Targeting Gli transcription activation by small molecule suppresses tumor growth.

Targeted inhibition of Hedgehog signaling at the cell membrane has been associated with anticancer activity in preclinical and early clinical studies. Hedgehog signaling involves activation of Gli transcription factors that can also be induced by alternative pathways. In this study, we identified an interaction between Gli proteins and a transcription coactivator TBP-associated factor 9 (TAF9), and validated its functional relevance in regulating Gli transactivation. We also describe a novel, synthetic small molecule, FN1-8, that efficiently interferes with Gli/TAF9 interaction and downregulate Gli/TAF9-dependent transcriptional activity. More importantly, FN1-8 suppresses cancer cell proliferation in vitro and inhibits tumor growth in vivo. Our results suggest that blocking Gli transactivation, an important control point of multiple oncogenic pathways, may be an effective anticancer strategy.

Identification of pY654-ß-catenin as a critical co-factor in hypoxia-inducible factor-1α signaling and tumor responses to hypoxia.

Hypoxia is linked to epithelial-mesenchymal transition (EMT) and tumor progression in numerous carcinomas. Responses to hypoxia are thought to operate via hypoxia-inducible factors (HIFs), but the importance of co-factors that regulate HIF signaling within tumors is not well understood. Here, we elucidate a signaling pathway that physically and functionally couples tyrosine phosphorylation of ß-catenin to HIF1α signaling and HIF1α-mediated tumor EMT. Primary human lung adenocarcinomas accumulate pY654-ß-catenin and HIF1α. All pY654-ß-catenin, and only the tyrosine phosphorylated form, was found complexed with HIF1α and active Src, both within the human tumors and in lung tumor cell lines exposed to hypoxia. Phosphorylation of Y654, generated by hypoxia mediated, reactive oxygen species (ROS)-dependent Src kinase activation, was required for ß-catenin to interact with HIF1α and Src, to promote HIF1α transcriptional activity, and for hypoxia-induced EMT. Mice bearing hypoxic pancreatic islet adenomas, generated by treatment with anti-vascular endothelial growth factor antibodies, accumulate HIF1α/pY654-ß-catenin complexes and develop an invasive phenotype. Concurrent administration of the ROS inhibitor N-acetylcysteine abrogated ß-catenin/HIF pathway activity and restored adenoma architecture. Collectively, the findings implicate accumulation of pY654-ß-catenin specifically complexed to HIF1α and Src kinase as critically involved in HIF1α signaling and tumor invasion. The findings also suggest that targeting ROS-dependent aspects of the pY654-ß-catenin/ HIF1α pathway may attenuate untoward biological effects of anti-angiogenic agents and tumor hypoxia.

The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance.

Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were 'primed for death'. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, ∼70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically.

EMX2 is epigenetically silenced and suppresses growth in human lung cancer.

Lung cancer is a common cancer and the leading cause of cancer-related death worldwide. Aberrant activation of WNT signaling is implicated in lung carcinogenesis. EMX2, a human homologue of the Drosophila empty spiracles gene is a homeodomain-containing transcription factor. The function of EMX2 has been linked to the WNT signaling pathway during embryonic patterning in mice. However, little is known about the role of EMX2 in human tumorigenesis. In this study, we found that EMX2 was dramatically downregulated in lung cancer tissue samples and this downregulation was associated with methylation of the EMX2 promoter. Restoration of EMX2 expression in lung cancer cells lacking endogenous EMX2 expression suppressed cell proliferation and invasive phenotypes, inhibited canonical WNT signaling, and sensitized lung cancer cells to the treatment of the chemo cytotoxic drug cisplatin. On the other hand, knockdown of EMX2 expression in lung cancer cells expressing endogenous EMX2 promoted cell proliferation, invasive phenotypes and canonical WNT signaling. Taken together, our study suggests that EMX2 may have important roles as a novel suppressor in human lung cancer.

Discovery of molecular subtypes in leiomyosarcoma through integrative molecular profiling.

Leiomyosarcoma (LMS) is a soft tissue tumor with a significant degree of morphologic and molecular heterogeneity. We used integrative molecular profiling to discover and characterize molecular subtypes of LMS. Gene expression profiling was performed on 51 LMS samples. Unsupervised clustering showed three reproducible LMS clusters. Array comparative genomic hybridization (aCGH) was performed on 20 LMS samples and showed that the molecular subtypes defined by gene expression showed distinct genomic changes. Tumors from the 'muscle-enriched' cluster showed significantly increased copy number changes (P=0.04). A majority of the muscle-enriched cases showed loss at 16q24, which contains Fanconi anemia, complementation group A, known to have an important role in DNA repair, and loss at 1p36, which contains PRDM16, of which loss promotes muscle differentiation. Immunohistochemistry (IHC) was performed on LMS tissue microarrays (n=377) for five markers with high levels of messenger RNA in the muscle-enriched cluster (ACTG2, CASQ2, SLMAP, CFL2 and MYLK) and showed significantly correlated expression of the five proteins (all pairwise P<0.005). Expression of the five markers was associated with improved disease-specific survival in a multivariate Cox regression analysis (P<0.04). In this analysis that combined gene expression profiling, aCGH and IHC, we characterized distinct molecular LMS subtypes, provided insight into their pathogenesis, and identified prognostic biomarkers.

Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis.

Heparan sulfate (HS) proteoglycans (HSPGs) bind to multiple growth factors/morphogens and regulate their signaling. 6-O-sulfation (6S) of glucosamine within HS chains is critical for many of these ligand interactions. Sulf-1 and Sulf-2, which are extracellular neutral-pH sulfatases, provide a novel post-synthetic mechanism for regulation of HSPG function by removing 6S from intact HS chains. The Sulfs can thereby modulate several signaling pathways, including the promotion of Wnt signaling. We found induction of SULF2 transcripts and Sulf-2 protein in human lung adenocarcinoma and squamous cell carcinoma, the two major classes of non-small-cell lung carcinomas (NSCLCs). We confirmed widespread Sulf-2 protein expression in tumor cells of 10/10 surgical specimens of human lung squamous carcinomas. We studied five Sulf-2(+) NSCLC cell lines, including two, which were derived by cigarette-smoke transformation of bronchial epithelial cells. shRNA-mediated Sulf-2 knockdown in these lines caused an increase in 6S on their cell surface and in parallel reversed their transformed phenotype in vitro, eliminated autocrine Wnt signaling and strongly blunted xenograft tumor formation in nude mice. Conversely, forced Sulf-2 expression in non-malignant bronchial epithelial cells produced a partially transformed phenotype. Our findings support an essential role for Sulf-2 in lung cancer, the leading cancer killer.

Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX vaccine in advanced-stage non-small-cell lung cancer.

Tumor vaccines composed of autologous tumor cells genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) (GVAX) have demonstrated clinical activity in advanced-stage non-small-cell lung cancer (NSCLC). In an effort to remove the requirement for genetic transduction of individual tumors, we developed a 'bystander' GVAX platform composed of autologous tumor cells mixed with an allogeneic GM-CSF-secreting cell line. We conducted a phase I/II trial of this vaccine (3-12 biweekly vaccinations) in advanced-stage NSCLC. Tumors were harvested from 86 patients, tumor cell processing was successful in 76, and 49 proceeded to vaccination. The most common toxicity was local vaccine injection site reactions. Serum GM-CSF pharmacokinetics were consistent with secretion of GM-CSF from vaccine cells for up to 4 days with associated transient leukocytosis confirming the bioactivity of vaccine-secreted GM-CSF. Evidence of vaccine-induced immune activation was demonstrated; however, objective tumor responses were not seen. Compared with autologous GVAX vaccines prepared by transduction of individual tumors with an adenoviral GM-CSF vector, vaccine GM-CSF secretion was approximately 25-fold higher with the bystander GVAX vaccine used in this trial. However, the frequency of vaccine site reactions, tumor response, time to disease progression, and survival were all less favorable in the current study.

Wnt signaling in stem cells and lung cancer.

The Wnt signal transduction pathway plays important roles during embryo development, regulating cell proliferation and survival of immature cells. However, its improper function can lead to harmful consequences for humans, such as aberrant cell proliferation and, therefore, cancer. Increasing evidence suggests that stem cells may be the source of mutant cells that cause cancers to develop and proliferate. Wnt signaling has been shown to promote self-renewal in both gut epithelial and hematopoietic stem cells (HSCs) and to trigger critical pathways in carcinogenesis. Although the function of stem cells in solid tumor development is unclear, the Wnt pathway's role in determining the fate and self-renewal potential of cancer stem cells suggests a critical role in carcinogenesis. The development of new inhibitors, such as antibodies or small molecules, to inhibit this pathway may be of great therapeutic utility against cancer.

Aberrations in the fragile histidine triad (FHIT) gene in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) seems to be closely associated with lung carcinogenesis. To identify the genetic characteristics of precancerous IPF lesions in the peripheral lung, we performed PCR-based microsatellite analysis with DNA extracted from microdissected tissues; fluorescent in situ hybridization (FISH) analysis of the fragile histidine triad (FHIT) gene and immunohistochemical analysis of Fhit protein expression in samples of metaplasias and bronchiolar epithelia obtained from patients with IPF. We used four microsatellite markers of the FHIT gene within or flanking the FHIT gene on chromosome 3p for loss of heterozygosity (LOH) analysis. LOH of the FHIT locus was frequently found among the lesions of metaplasias and bronchiolar epithelia in the patients with IPF [62 (52%) of 119 informative lesions]. Fifty-four (73%) of the 74 lesions of metaplasias and bronchiolar epithelia obtained from the IPF patients with lung carcinoma and 8 (17%) of the 46 samples obtained from the IPF patients without lung carcinoma showed LOH at the FHIT gene (P < 0.0001). We confirmed allelic loss in the metaplasias and bronchiolar epithelia of IPF by FISH analysis of the FHIT gene. Additionally, the level of Fhit protein expression in the metaplastic cells of IPF was frequently reduced. Our findings suggest that allelic loss of the FHIT gene may be involved in carcinogenesis in the peripheral lung of patients with IPF.

p14(ARF) modulates the cytolytic effect of ONYX-015 in mesothelioma cells with wild-type p53.

ONYX-015 has been reported to kill selectively tumor cells lacking functional p53. Genetic alterations of INK4a/ARF locus, which is a predominant event in malignant pleural mesothelioma, may result in loss of p14(ARF) and subsequent disruption of p53 pathway in cancer cells. In the present study, ONYX-015 was able to kill three mesothelioma cell lines (H28, H513, and 211H) with wild-type p53 but lacking p14(ARF). In contrast, MS-1 mesothelioma cells, which expressed both p53 and p14(ARF), were resistant to ONYX-015. Introducing p14(ARF) gene into the H28 cell, a mesothelioma cell without p14(ARF) expression, significantly increased the resistance of this cell line to the cytolytic effect of ONYX-015. Our results suggest that human mesotheliomas with wild-type p53 yet lacking p14(ARF) are potential candidates for ONYX-015 therapy.

Stage II (N1) lung cancer.

As therapies evolve and mature, the authors predict that the next 20 years will see significant advances in the understanding of non-small-cell lung cancer (NSCLC), in molecular stratification of NSCLC, and significant improvement in survival and cure rates. This survival will be achieved through early detection and combined treatments using effective surgical interventions, improved radiotherapeutics, and especially significantly enhanced, rationally designed systemic therapies.

Expression sequence tag-specific full-length cDNA cloning: actin cDNAs.

Current strategies for cDNA cloning are based on construction of cDNA libraries and colony screening. The process of obtaining a full-length cDNA clone can be highly time and labor intensive. Using the human actin gene as a model target cDNA, we have developed an RNA-capture method for rapid cloning of full-length cDNAs. The approach involves the capture of mRNA with expressed sequence tag (EST)-derived, biotin labeled antisense "capture" primers and streptavidin-coated magnetic beads. Full-length cDNA is then synthesized from purified EST-specific mRNA and cloned directly into plasmid vectors. The results of using beta-actin-based capture primers on cytoplasmic RNA were the isolation of both beta- and gamma-actin cDNA clones. Of the 16 actin-specific cDNA clones analyzed, 15 (93%) were full-length. This approach for cloning full-length cDNAs from available ESTs or partial cDNA sequences will facilitate a more rapid and efficient characterization of gene structure and function.

Prostaglandin E2 and vasoactive intestinal peptide increase vascular endothelial cell growth factor mRNAs in lung cancer cells.

The effects of prostaglandin E2 (PGE2) and vasoactive intestinal peptide (VIP) on vascular endothelial cell growth factor (VEGF) mRNAs were investigated using lung cancer cells. By RT-PCR, VEGF(121), VEGF(165), and VEGF(189), but not VEGF(206) isoforms were detected in all lung cancer cell lines and biopsy specimens examined. By Northern blot, VEGF mRNA was detected in all small cell lung cancer (SCLC) and non-SCLC (NSCLC) cell lines examined. PGE2, VIP and forskolin caused increased VEGF expression in a time- and concentration-dependent manner using NSCLC cell line NCI-H157. Approximately 1 microM PGE2, 0.1 microM VIP and 50 microM forskolin caused cAMP elevation, 64-, 33- and 128-fold, respectively, using NCI-H157 cells after 5 min. The increase in cAMP caused by PGE(2) and VIP was reversed by somatostatin (SST). Also 1 microM PGE2, 0.1 microM VIP and 50 microM forskolin increased the VEGF mRNA 2.0-, 1.5- and 2.3-fold, respectively, after 4 h. The increase in VEGF mRNA caused by PGE2, VIP and forskolin was inhibited by H-89, a protein kinase A inhibitor. A VIP receptor antagonist, VIPhybrid, inhibited the increase in cAMP and VEGF mRNA caused by VIP. By ELISA, VEGF was detected in the conditioned media exposed to the lung cancer cell lines. These results suggest that VEGF synthesis in and secretion from lung cancer cells can be regulated by agents, which cause adenylyl cyclase activation.

Positron emission tomography with f18-fluorodeoxyglucose in the staging and preoperative evaluation of malignant pleural mesothelioma.

OBJECTIVES: The purpose of this study was to evaluate the utility of positron emission tomography with F18-fluorodeoxyglucose in the preoperative evaluation and staging of malignant mesothelioma in patients who were candidates for aggressive combined modality therapy. METHODS: Eighteen consecutive patients with biopsy-proven malignant mesothelioma underwent positron emission tomographic scanning. The results of positron emission tomographic imaging were compared with results obtained by computed tomography, mediastinoscopy, thoracoscopy, and pathologic examination of surgical specimens. All patients fasted and received an average of 14.5 +/- 2.7 mCi of F18-fluorodeoxyglucose for positron emission tomographic scanning. Attenuation-corrected whole-body and regional emission images of the chest and upper abdomen were acquired and formatted into transaxial, coronal, and sagittal images. RESULTS: All primary malignant mesotheliomas accumulated F18-fluorodeoxyglucose, and the mean standardized uptake value was 7. 6 (range, 3.33-14.85; n = 9). There were no false-negative results of positron emission tomography. Identification of occult extrathoracic metastases by positron emission tomography was the basis for excluding two patients from surgical therapy. There were two false-positive results of positron emission tomography: increased F18-fluorodeoxyglucose uptake in the contralateral chest that was negative by thoracoscopic biopsy (n = 1) and increased abdominal F18-fluorodeoxyglucose uptake after partial colectomy for diverticular disease (n = 1). CONCLUSIONS: Positron emission tomography can identify malignant pleural mesothelioma and appears to be a useful noninvasive staging modality for patients being considered for aggressive combined modality therapy.

Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma cells.

BACKGROUND: The p14(ARF) protein encoded by the INK4a/ARF locus promotes degradation of the MDM2 protein and thus prevents the MDM2-mediated inhibition of p53. Homozygous deletion of the INK4a/ARF locus is common in human mesothelioma and may result in the loss of p14(ARF) and the inactivation of p53. We designed this study to evaluate the biologic and potential therapeutic roles of p14(ARF) expression in mesothelioma cells. METHODS AND RESULTS: We constructed Adp14, an adenoviral vector carrying human p14(ARF) complementary DNA, and used it to transfect human mesothelioma cell lines H28, H513, H2052, and MSTO-211H. Overexpression of p14(ARF) led to increased amounts of p53 and the p21(WAF) proteins and dephosphorylation of the retinoblastoma protein. The growth rate of mesothelioma cells was inhibited markedly by infection with Adp14 compared with mock infection or infection with a control adenovirus vector, AdCtrl. Overexpression of p14(ARF) induced G(1)-phase cell cycle arrest and apoptotic cell death. Cytotoxicity assays showed that Adp14 had a statistically significantly (P =.002) greater effect on colon cancer (HCT116) cell lines containing two copies of the wild-type p53 gene than on p53-null cells, suggesting that functional p53 is a critical determinant of p14(ARF)-mediated cytotoxicity. CONCLUSIONS: The transfection of p14(ARF) into mesothelioma cells led to the overexpression of p14(ARF), which resulted in G(1)-phase arrest and apoptotic cell death. These results suggest that this gene therapy-based approach may be of use in the treatment of mesothelioma.

ONYX-015 works synergistically with chemotherapy in lung cancer cell lines and primary cultures freshly made from lung cancer patients.

p53 mutations and loss of heterozygosity (LOH) have been detected in >50% of lung cancers. Wild-type p53 can prevent replication of damaged DNA and promote apoptosis of cells with abnormal DNA. A human adenovirus, ONYX-015, which has a deletion in the E1B region, has shown tumor-specific cytolytic effect in tumor cells with nonfunctional p53 and antitumor efficacy that can be augmented by chemotherapeutic agents. A recent report from an independent group, however, indicates that wild-type p53 is necessary for the infection of this replicating virus, and it is in direct contradiction to previous observations of the ONYX group. In this study, we carried out cytopathic effect (CPE) assays using ONYX-015 on five human lung cancer cell lines with known p53 status. Two of these cell lines, NCI-H522 and NCI-H1703, have mutations and LOH in their p53 gene. Both lines were lysed in a dose-dependent manner and showed 100% cytolysis at a multiplicity of infection of 0.1. Two additional cell lines, NCI-H2347 and NCI-H838, both of which have wild-type p53 gene, showed near complete lysis at a multiplicity of infection of 1. We demonstrate here that the lung cancer cells with nonfunctional p53 are at least 10 times more sensitive to ONYX-015 cytolysis than the lung cancer cells with wild-type p53. In addition, standard chemotherapeutic agents (paclitaxol and cisplatin) showed a synergistic effect when combined with ONYX-015, and this effect was p53 mutant dependent. Furthermore, we tested the cytolytic effect of ONYX-015 on a panel (n = 7) of primary first-passage cultures made from freshly resected lung cancers. ONYX-015 lysed primary lung cancer cells in six of seven (86 %) primary cultures. Two of four primary cultures treated with chemotherapeutic agents had a synergistic effect with ONYX-015. Our data indicate that wild-type p53 is not required for the infection of this replicating virus, and also we demonstrate that ONYX-015 is effective alone and works synergistically with chemotherapeutic agents in lung cancer cell lines and primary cultures. This study suggests that ONYX-015 may be effective, especially in combination with conventional chemotherapy, in the treatment of patients with lung cancer.

Giant tumors of the chest: preoperative embolization and resection.

Giant tumors of the chest are rare. These tumors comprise a spectrum of disease from benign lesions to highly aggressive malignant tumors with cells of origin in the pleura, pulmonary parenchyma, blood vessels, thymus, and connective tissues. We report four cases of giant tumors of the thorax treated with preoperative arterial embolization followed by complete surgical resection. Their diagnostic and treatment courses, imaging, and pathology are described.