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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.)

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.)

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.)

Elevated tumor cyclooxygenase-2 (COX-2) expression is associated with tumor invasion, metastasis, and poor prognosis in non-small cell lung cancer (NSCLC). Here, we report that COX-2-dependent pathways contribute to the modulation of E-cadherin expression in NSCLC. First, whereas genetically modified COX-2-sense (COX-2-S) NSCLC cells expressed low E-cadherin and showed diminished capacity for cellular aggregation, genetic or pharmacologic inhibition of tumor COX-2 led to increased E-cadherin expression and resulted in augmented homotypic cellular aggregation among NSCLC cells in vitro. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human lung adenocarcinoma tissue sections. Second, treatment of NSCLC cells with exogenous prostaglandin E(2) (PGE(2)) significantly decreased the expression of E-cadherin, whereas treatment of COX-2-S cells with celecoxib (1 mumol/L) led to increased E-cadherin expression. Third, the transcriptional suppressors of E-cadherin, ZEB1 and Snail, were up-regulated in COX-2-S cells or PGE(2)-treated NSCLC cells but decreased in COX-2-antisense cells. PGE(2) exposure led to enhanced ZEB1 and Snail binding at the chromatin level as determined by chromatin immunoprecipitation assays. Small interfering RNA-mediated knockdown of ZEB1 or Snail interrupted the capacity of PGE(2) to down-regulate E-cadherin. Fourth, an inverse relationship between E-cadherin and ZEB1 and a direct relationship between COX-2 and ZEB1 were shown by immunohistochemical staining of human lung adenocarcinoma tissue sections. These findings indicate that PGE(2), in autocrine or paracrine fashion, modulates transcriptional repressors of E-cadherin and thereby regulates COX-2-dependent E-cadherin expression in NSCLC. Thus, blocking PGE(2) production or activity may contribute to both prevention and treatment of NSCLC.

The antitumor efficiency of dendritic cells transduced with an adenovirus vector expressing secondary lymphoid chemokine (CCL21) was evaluated in a murine model of spontaneous bronchoalveolar cell carcinoma. The transgenic mice (CC-10 TAg) express the SV40 large T antigen (TAg) under the Clara cell promoter, develop bilateral, multifocal, and pulmonary adenocarcinomas, and die at 4 months as a result of progressive pulmonary tumor burden. A single intratracheal administration of CCL21 gene-modified dendritic cells (DC-AdCCL21) led to a marked reduction in tumor burden with extensive mononuclear cell infiltration of the tumors. The reduction in tumor burden was accompanied by the enhanced elaboration of type 1 cytokines [IFN-gamma, interleukin (IL)-12, and granulocyte macrophage colony-stimulating factor] and antiangiogenic chemokines (CXCL9 and CXCL10) but a concomitant decrease in the immunosuppressive molecules (IL-10, transforming growth factor-beta, prostaglandin E(2)) in the tumor microenvironment. The DC-AdCCL21 therapy group revealed a significantly greater frequency of tumor-specific T cells releasing IFN-gamma compared with the controls. Continuous therapy with weekly intranasal delivery of DC-AdCCL21 significantly prolonged median survival by >7 weeks in CC-10 TAg mice. Both innate natural killer and specific T-cell antitumor responses significantly increased following DC-AdCCL21 therapy. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for further evaluation of intrapulmonary-administered DC-AdCCL21 in regulation of tumor immunity and genetic immunotherapy for lung cancer.

Cyclooxygenase 2 (COX-2) overexpression is found in a wide variety of human cancers and is linked to all stages of tumorigenesis. Elevated tumor COX-2 expression is associated with increased angiogenesis, tumor invasion, suppression of host immunity and promotes tumor cell resistance to apoptosis. Previous reports have linked the COX-2 product prostaglandin E2 (PGE2) to the abnormal activation of the mitogen-activated protein kinase/Erk kinase pathway. Here we show that PGE2 is able to rapidly stimulate Erk phosphorylation in a subset of non-small cell lung cancer (NSCLC) cell lines. This effect is not evident in bronchial epithelial cells. In contrast to previous reports in colon cancer, we found that Erk activation as well as cellular proliferation induced by PGE2 was not inhibited by pretreatment of the cells with epidermal growth factor receptor (EGFR) inhibitors. Activation of the Erk pathway by PGE2 was also resistant to src kinase inhibitors but sensitive to the protein kinase C inhibition. PGE2 effects are mediated through four G protein-coupled receptors. Selective inhibition of EP receptors revealed the possible involvement of Ca2+-dependent signaling in PGE2-mediated activation of Erk. Our data indicate the presence of an EGFR-independent activation of the mitogen-activated protein kinase/Erk pathway by PGE2 in NSCLC cells. These findings provide evidence for the possible link between tumor COX-2 overexpression and elevated Erk-mediated cancer cell proliferation and migration. Importantly, these findings suggest that COX-2 overexpression may contribute to EGFR inhibitor resistance in NSCLC.

Cyclooxygenase (COX)-2 and its product prostaglandin (PG) E2 underlie an immunosuppressive network that is important in the pathogenesis of non-small cell lung cancer. CD4+ CD25+ T regulatory (Treg) cells play an important role in maintenance of immunologic self-tolerance. CD4+ CD25+ Treg cell activities increase in lung cancer and appear to play a role in suppressing antitumor immune responses. Definition of the pathways controlling Treg cell activities will enhance our understanding of limitation of the host antitumor immune responses. Tumor-derived COX-2/PGE2 induced expression of the Treg cell-specific transcription factor, Foxp3, and increased Treg cell activity. Assessment of E-prostanoid (EP) receptor requirements revealed that PGE2-mediated induction of Treg cell Foxp3 gene expression was significantly reduced in the absence of the EP4 receptor and ablated in the absence of the EP2 receptor expression. In vivo, COX-2 inhibition reduced Treg cell frequency and activity, attenuated Foxp3 expression in tumor-infiltrating lymphocytes, and decreased tumor burden. Transfer of Treg cells or administration of PGE2 to mice receiving COX-2 inhibitors reversed these effects. We conclude that inhibition of COX-2/PGE2 suppresses Treg cell activity and enhances antitumor responses.

Constitutive overexpression of cyclooxygenase-2 (COX-2) occurs frequently in several different malignancies, including lung, colon, breast, and prostate cancer. Clinical studies have established elevated serum insulin-like growth factor (IGF-I) content and IGF-I:IGF-binding protein 3 (IGFBP-3) ratio as risk factors for these same malignancies. Therefore, we sought to determine the link between COX-2 expression and the IGF axis in COX-2 gene-modified human non-small-cell lung cancer (NSCLC) cells. Overexpression of COX-2 in NSCLC cells enhanced the antiapoptotic and mitogenic effects of IGF-I and IGF-II, facilitated the autophosphorylation of the type 1 IGF receptor, increased class IA phosphatidylinositol 3'-kinase activity, and decreased expression of IGFBP-3. Thus, these findings show that COX-2 augments the stimulatory arm of the IGF axis.

Elevated tumor cyclooxygenase 2 (COX-2) expression is associated with increased angiogenesis, tumor invasion, and promotion of tumor cell resistance to apoptosis. In our previous studies using non-small cell lung cancer (NSCLC) cell lines constitutively expressing COX-2 cDNA in sense and antisense orientations, we demonstrated that constitutive overexpression of COX-2 leads to stabilization of the inhibitor of apoptosis protein survivin resulting in the elevated apoptosis resistance of COX-2-overexpressing cells. Genetic or pharmacologic suppression of COX-2 activity increased proteasomal degradation of survivin and cellular response to apoptosis induction. Our data show that expression of survivin in non-small cell lung cancer cells can be significantly down-regulated by RNA interference. Whereas COX-2-overexpressing NSCLC cells have significantly higher apoptosis resistance than the parental cells, inhibition of survivin expression by small interfering RNA decreases apoptosis resistance to the level of the parental non-small cell lung cancer. We conclude that COX-2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer.

The Coxsackie Adenovirus Receptor (CAR) has primarily been studied in its role as the initial cell surface attachment receptor for Coxsackie and group C adenoviruses. Recent reports suggest that CAR mediates homotypic intercellular adhesion as part of the tight and/or adherens junction. Thus, CAR is well positioned to participate in intercellular interactions and signaling. Using an antisense (AS)-CAR plasmid vector, we silenced surface CAR expression in lung cancer cells that possessed a high basal expression of this molecule and monitored the resultant tumorigenesis. AS-CAR transfectants exhibit a profound loss in the ability to generate xenografts in scid/scid mice. The emergence of delayed-onset tumors in animals that received injection with AS-CAR transfectants correlates with the resurfacing of CAR expression, suggesting that such expression and tumor emergence are temporally related. To study the mechanism underlying the differences in tumorigenicity, control and AS-CAR cells were compared in terms of their in vitro growth potential. Whereas only subtle differences in the proliferative capacity of the two populations were evident when assayed with growth on plastic, significant differences became apparent when one compared the relative ability of these populations to form colonies in soft agar. These data indicate that silencing surface CAR expression abrogates xenograft tumorigenesis in vivo and colony formation in vitro and invoke the novel possibility that CAR expression is needed for the efficient formation of tumors by a subset of lung cancer cells.

Elevated tumor cyclooxygenase (COX)-2 activity plays a multifaceted role in non-small cell lung cancer (NSCLC). To elucidate the role of COX-2 in the in vitro and in vivo expression of two known NSCLC angiogenic peptides, CXC ligand (CXCL) 8 and CXCL5, we studied two COX-2 gene-modified NSCLC cell lines, A549 and H157. COX-2 overexpression enhanced the in vitro expression of both CXCL8 and CXCL5. In contrast, specific COX-2 inhibition decreased the production of both peptides as well as nuclear translocation of nuclear factor kappaB. In a severe combined immunodeficient mouse model of human NSCLC, the enhanced tumor growth of COX-2-overexpressing tumors was inhibited by neutralizing anti-CXCL5 and anti-CXCL8 antisera. We conclude that COX-2 contributes to the progression of NSCLC tumorigenesis by enhancing the expression of angiogenic chemokines CXCL8 and CXCL5.

We assessed a nonradioactive approach to induce apoptosis in non-small cell lung cancer by a novel iodide uptake and retention mechanism. To enhance tumor apoptosis, we transduced non-small cell lung cancer cells with retroviral vectors containing the sodium iodide symporter (NIS) and thyroperoxidase (TPO) genes. Expression of NIS and TPO facilitated concentration of iodide in tumors. As a consequence of the marked increase in intracellular levels of iodide, apoptosis was seen in >95% of NIS/TPO-modified lung cancer cells. Intraperitoneal injection of potassium iodide resulted in significant tumor volume reduction in NIS/TPO-modified tumor xenografts without apparent adverse effects in SCID mice. Iodide induced an increase in the level of reactive oxygen species. Iodide-induced apoptosis is sensitive to N-acetylcysteine inhibition, suggesting an important role by reactive oxygen species in this apoptotic process. In addition, iodide-induced apoptosis is associated with overexpression of CDKN1A (p21/Waf1)and down-regulation of survivin at both mRNA and protein levels. This is the first report demonstrating that a therapeutic dose of nonradioactive iodide has potent efficacy and high selectivity against lung cancer when used in combination with genetic modification of cancer cells to express the NIS/TPO genes.

Non-small cell lung cancer (NSCLC) cells are known to constitutively overexpress cyclooxygenase (COX)-2. Tumor COX-2-dependent production of PGE(2) triggers the synthesis of lymphocyte and macrophage interleukin (IL)-10 that, in turn, is known to potently suppress COX-2 in normal cells. Thus, we investigated the capacity of IL-10 to down-regulate COX-2 expression in NSCLC cells. Western blotting and ELISA analyses revealed that IL-10 did not affect COX-2 expression and subsequent PGE(2) production in NSCLC cells. Although normal human bronchial epithelial cells expressed both intracellular and membrane IL-10Ralpha, NSCLC cells only expressed intracellular but not cell surface membrane IL-10Ralpha. Unresponsiveness of COX-2 to IL-10 is due to the deficiency of IL-10Ralpha on the surface of NSCLC cells. Our findings highlight a novel mechanism contributing to maintenance of elevated COX-2 and PGE(2) in the lung tumor environment.

T lymphocyte survival is critical for the development and maintenance of an effective host antitumor immune response; however, the tumor environment can negatively impact T-cell survival. Lymphocytes exposed to tumor supernatants (TSNs) were evaluated for apoptosis after mitogen stimulation. TSN was observed to significantly enhance phorbol 12-myristate 13-acetate/ionomycin- and anti-CD3-stimulated lymphocyte apoptosis. Enhanced lymphocyte apoptosis was associated with an impairment of nuclear factor kappa B nuclear translocation and diminished I kappa B alpha degradation. In lymphocytes stimulated after exposure to TSNs, cytoplasmic I kappa B alpha persisted as a result of alterations in I kappa B kinase (IKK) activity. Accordingly, although there were no apparent differences in IKK component concentrations, lymphocytes preexposed to TSNs exhibited markedly reduced IKK activity. We conclude that non-small cell lung cancer-derived soluble factors promote apoptosis in activated lymphocytes by an IKK-dependent pathway.