Adenovirus and Oxaliplatin cooperate as agnostic sensitizers for immunogenic cell death in colorectal carcinoma.

Treatments with cytotoxic agents or viruses may cause Immunogenic Cell Death (ICD) that immunize tumor-bearing hosts but do not cause complete regression of tumor. We postulate that combining two ICD inducers may cause durable regression in immunocompetent mice. ICD was optimized in vitro by maximizing calreticulin externalization in human colorectal carcinoma (CRC) cells by exposure to mixtures of Oxaliplatin (OX) and human adenovirus (AdV). Six mm diameter CT26 or 4T1 carcinomas in flanks of BALB/c mice were injected once intratumorally (IT) with OX, AdV or their mixture. Tumor growth, Tumor-Infiltrating Lymphocytes (TIL), nodal cytotoxicity, and rejection of a viable cell challenge were measured. Tumors injected IT once with an optimum mixture of 80 µM OX - AdV 25 Multiplicity of Infection (MOI) in PBS buffer were 17-29% the volume of control tumors. When buffer was changed from PBS to 5% dextrose in water (D5W), volumes of tumors injected IT with 80 µM OX-AdV 25 MOI were 10% while IT OX or AdV alone were 32% and 40% the volume of IT buffer-treated tumors. OX-AdV IT increased CD3+ TIL by 4-fold, decreased CD8+ PD-1+ TIL from 79% to 19% and induced cytotoxicity to CT26 cells in draining node lymphocytes while lymphocytes from CT26-bearing untreated mice were not cytotoxic. OX-AdV IT in D5W caused complete regression in 40% of mice. Long-term survivors rejected a contralateral challenge of CT26. The buffer for Oxaliplatin is critical. The two ICD inducer mixture is promising as an agnostic sensitizer for carcinomas like colorectal carcinoma.

Epigenetic re-wiring of breast cancer by pharmacological targeting of C-terminal binding protein.

The C-terminal binding protein (CtBP) is an NADH-dependent dimeric family of nuclear proteins that scaffold interactions between transcriptional regulators and chromatin-modifying complexes. Its association with poor survival in several cancers implicates CtBP as a promising target for pharmacological intervention. We employed computer-assisted drug design to search for CtBP inhibitors, using quantitative structure-activity relationship (QSAR) modeling and docking. Functional screening of these drugs identified 4 compounds with low toxicity and high water solubility. Micro molar concentrations of these CtBP inhibitors produces significant de-repression of epigenetically silenced pro-epithelial genes, preferentially in the triple-negative breast cancer cell line MDA-MB-231. This epigenetic reprogramming occurs through eviction of CtBP from gene promoters; disrupted recruitment of chromatin-modifying protein complexes containing LSD1, and HDAC1; and re-wiring of activating histone marks at targeted genes. In functional assays, CtBP inhibition disrupts CtBP dimerization, decreases cell migration, abolishes cellular invasion, and improves DNA repair. Combinatorial use of CtBP inhibitors with the LSD1 inhibitor pargyline has synergistic influence. Finally, integrated correlation of gene expression in breast cancer patients with nuclear levels of CtBP1 and LSD1, reveals new potential therapeutic vulnerabilities. These findings implicate a broad role for this class of compounds in strategies for epigenetically targeted therapeutic intervention.

MicroRNA 17-92 cluster mediates ETS1 and ETS2-dependent RAS-oncogenic transformation.

The ETS-family transcription factors Ets1 and Ets2 are evolutionarily conserved effectors of the RAS/ERK signaling pathway, but their function in Ras cellular transformation and biology remains unclear. Taking advantage of Ets1 and Ets2 mouse models to generate Ets1/Ets2 double knockout mouse embryonic fibroblasts, we demonstrate that deletion of both Ets1 and Ets2 was necessary to inhibit HrasG12V induced transformation both in vitro and in vivo. HrasG12V expression in mouse embryonic fibroblasts increased ETS1 and ETS2 expression and binding to cis-regulatory elements on the c-Myc proximal promoter, and consequently induced a robust increase in MYC expression. The expression of the oncogenic microRNA 17-92 cluster was increased in HrasG12V transformed cells, but was significantly reduced when ETS1 and ETS2 were absent. MYC and ETS1 or ETS2 collaborated to increase expression of the oncogenic microRNA 17-92 cluster in HrasG12V transformed cells. Enforced expression of exogenous MYC or microRNA 17-92 rescued HrasG12V transformation in Ets1/Ets2-null cells, revealing a direct function for MYC and microRNA 17-92 in ETS1/ETS2-dependent HrasG12V transformation.

Transcriptomic architecture of the adjacent airway field cancerization in non-small cell lung cancer.

BACKGROUND: Earlier work identified specific tumor-promoting abnormalities that are shared between lung cancers and adjacent normal bronchial epithelia. We sought to characterize the yet unknown global molecular and adjacent airway field cancerization (FC) in early-stage non-small cell lung cancer (NSCLC). METHODS: Whole-transcriptome expression profiling of resected early-stage (I-IIIA) NSCLC specimens (n = 20) with matched tumors, multiple cytologically controlled normal airways with varying distances from tumors, and uninvolved normal lung tissues (n = 194 samples) was performed using the Affymetrix Human Gene 1.0 ST platform. Mixed-effects models were used to identify differentially expressed genes among groups. Ordinal regression analysis was performed to characterize site-dependent airway expression profiles. All statistical tests were two-sided, except where noted. RESULTS: We identified differentially expressed gene features (n = 1661) between NSCLCs and airways compared with normal lung tissues, a subset of which (n = 299), after gene set enrichment analysis, statistically significantly (P < .001) distinguished large airways in lung cancer patients from airways in cancer-free smokers. In addition, we identified genes (n = 422) statistically significantly and progressively differentially expressed in airways by distance from tumors that were found to be congruently modulated between NSCLCs and normal lung tissues. Furthermore, LAPTM4B, with statistically significantly increased expression (P < .05) in airways with shorter distance from tumors, was upregulated in human immortalized cells compared with normal bronchial epithelial cells (P < .001) and promoted anchorage-dependent and -independent lung cancer cell growth. CONCLUSIONS: The adjacent airway FC comprises both site-independent profiles as well as gradient and localized airway expression patterns. Profiling of the airway FC may provide new insights into NSCLC oncogenesis and molecular tools for detection of the disease.

ETS2 mediated tumor suppressive function and MET oncogene inhibition in human non-small cell lung cancer.

PURPOSE: The ETS2 transcription factor is an evolutionarily conserved gene that is deregulated in cancer. We analyzed the transcriptome of lung adenocarcinomas and normal lung tissue by expression profiling and found that ETS2 was significantly downregulated in adenocarcinomas. In this study, we probed the yet unknown functional role of ETS2 in lung cancer pathogenesis. EXPERIMENTAL DESIGN: Lung adenocarcinomas (n = 80) and normal lung tissues (n = 30) were profiled using the Affymetrix Human Gene 1.0 ST platform. Immunohistochemical (IHC) analysis was conducted to determine ETS2 protein expression in non-small cell lung cancer (NSCLC) histologic tissue specimens (n = 201). Patient clinical outcome, based on ETS2 IHC expression, was statistically assessed using the log-rank and Kaplan-Meier tests. RNA interference and overexpression strategies were used to assess the effects of ETS2 expression on the transcriptome and on various malignant phenotypes. RESULTS: ETS2 expression was significantly reduced in lung adenocarcinomas compared with normal lung (P < 0.001). Low ETS2 IHC expression was a significant predictor of shorter time to recurrence in NSCLC (P = 0.009, HR = 1.89) and adenocarcinoma (P = 0.03, HR = 1.86). Moreover, ETS2 was found to significantly inhibit lung cancer cell growth, migration, and invasion (P < 0.05), and microarray and pathways analysis revealed significant (P < 0.001) activation of the HGF pathway following ETS2 knockdown. In addition, ETS2 was found to suppress MET phosphorylation and knockdown of MET expression significantly attenuated (P < 0.05) cell invasion mediated by ETS2-specific siRNA. Furthermore, knockdown of ETS2 augmented HGF-induced MET phosphorylation, cell migration, and invasion. CONCLUSION(S): Our findings point to a tumor suppressor role for ETS2 in human NSCLC pathogenesis through inhibition of the MET proto-oncogene.

Characterizing the molecular spatial and temporal field of injury in early-stage smoker non-small cell lung cancer patients after definitive surgery by expression profiling.

Gene expression alterations in response to cigarette smoke have been characterized in normal-appearing bronchial epithelium of healthy smokers, and it has been suggested that adjacent histologically normal tissue displays tumor-associated molecular abnormalities. We sought to delineate the spatial and temporal molecular lung field of injury in smoker patients with early-stage non-small cell lung cancer (NSCLC; n = 19) who were accrued into a surveillance clinical trial for annual follow-up and bronchoscopies within 1 year after definitive surgery. Bronchial brushings and biopsies were obtained from six different sites in the lung at the time of inclusion in the study and at 12, 24, and 36 months after the first time point. Affymetrix Human Gene 1.0 ST arrays were used for whole-transcript expression profiling of airways (n = 391). Microarray analysis identified gene features (n = 1,165) that were nonuniform by site and differentially expressed between airways adjacent to tumors relative to more distant samples as well as those (n = 1,395) that were significantly altered with time up to 3 years. In addition, gene interaction networks mediated by phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK)1/2 were modulated in adjacent compared with contralateral airways and the latter network with time. Furthermore, phosphorylated AKT and ERK1/2 immunohistochemical expression were significantly increased with time (nuclear pAKT, P = 0.03; cytoplasmic pAKT, P < 0.0001; pERK1/2, P = 0.02) and elevated in adjacent compared with more distant airways (nuclear pAKT, P = 0.04; pERK1/2, P = 0.03). This study highlights spatial and temporal cancer-associated expression alterations in the molecular field of injury of patients with early-stage NSCLCs after definitive surgery that warrant further validation in independent studies.

G-protein coupled receptor family C, group 5, member A (GPRC5A) expression is decreased in the adjacent field and normal bronchial epithelia of patients with chronic obstructive pulmonary disease and non-small-cell lung cancer.

INTRODUCTION: Understanding oncogenes and tumor suppressor genes expression patterns is essential for characterizing lung cancer pathogenesis. We have previously demonstrated that mGprc5a/hGPRC5A is a lung-specific tumor suppressor evidenced by inflammation-mediated tumorigenesis in Gprc5a-knockout mice. The implication of GPRC5A in human lung cancer pathogenesis, including that associated with inflammatory chronic obstructive pulmonary disease (COPD), a risk factor for the malignancy, remains elusive. METHODS: We sought to examine GPRC5A immunohistochemical expression in histologically normal bronchial epithelia (NBE) from lung disease-free never- and ever-smokers (n = 13 and n = 18, respectively), from COPD patients with (n = 26) and without cancer (n = 24) and in non-small cell lung cancers (NSCLCs) (n = 474). Quantitative assessment of GPRC5A transcript expression in airways (n = 6), adjacent NBEs (n = 29) and corresponding tumors (n = 6) from 6 NSCLC patients was also performed. RESULTS: GPRC5A immunohistochemical expression was significantly lower in tumors compared to uninvolved NBE (p < 0.0001) and was positively associated with adenocarcinoma histology (p < 0.001). GPRC5A airway expression was highest in lung disease-free NBE, decreased and intermediate in NBE of cancer-free COPD patients (p = 0.004) and further attenuated and lowest in epithelia of COPD patients with adenocarcinoma and SCC (p < 0.0001). Furthermore, GPRC5A mRNA was significantly decreased in NSCLCs and corre sponding NBE compared to uninvolved normal lung (p = 0.03). CONCLUSIONS: Our findings highlight decreased GPRC5A expression in the field cancerization of NSCLC, including that associated with lung inflammation. Assessment of the use of GPRC5A expression as a risk factor for NSCLC development in COPD patients is warranted.

Pulmonary adenocarcinoma: a renewed entity in 2011.

Lung cancer, of which non-small-cell lung cancer comprises the majority, is the leading cause of cancer-related deaths in the United States and worldwide. Lung adenocarcinomas are a major subtype of non-small-cell lung cancers, are increasing in incidence globally in both males and females and in smokers and non-smokers, and are the cause for almost 50% of deaths attributable to lung cancer. Lung adenocarcinoma is a tumour with complex biology that we have recently started to understand with the advent of various histological, transcriptomic, genomic and proteomic technologies. However, the histological and molecular pathogenesis of this malignancy is still largely unknown. This review will describe advances in the molecular pathology of lung adenocarcinoma with emphasis on genomics and DNA alterations of this disease. Moreover, the review will discuss recognized lung adenocarcinoma preneoplastic lesions and current concepts of the early pathogenesis and progression of the disease. We will also portray the field cancerization phenomenon and lineage-specific oncogene expression pattern in lung cancer and how both remerging concepts can be exploited to increase our understanding of lung adenocarcinoma pathogenesis for subsequent development of biomarkers for early detection of adenocarcinomas and possibly personalized prevention.

Transcription factor ets-2 plays an important role in the pathogenesis of pulmonary fibrosis.

Ets-2 is a ubiquitous transcription factor activated after phosphorylation at threonine-72. Previous studies highlighted the importance of phosphorylated ets-2 in lung inflammation and extracellular matrix remodeling, two pathways involved in pulmonary fibrosis. We hypothesized that phosphorylated ets-2 played an important role in pulmonary fibrosis, and we sought to determine the role of ets-2 in its pathogenesis. We challenged ets-2 (A72/A72) transgenic mice (harboring a mutated form of ets-2 at phosphorylation site threonine-72) and ets-2 (wild-type/wild-type [WT/WT]) control mice with sequential intraperitoneal injections of bleomycin, followed by quantitative measurements of lung fibrosis and inflammation and primary cell in vitro assays. Concentrations of phosphorylated ets-2 were detected via the single and dual immunohistochemical staining of murine lungs and lung sections from patients with idiopathic pulmonary fibrosis. Ets-2 (A72/A72) mice were protected from bleomycin-induced pulmonary fibrosis, compared with ets-2 (WT/WT) mice. This protection was characterized by decreased lung pathological abnormalities and the fibrotic gene expression of Type I collagen, Type III collagen, α-smooth muscle actin, and connective tissue growth factor. Immunohistochemical staining of lung sections from bleomycin-treated ets-2 (WT/WT) mice and from patients with idiopathic pulmonary fibrosis demonstrated increased staining of phosphorylated ets-2 that colocalized with Type I collagen expression and to fibroblastic foci. Lastly, primary lung fibroblasts from ets-2 (A72/A72) mice exhibited decreased expression of Type I collagen in response to stimulation with TGF-ß, compared with fibroblasts from ets-2 (WT/WT) mice. These data indicate the importance of phosphorylated ets-2 in the pathogenesis of pulmonary fibrosis through the expression of Type I collagen and (myo)fibroblast activation.

Stage-specific effect of N-(4-hydroxyphenyl)retinamide on cell growth in squamous cell carcinogenesis.

Squamous cell carcinoma (SCC) is the most prevalent form of epithelial cancer. SCC results when normal epithelial cells undergo multiple neoplastic changes that culminate in the evolution of an invasive cancer. Retinoids are commonly used as chemopreventive and treatment agents in skin cancer; however, SCC progression is accompanied by a gradual loss of retinoid responsiveness. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has shown promising anti-neoplastic activity in a variety of tumor cells, including those that are resistant to all-trans retinoic acid (t-RA). We investigated the effect of HPR on growth and apoptosis of squamous cells at different stages of carcinogenesis. We then determined if retinoic acid receptor (RAR) overexpression affected the outcome of HPR treatment. To model SCC malignant progression, we used a panel of murine keratinocytes representing different stages of squamous cell carcinogenesis. This panel consisted of primary keratinocytes, SP1 and 308 papilloma cell lines, the PAM-212 squamous carcinoma cell line, and the spindle I7 cell line. With the exception of the primary keratinocytes, all cells were unresponsive to t-RA treatment. Pharmacological concentrations of HPR were non-cytotoxic to all keratinocytes tested and HPR sensitivity was stage-dependent, with the papilloma cell lines being the most sensitive, and the spindle cells being the most resistant. Overexpression of RARgamma in SP1 papilloma cells enhanced growth suppression and apoptosis induction by HPR. HPR-induced growth suppression was accompanied by a simultaneous block in the G(1) phase of the cell cycle in RAR-transduced and control SP1 cells and differential regulation of cell cycle and apoptotic mediators.