 but reduced IL-10 levels in tumors. Furthermore, increased anti-angiogenic chemokines corresponding with decreased pro-angiogenic chemokine levels correlated with reduced expression of the endothelial cell marker Meca 32 in the tumors of DTA-1 treated mice. In accordance, there was reduced tumor growth (8-fold by weight) in the DTA-1 treatment group. NK cell depletion markedly inhibited the antitumor response elicited by DTA-1. DTA-1 combined with therapeutic vaccination caused tumor rejection in 38% of mice and a 20-fold reduction in tumor burden in the remaining mice relative to control. Mice that rejected tumors following therapy developed immunological memory against subsequent re-challenge. Our data demonstrates GITR agonist antibody activated NK cell and T lymphocyte activity, and enhanced therapeutic vaccination responses against lung cancer.

T helper 17 (Th17), a distinct subset of CD4(+) T cells with IL-17 as their major cytokine, orchestrate the pathogenesis of inflammatory and autoimmune diseases. Dysregulated Th17 cells contribute to inflammatory and autoimmune diseases. Candidate biologics are in development for targeting IL-17, IL-17 receptors or IL-17 pathways. Several drugs that impact the IL-17 pathway are already in clinical trials for the treatment of autoimmune diseases. In this review we provide evidence for the role of Th17 cells in immune-mediated diseases. An understanding of the role of Th17 in these conditions will provide important insights and unravel novel targets for therapeutic intervention., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition (EMT) in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway., Objective: The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling., Methods and Results: Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer (NSCLC) cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers (N-cadherin and vimentin) and a concomitant increase in expression of epithelial markers (E-cadherin, β-and γ-catenins), and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment., Conclusion: Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell migration in human lung cancer cells through a STAT1 dominant pathway.

Background and Purpose: Doxorubicin-based chemotherapy induces cardiotoxicity, which limits its clinical application. We previously reported the protective effects of quercetin against doxorubicin-induced hepatotoxicity. In this study, we tested the effects of quercetin on the expression of Bmi-1, a protein regulating mitochondrial function and ROS generation, as a mechanism underlying quercetin-mediated protection against doxorubicin-induced cardiotoxicity., Experimental Approach: Effects of quercetin on doxorubicin-induced cardiotoxicity was evaluated using H9c2 cardiomyocytes and C57BL/6 mice. Changes in apoptosis, mitochondrial function, oxidative stress and related signalling were evaluated in H9c2 cells. Cardiac function, serum enzyme activity and reactive oxygen species (ROS) generation were measured in mice after a single injection of doxorubicin with or without quercetin pre-treatment., Key Results: In H9c2 cells, quercetin reduced doxorubicin-induced apoptosis, mitochondrial dysfunction, ROS generation and DNA double-strand breaks. The quercetin-mediated protection against doxorubicin toxicity was characterized by decreased expression of Bid, p53 and oxidase (p47 and Nox1) and by increased expression of Bcl-2 and Bmi-1. Bmi-1 siRNA abolished the protective effect of quercetin against doxorubicin-induced toxicity in H9c2 cells. Furthermore, quercetin protected mice from doxorubicin-induced cardiac dysfunction that was accompanied by reduced ROS levels and lipid peroxidation, but enhanced the expression of Bmi-1 and anti-oxidative superoxide dismutase., Conclusions and Implications: Our results demonstrate that quercetin decreased doxorubicin-induced cardiotoxicity in vitro and in vivo by reducing oxidative stress by up-regulation of Bmi-1 expression. The findings presented in this study have potential applications in preventing doxorubicin-induced cardiomyopathy., (© 2014 The British Pharmacological Society.)

Objective: This study aimed to evaluate the therapeutic efficacy of a novel polymer platform delivering cisplatin and cytokines in the treatment of head and neck squamous cell carcinoma (HNSCC)., Study Design: In vivo study., Setting: Academic research laboratory., Subjects and Methods: Mice were randomized to receive implantation of (1) no polymer, (2) plain polymer, (3) plain polymer with local cisplatin injection, or (4) cisplatin polymer. The 2 groups of mice implanted with cisplatin polymer or no polymer were further randomized to receive (1) 4 Grays external beam radiation for 4 days or (2) no radiation. For cytokine studies, mice were grouped into (1) no polymer, (2) plain polymer, (3) plain polymer with intratumoral injection of recombinant CCL21 twice a week, (4) polymer containing parental dendritic cells, or (5) polymer containing dendritic cells secreting CCL21 (DC-CCL21)., Results: The cisplatin-secreting polymer effectively reduced tumors in the mice by more than 16-fold (P < .01). We also observed a statistically significant lower tumor weight among mice treated with cisplatin polymer and concomitant radiation compared to control groups. The DC-CCL21 polymer reduced SCCVII/SF tumors in the C3H/HeJ mice by more than 41% (P < .01)., Conclusion: Herein, we demonstrate the efficacy of a novel polymer platform in delivering cisplatin and cytokines. We also demonstrate that we can effectively grow dendritic cells in the polymer that can actively secrete CCL21 for a minimum of 5 days. This polymer may represent a new therapeutic modality for patients with HNSCC. Once this polymer platform is optimized, we will plan to pursue prospective trials in patients with HNSCC., (© American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014.)

Cancer, a major health problem, affects 12 million people worldwide every year. With surgery and chemo-radiation the long term survival rate for the majority of cancer patients is dismal. Thus novel treatments are urgently needed. Immunotherapy, the harnessing of the immune system to destroy cancer cells is an attractive option with potential for long term anti-tumor benefit. Cytokines are biological response modifiers that stimulate anti-tumor immune responses. In this review, we discuss the anti-tumor efficacy of the chemotactic cytokine CCL21 and its pre-clinical and clinical application in cancer.

Malignant pleural mesothelioma (MPM) is a rare malignancy of the pleura that is frequently resistant to conventional therapies. Immunotherapy is a promising investigational approach for MPM that has shown some evidence of clinical benefit in select patients. However, tumor-induced immunosuppression is likely a major impediment to achieving optimal clinical responses to immunotherapeutic intervention. MPM contains a variable degree of infiltrating T-regulatory cells and M2 macrophages, which are believed to facilitate tumor evasion from the host immune system. Additional immunosuppressive factors identified in other human tumor types, such as tumor-associated programmed death ligand-1 expression, may be relevant for investigation in MPM. Conventional cytoreductive therapies, such as radiation, chemotherapy, and surgery, may play a critical role in successful immunotherapeutic strategies by ablating intratumoral and/or systemic immunosuppressive factors, thus creating a host environment more amenable to immunotherapy. This article reviews the immunotherapeutic approaches being evaluated in patients with MPM and discusses how immunotherapy might be rationally combined with conventional tumor cytoreductive therapies for this disease.

Unlabelled: Aberrant expression of microRNAs (miRNA) with oncogenic capacities (oncomiRs) has been described for several different malignancies. The first identified oncomiR, miR-17-92, is frequently overexpressed in a variety of cancers and its targets include the tumor suppressor PTEN. The transcription factor c-Myc (MYC) plays a central role in proliferative control and is rapidly upregulated upon mitogenic stimulation. Expression of c-Myc is frequently deregulated in tumors, facilitating proliferation and inhibiting terminal differentiation. The c-Myc-regulated network comprises a large number of transcripts, including those encoding miRNAs. Here, prostaglandin E2 (PGE2) exposure rapidly upregulates the expression of the MYC gene followed by the elevation of miR-17-92 levels, which in turn suppresses PTEN expression, thus enhancing apoptosis resistance in non-small cell lung cancer (NSCLC) cells. Knockdown of MYC expression or the miR-17-92 cluster effectively reverses this outcome. Similarly, miR-17-92 levels are significantly elevated in NSCLC cells ectopically expressing COX-2. Importantly, circulating miR-17-92 was elevated in the blood of patients with lung cancer as compared with subjects at risk for developing lung cancer. Furthermore, in patients treated with celecoxib, miR-17-92 levels were significantly reduced. These data demonstrate that PGE2, abundantly produced by NSCLC and inflammatory cells in the tumor microenvironment, is able to stimulate cell proliferation and promote resistance to pharmacologically induced apoptosis in a c-Myc and miR-17-92-dependent manner., Implications: This study describes a novel mechanism, involving c-Myc and miR-17-92, which integrates cell proliferation and apoptosis resistance., (©2014 AACR.)

Background: Non-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC., Methods: Using IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay., Results: Relative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0-3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU11274, had marginal additive effect on loss of NSCLC cell viability., Conclusion: PAX8 provides signals for growth and motility of NSCLC cells and is necessary for MET and RON expression. Further investigations are necessary to investigate the therapeutic potential of PA8 in NSCLC.

Tumors treated with fractionated doses of ionizing radiation (IR) often acquire radioresistance. Although histone deacetylase inhibitors (HDIs) have been demonstrated to sensitize intrinsic radioresistant cancer cell lines to IR, little is known on the impact of HDIs on the effects of IR in acquired radioresistant cancer cells. This study evaluates the mechanisms by which HDIs sensitize acquired radioresistant esophageal squamous cell carcinoma cells to IR. The HDIs trichostatin A and sodium butyrate were tested for the irability to sensitize acquired radioresistant KYSE-150R and radiosensitive KYSE-150 parental cells to IR. Although the HDIs induced similar levels of cytotoxicity in the KYSE-150 and the KYSE-150R cells, HDIs increased the: (i) radiosensitivity, (ii) IR-induced ROS generation, and (iii) IR-induced G2/M arrest and apoptosis of KYSE-150R cells compared with those of KYSE-150 cells. These changes were accompanied by increased p21 expression and decreased mitochondrial membrane potential. When combined with IR, HDIs inhibited Bmi-1 expression in KYSE-150R cells and their ability to repair DNA damage. The results demonstrate the potential utility of HDIs in augmenting the efficacy of fractionated radiotherapy.

Background: Interleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial-mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells., Methods: STAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors., Results: Our results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27-treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype., Conclusion: IL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1-dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27.

miRNAs are small non-coding RNA molecules that modulate the expression of multiple protein-encoding genes at the post-transcriptional level. They have recently been recognized as powerful regulators of numerous genes and pathways in the pathogenesis of inflammatory and autoimmune diseases. The targets of most miRNAs remain unknown and their roles in biological processes such as cell differentiation, proliferation, and death (apoptosis) are not clearly understood. In this review we will discuss how certain candidate miRNAs affect inflammatory and immune mediated diseases by regulating their cellular and molecular targets. We focused the influence of gender and sex hormones on miRNA. We believe that understanding the role of miRNAs could shed light on the cause and progression of many inflammatory and autoimmune diseases and eventually lay the groundwork for therapeutic options., (© 2013.)

Purpose: The EGFR tyrosine kinase inhibitors (TKIs) demonstrate efficacy in NSCLC patients whose tumors harbor activating EGFR mutations. However, patients who initially respond to EGFR TKI treatment invariably develop resistance to the drugs. Known mechanisms account for approximately 70% of native and acquired EGFR TKI resistance. In the current study we investigated a novel mechanism of NSCLC resistance to erlotinib., Experimental Design: The mechanisms of acquired erlotinib resistance were evaluated by microarray analysis in thirteen NSCLC cell lines and in vivo in mice. Correlations between plasma neutrophil gelatinase associated lipocalin (NGAL) levels, erlotinib response and the EGFR mutational status were assessed in advanced stage NSCLC patients treated with erlotinib., Results: In 5 of 13 NSCLC cell lines NGAL was significantly upregulated. NGAL knockdown in erlotinib-resistant cells increased erlotinib sensitivity in vitro and in vivo. NGAL overexpression in erlotinib-sensitive cells augmented apoptosis resistance. This was mediated by NGAL-dependent modulation of the pro-apoptotic protein Bim levels. Evaluation of the plasma NGAL levels in NSCLC patients that received erlotinib revealed that patients with lower baseline NGAL demonstrated a better erlotinib response. Compared to patients with wild type EGFR, patients with activating EGFR mutations had lower plasma NGAL at baseline and weeks 4 and 8., Conclusions: Our studies uncover a novel mechanism of NGAL-mediated modulation of Bim levels in NSCLC that might contribute to TKI resistance in lung cancer patients. These findings provide the rationale for the further investigations of the utility of NGAL as a potential therapeutic target or diagnostic biomarker.

We have identified an alternatively spliced, non-functional aberrant E-cadherin transcript that lacks exon 11 and is over expressed in malignant cells as compared to the normal non-malignant cells. This increase in the aberrant transcript is a mechanism of loss of E-cadherin gene expression as it is rapidly degraded by the nonsense mediated decay pathway. To study the mechanism of this gene missplicing we analyzed the role of histone epigenetic modifications in lung cancer cell lines. The treatment of low E-cadherin lung cancer cell lines with histone deacetylase inhibitor (HDACi, MS-275) resulted in the preferential expression of the correctly spliced transcripts in the low E-cadherin expressing cell lines only. Chromatin immunoprecipitation (ChIP) assays revealed that the histone hypoacetylation levels correlate with aberrant exon 11 splicing as there is more aberrant splicing in cell lines with E-cadherin promoter hypoacetylation. Inactivation of histone deacetylases (HDAC) 1, 2 and 3 resulted in an increase in E-cadherin expression and an increase in the ratio of the correctly spliced E-cadherin transcript. As transcription of the gene is closely linked to splicing, we considered the possibility that change in E-cadherin transcription correlates with splicing. The Zeb1 epithelial-mesenchymal transformation (EMT) inducer silences E-cadherin expression and could also alter the splicing of this exon. Inhibition of the E-cadherin promoter transcription with Zeb1 expression increases aberrant splicing and the reverse is observed when Zeb1 is knocked down. The role of HDAC inhibitors was also studied in vivo in a immunodeficient mouse xenograft model. Exposure of mice to HDACi resulted in growth inhibition, increase in E-cadherin expression, alteration of aberrant splicing and the reversal of EMT in mouse tumors. The findings support the modulation of E-cadherin exon 11 inclusion or exclusion by histone epigenetic modifications as they change the overall chromatin structure. The results provide an interesting link between epigenetic alterations in cancer cells and gene splicing in addition to their effect on gene silencing.

Lung cancer remains a challenging health problem with more than 1.1 million deaths worldwide annually. With current therapy, the long term survival for the majority of lung cancer patients remains low, thus new therapeutic strategies are needed. One such strategy would be to develop immune therapy for lung cancer. Immune approaches remain attractive because although surgery, chemotherapy, and radiotherapy alone or in combination produce response rates in all histological types of lung cancer, relapse is frequent. Strategies that harness the immune system to react against tumors can be integrated with existing forms of therapy for optimal responses toward this devastating disease. Both antigen presenting cell (APC) and T cell activities are reduced in the lung tumor microenvironment. In this review we discuss our experience with efforts to restore host APC and T cell activities in the lung cancer microenvironment by intratumoral administration of dendritic cells (DC) expressing the CCR7 receptor ligand CCL21 (secondary lymphoid chemokine, SLC). Based on the results demonstrating that CCL21 is an effective anti cancer agent in the pre-clinical lung tumor model systems, a phase I clinical trial was initiated using intratumoral injection of CCL21 gene modified autologous DC in lung cancer. Results from the trial thus far indicate tolerability, immune enhancement and tumor shrinkage via this approach.

Myeloid-derived suppressor cells (MDSCs) are important regulators of immune responses. These cells suppress the cytotoxic activities of natural killer (NK)-cell and T-cell effectors and promote tumor growth. We demonstrated that depleting MDSCs improve therapeutic responses to vaccination in a murine model of lung cancer. This approach may prove beneficial against tumors in which MDSC exert prominent immunosuppressive effects.

Objective: The presence of regional metastases in patients with head and neck squamous cell carcinoma (HNSCC) is a common and adverse event associated with poor prognosis. The authors' recent work on human HNSCC tissues underlies Snail's role as a molecular prognostic marker for HNSCC. Snail positivity is significantly predictive of poorly differentiated, lymphovascular invasive, and regionally metastatic tumors. Here, the authors investigate the capacity of Snail to drive epithelial-mesenchymal transition (EMT) in human oral epithelial cell lines and its ability to confer drug resistance., Study Design: Snail was overexpressed in HNSCC and oral epithelial cell lines. Anchorage independent growth assays, wound healing assays, invasion and migration assays, spheroid modeling, and cell survival assays were performed., Setting: Academic tertiary medical center., Subjects and Methods: Snail overexpressing HNSCC (OSC, Tu212, Tu686) and oral epithelial cell lines (HOK 16-B, OKF-6) were evaluated using assays for wound healing, invasion and migration, 3-dimensional growth, Western blot, and immunofluorescence., Results: The overexpression of Snail in human HNSCC and oral epithelial cell lines drives EMT. The transfection of Snail confers the expression of a mesenchymal molecular signature, including downregulation of the epithelial adherens, such as E-cadherin and β-catenin, and induction of mesenchymal markers. Snail-overexpressing cell lines demonstrate rapid growth in Anchorage-independent growth assays, a decreased capacity to form tight spheroids, an increased resistance to erlotinib, and an increased capacity for invasion., Conclusion: Snail controls the mesenchymal phenotype and drives erlotinib resistance in HNSCC cells. Snail may prove to be a useful marker in predicting epidermal growth factor receptor inhibitor responsiveness.

Lung cancer evades host immune surveillance by dysregulating inflammation. Tumors and their surrounding stromata produce growth factors, cytokines, and chemokines that recruit, expand, and/or activate myeloid-derived suppressor cells (MDSCs). MDSCs regulate immune responses and are frequently found in malignancy. In this review the authors discuss tumor-MDSC interactions that suppress host antitumor activities and the authors' recent findings regarding MDSC depletion that led to improved therapeutic vaccination responses against lung cancer. Despite the identification of a repertoire of tumor antigens, hurdles persist for immune-based anticancer therapies. It is likely that combined therapies that address the multiple immune deficits in cancer patients will be required for effective therapy. MDSCs play a major role in the suppression of T-cell activation and they sustain tumor growth, proliferation, and metastases. Regulation of MDSC recruitment, differentiation or expansion, and inhibition of the MDSC suppressive function with pharmacologic agents will be useful in the control of cancer growth and progression. Pharmacologic agents that regulate MDSCs may be more effective when combined with immunotherapies. Optimization of combined approaches that simultaneously downregulate MDSC suppressor pathways, restore APC immune-stimulating activity, and expand tumor-reactive T cells will be useful in improving therapy.

Radiotherapy (RT) is a major modality of cancer treatment. However, tumors often acquire radioresistance, which causes RT to fail. The exact mechanisms by which tumor cells subjected to fractionated irradiation (FIR) develop an adaptive radioresistance are largely unknown. Using the radioresistant KYSE-150R esophageal squamous cell carcinoma (ESCC) model, which was derived from KYSE-150 parental cells using FIR, the role of Bmi-1 in mediating the radioadaptive response of ESCC cells to RT was investigated. The results showed that the level of Bmi-1 expression was significantly higher in KYSE-150R cells than in the KYSE-150 parental cells. Bmi-1 depletion sensitized the KYSE-150R cells to RT mainly through the induction of apoptosis, partly through the induction of senescence. A clonogenic cell survival assay showed that Bmi-1 depletion significantly decreased the radiation survival fraction in KYSE-150R cells. Furthermore, Bmi-1 depletion increased the generation of reactive oxygen species (ROS) and the expression of oxidase genes (Lpo, Noxo1 and Alox15) in KYSE-150R cells exposed to irradiation. DNA repair capacities assessed by γ-H2AX foci formation were also impaired in the Bmi-1 down-regulated KYSE-150R cells. These results suggest that Bmi-1 plays an important role in tumor radioadaptive resistance under FIR and may be a potent molecular target for enhancing the efficacy of fractionated RT., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Chronic neuroinflammation is an important component of Alzheimer's disease and could contribute to neuronal dysfunction, injury and loss that lead to disease progression. Multiple clinical studies implicate tumor necrosis factor-α as an inflammatory mediator of neurodegeneration in patients with Alzheimer's because of elevated levels of this cytokine in the cerebrospinal fluid, hippocampus and cortex. Current Alzheimer's disease interventions are symptomatic treatments with limited efficacy that do not address etiology. Thus, a critical need exists for novel treatments directed towards modifying the pathophysiology and progression., Methods: To investigate the effect of early immune modulation on neuroinflammation and cognitive outcome, we treated triple transgenic Alzheimer's disease mice (harboring PS1(M146V), APP(Swe), and tau(P301L) transgenes) with the small molecule tumor necrosis factor-α inhibitors, 3,6'-dithiothalidomide and thalidomide, beginning at four months of age. At this young age, mice do not exhibit plaque or tau pathology but do show mild intraneuronal amyloid beta protein staining and a robust increase in tumor necrosis factor-α. After 10 weeks of treatment, cognitive performance was assessed using radial arm maze and neuroinflammation was assessed using biochemical, stereological and flow cytometric endpoints., Results: 3,6'-dithiothalidomide reduced tumor necrosis factor-α mRNA and protein levels in the brain and improved working memory performance and the ratio of resting to reactive microglia in the hippocampus of triple transgenic mice. In comparison to non-transgenic controls, triple transgenic Alzheimer's disease mice had increased total numbers of infiltrating peripheral monomyelocytic/granulocytic leukocytes with enhanced intracytoplasmic tumor necrosis factor-α, which was reduced after treatment with 3,6'-dithiothalidomide., Conclusions: These results suggest that modulation of tumor necrosis factor-α with small molecule inhibitors is safe and effective with potential for the long-term prevention and treatment of Alzheimer's disease.

Objective: To evaluate the therapeutic efficacy of a novel modular polymer platform in the treatment of head and neck squamous cell carcinoma (HNSCC)., Design: In vivo study., Setting: Academic research laboratory. Subjects and, Methods: C3H/HeJ mice and SCID/beige mice were randomized to receive implantation of no polymer, plain polymer, plain polymer with local cisplatin injection, or cisplatin polymer. The 2 groups of mice implanted with cisplatin polymer or no polymer were further randomized to receive 4 Gy of external beam radiation for 4 days or no radiation. Tumor size was measured until the mice were humanely killed. At necropsy, the tumors were excised and weighed., Results: There was a significant reduction in tumor growth using this novel polymer platform. The cisplatin-secreting polymer effectively reduced human head and neck tumor growth in SCID mice by 17-fold and SCC VII/SF tumors in C3H/HeJ mice by more than 16-fold compared with the control, plain polymer, and plain polymer + intratumoral cisplatin injection groups (P = .01 for both). We also observed a statistically significant lower tumor weight in mice treated with cisplatin polymer and concomitant radiation compared with the radiation alone and control groups., Conclusions: We demonstrate the efficacy of a novel polymer platform in delivering cisplatin to a partially resected SCC in a murine model. This polymer may represent a new therapeutic modality for patients with HNSCC. Once this polymer platform is optimized, we will plan for validation in the context of a prospective trial in patients with unresectable advanced or recurrent HNSCC.

Many tumors, including lung cancers, promote immune tolerance to escape host immune surveillance and facilitate tumor growth. Tumors utilize numerous pathways to inhibit immune responses, including the elaboration of immune-suppressive mediators such as PGE2, TGF-β, IL-10, VEGF, GM-CSF, IL-6, S100A8/A9 and SCF, which recruit and/or activate myeloid-derived suppressor cells (MDSCs). MDSCs, a subset of heterogeneous bone marrow-derived hematopoietic cells, are found in the peripheral blood of cancer patients and positively correlate to malignancy. Solid tumors contain MDSCs that maintain an immune-suppressive network in the tumor microenvironment. This review will focus on the interaction of tumors with MDSCs that lead to dysregulation of antigen presentation and T-cell activities in murine tumor models. Specific genetic signatures in lung cancer modulate the activities of MDSCs and impact tumor progression. Targeting MDSCs may have a long-term antitumor benefit and is at the forefront of anticancer therapeutic strategies.

The role of c-Crk (CRK) in promoting metastasis is well described however the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have observed CRK-II serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK-II serine 41 phosphorylation in the down-regulation of p120-catenin, cell motility and cell invasiveness in NSCLC cells. For this purpose, we expressed phosphomimetic and phosphodeficient CRK-II serine 41 mutants in NSCLC cells. NSCLC cells expressing phosphomimetic CRK-II seine 41 mutant showed lower p120-catenin level while CRK-II seine 41 phosphodeficient mutant expression resulted in higher p120-catenin. In addition, A549 cells expressing CRK-II serine 41 phosphomimetic mutant demonstrated more aggressive behavior in wound healing and invasion assays and, on the contrary, expression of phosphodeficient CRK-II serine 41 mutant in A549 cells resulted in reduced cell motility and invasiveness. We also provide evidence that PAK1 mediates CRK-II serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level in A549 and H157 cells. Furthermore, PAK1 silencing decreased cell motility and invasiveness in A549 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK-II serine 41. In summary, these data provide evidence for the role of PAK1 in the promotion of cell motility, cell invasiveness and the down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells.

Background: Myeloid derived suppressor cells (MDSC) are important regulators of immune responses. We evaluated the mechanistic role of MDSC depletion on antigen presenting cell (APC), NK, T cell activities and therapeutic vaccination responses in murine models of lung cancer., Principal Findings: Individual antibody mediated depletion of MDSC (anti-Gr1 or anti-Ly6G) enhanced the antitumor activity against lung cancer. In comparison to controls, MDSC depletion enhanced the APC activity and increased the frequency and activity of the NK and T cell effectors in the tumor. Compared to controls, the anti-Gr1 or anti-Ly6G treatment led to increased: (i) CD8 T cells, (ii) NK cells, (iii) CD8 T or NK intracytoplasmic expression of IFNγ, perforin and granzyme (iv) CD3 T cells expressing the activation marker CD107a and CXCR3, (v) reduced CD8 T cell IL-10 production in the tumors (vi) reduced tumor angiogenic (VEGF, CXCL2, CXCL5, and Angiopoietin1&2) but enhanced anti-angiogenic (CXCL9 and CXCL10) expression and (vii) reduced tumor staining of endothelial marker Meca 32. Immunocytochemistry of tumor sections showed reduced Gr1 expressing cells with increased CD3 T cell infiltrates in the anti-Gr1 or anti-Ly6G groups. MDSC depletion led to a marked inhibition in tumor growth, enhanced tumor cell apoptosis and reduced migration of the tumors from the primary site to the lung compared to controls. Therapeutic vaccination responses were enhanced in vivo following MDSC depletion with 50% of treated mice completely eradicating established tumors. Treated mice that rejected their primary tumors acquired immunological memory against a secondary tumor challenge. The remaining 50% of mice in this group had 20 fold reductions in tumor burden compared to controls., Significance: Our data demonstrate that targeting MDSC can improve antitumor immune responses suggesting a broad applicability of combined immune based approaches against cancer. This multifaceted approach may prove useful against tumors where MDSC play a role in tumor immune evasion.

Background: Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier., Methodology and Principal Findings: We characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA) encased in vault nanocapsules and liposomes. We measured OVA responsive CD8(+) and CD4(+) memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8(+) memory T cells and production of IFNγ plus CD4(+) memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes., Conclusions/significance: These experiments show that vault nanocapsules induced strong anti-OVA CD8(+) and CD4(+) T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8(+) and CD4(+) T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer.

Background: The dose-dependent toxicities of doxorubicin (DOX) limit its clinical applications, particularly in drug-resistant cancers, such as liver cancer. In this study, we investigated the role of quercetin on the antitumor effects of DOX on liver cancer cells and its ability to provide protection against DOX-mediated liver damage in mice., Methodology and Results: The MTT and Annexin V/PI staining assay demonstrated that quercetin selectively sensitized DOX-induced cytotoxicity against liver cancer cells while protecting normal liver cells. The increase in DOX-mediated apoptosis in hepatoma cells by quercetin was p53-dependent and occurred by downregulating Bcl-xl expression. Z-VAD-fmk (caspase inhibitor), pifithrin-α (p53 inhibitor), or overexpressed Bcl-xl decreased the effects of quercetin on DOX-mediated apoptosis. The combined treatment of quercetin and DOX significantly reduced the growth of liver cancer xenografts in mice. Moreover, quercetin decreased the serum levels of alanine aminotransferase and aspartate aminotransferase that were increased in DOX-treated mice. Quercetin also reversed the DOX-induced pathological changes in mice livers., Conclusion and Significance: These results indicate that quercetin potentiated the antitumor effects of DOX on liver cancer cells while protecting normal liver cells. Therefore, the development of quercetin may be beneficial in a combined treatment with DOX for increased therapeutic efficacy against liver cancer.

Purpose: We evaluated the utility of chimeric γc homeostatic cytokine, IL-7/IL-7Rα-Fc, to restore host APC (antigen presenting cell) and T cell activities in lung cancer., Experimental Design: Utilizing murine lung cancer models we determined the antitumor efficacy of IL-7/IL-7Rα-Fc. APC, T cell, cytokine analyses, neutralization of CXCL9, CXCL10, and IFNγ were carried out to evaluate the mechanistic differences in the antitumor activity of IL-7/IL-7Rα-Fc in comparison to controls., Results: IL-7/IL-7Rα-Fc administration inhibited tumor growth and increased survival in lung cancer. Accompanying the tumor growth inhibition were increases in APC and T cell activities. In comparison to controls, IL-7/IL-7Rα-Fc treatment of tumor bearing mice led to increased: (i) levels of CXCL9, CXCL10, IFNγ, IL-12 but reduced IL-10 and TGFβ, (ii) tumor macrophage infiltrates characteristic of M1 phenotype with increased IL-12, iNOS but reduced IL-10 and arginase, (iii) frequencies of T and NK cells, (iv) T cell activation markers CXCR3, CD69 and CD127(low), (v) effector memory T cells, and (vi) T cell cytolytic activity against parental tumor cells. IL-7/IL-7Rα-Fc treatment abrogated the tumor induced reduction in splenic functional APC activity to T responder cells. The CXCR3 ligands played an important role in IL-7/IL-7Rα-Fc-mediated antitumor activity. Neutralization of CXCL9, CXCL10, or IFNγ reduced CXCR3 expressing activated T cells infiltrating the tumor and abrogated IL-7/IL-7Rα-Fc-mediated tumor growth inhibition., Conclusions: Our findings show that IL-7/IL-7Rα-Fc promotes afferent and efferent antitumor responses in lung cancer., (©2011 AACR.)

Background: Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer., Principal Findings: Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3(+)T, CCR7(+)T, IFNγ(+)T lymphocytes, DEC205(+) DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells., Significance: This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an "off the shelf" approach to deliver antitumor cytokines to treat a broad range of malignancies.

With their resistance to genotoxic and anti-proliferative drugs and potential to grow tumors and metastases from very few cells, cancer stem or tumor-initiating cells (TICs) are a severe limitation for the treatment of cancer by conventional therapies. Here, we explored whether human T cells that are redirected via an EpCAM/CD3-bispecific antibody called MT110 can lyse colorectal TICs and prevent tumor growth from TICs. MT110 recognizes EpCAM, a cell adhesion molecule expressed on TICs from diverse human carcinoma, which was recently shown to promote tumor growth through engagement of elements of the wnt pathway. MT110 was highly potent in mediating complete redirected lysis of KRAS-, PI3 kinase- and BRAF-mutated colorectal TICs, as demonstrated in a soft agar assay. In immunodeficient mice, MT110 prevented growth of tumors from a 5,000-fold excess of a minimally tumorigenic TIC dose. T cells engaged by MT110 may provide a potent therapeutic means to eradicate TICs and bulk tumor cells derived thereof.

Chemokines (ie, chemoattractant cytokines) are a family of small secreted molecules that mediate leukocyte migration. It is becoming increasingly more evident that chemokines play an integral role in the initiation of a specific immune response. With respect to cancer, chemokines are being studied for both their role in tumor biology and as promising immunotherapy candidates. We review several areas of chemokine importance in tumor immunity and discuss the experimental evidence that is leading to the clinical use of this cytokine family in new treatment approaches for patients with cancer.

CD4(+)CD25(bright) regulatory T cells (T(reg)) play an important role in cancer-mediated immunosuppression. We and others have previously shown that prostaglandin E2 (PGE(2)) and transforming growth factor beta (TGF-beta) induce CD4(+)CD25(bright)FOXP3(+)T(reg). Based on these studies, we investigated the requirement for PGE(2) in Treg induction by TGF-beta. TGF-beta stimulation of human CD4(+) T cells induced COX-2-dependent production of PGE(2). PGE(2)-neutralizing antibody treatment significantly reduced the suppressive function of TGF-beta-induced T(reg) (TGF-beta-T(reg)) in vitro. TGF-beta concentration measured in the plasma of non-small cell lung cancer (NSCLC) patients directly correlated with the frequency of circulating CD4(+)CD25(bright)FOXP3(+)T cells. Flow cytometry analysis showed increased FOXP3 expression in circulating CD4(+)CD25(+)HLA-DR- cells of lung cancer patients compared to control subjects. Immunohistochemical analysis revealed co-expression of TGF-beta, COX-2, and FOXP3 in serial sections from resected lung tumor tissues. All together these observations suggest interplay between TGF-beta and COX-2 in the induction of T(reg) activities. Interrupting TGF-beta and PGE(2) signaling may be important in therapeutic interventions that aim to limit T(reg)function in lung cancer.

Purpose: As a transcriptional repressor of E-cadherin, Snail has predominantly been associated with epithelial-mesenchymal transition, invasion, and metastasis. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the contributions of Snail to the progression of non-small cell lung cancer (NSCLC)., Experimental Design: Immunohistochemistry was done to quantify and localize Snail in human lung cancer tissues, and tissue microarray analysis was used to correlate these findings with survival. NSCLC cell lines gene-modified to stably overexpress Snail were evaluated in vivo in two severe combined immunodeficiency murine tumor models. Differential gene expression between Snail-overexpressing and control cell lines was evaluated using gene expression microarray analysis., Results: Snail is upregulated in human NSCLC tissue, and high levels of Snail expression correlate with decreased survival (P < 0.026). In a heterotopic model, mice bearing Snail-overexpressing tumors developed increased primary tumor burden (P = 0.008). In an orthotopic model, mice bearing Snail-overexpressing tumors also showed a trend toward increased metastases. In addition, Snail overexpression led to increased angiogenesis in primary tumors as measured by MECA-32 (P < 0.05) positivity and CXCL8 (P = 0.002) and CXCL5 (P = 0.0003) concentrations in tumor homogenates. Demonstrating the importance of these proangiogenic chemokines, the Snail-mediated increase in tumor burden was abrogated with CXCR2 blockade. Gene expression analysis also revealed Snail-associated differential gene expression with the potential to affect angiogenesis and diverse aspects of lung cancer progression., Conclusion: Snail upregulation plays a role in human NSCLC by promoting tumor progression mediated by CXCR2 ligands.