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Accurate diagnosis of lung cancer is important for treatment decision-making. Tumor biopsy and histologic examination are the standard for determining histologic lung cancer subtypes. Liquid biopsy, particularly cell-free DNA (cfDNA), has recently shown promising results in cancer detection and classification. In this study, we investigate the potential of cfDNA methylome for the noninvasive classification of lung cancer histologic subtypes. We focused on the two most prevalent lung cancer subtypes, lung adenocarcinoma and lung squamous cell carcinoma. Using a fragment-based marker discovery approach, we identified robust subtype-specific methylation markers from tumor samples. These markers were successfully validated in independent cohorts and associated with subtype-specific transcriptional activity. Leveraging these markers, we constructed a subtype classification model using cfDNA methylation profiles, achieving an AUC of 0.808 in cross-validation and an AUC of 0.747 in the independent validation. Tumor copy-number alterations inferred from cfDNA methylome analysis revealed potential for treatment selection. In summary, our study demonstrates the potential of cfDNA methylome analysis for noninvasive lung cancer subtyping, offering insights for cancer monitoring and early detection.SignificanceThis study explores the use of cfDNA methylomes for the classification of lung cancer subtypes, vital for effective treatment. By identifying specific methylation markers in tumor tissues, we developed a robust classification model achieving high accuracy for noninvasive subtype detection. This cfDNA methylome approach offers promising avenues for early detection and monitoring.

Immune checkpoint blockade (ICB) with PD-1/PD-L1 inhibition has revolutionized the treatment of non-small cell lung cancer (NSCLC). Durable responses, however, are observed only in a subpopulation of patients. Defective antigen presentation and an immunosuppressive tumor microenvironment (TME) can lead to deficient T cell recruitment and ICB resistance. We evaluate intratumoral (IT) vaccination with CXCL9- and CXCL10-engineered dendritic cells (CXCL9/10-DC) as a strategy to overcome resistance. IT CXCL9/10-DC leads to enhanced T cell infiltration and activation in the TME and tumor inhibition in murine NSCLC models. The antitumor efficacy of IT CXCL9/10-DC is dependent on CD4+ and CD8+ T cells, as well as CXCR3-dependent T cell trafficking from the lymph node. IT CXCL9/10-DC, in combination with ICB, overcomes resistance and establishes systemic tumor-specific immunity in murine models. These studies provide a mechanistic understanding of CXCL9/10-DC-mediated host immune activation and support clinical translation of IT CXCL9/10-DC to augment ICB efficacy in NSCLC.

A greater understanding of molecular, cellular, and immunological changes during the early stages of lung adenocarcinoma development could improve diagnostic and therapeutic approaches in patients with pulmonary nodules at risk for lung cancer. To elucidate the immunopathogenesis of early lung tumorigenesis, we evaluated surgically resected pulmonary nodules representing the spectrum of early lung adenocarcinoma as well as associated normal lung tissues using single-cell RNA sequencing and validated the results by flow cytometry and multiplex immunofluorescence (MIF). Single-cell transcriptomics revealed a significant decrease in gene expression associated with cytolytic activities of tumor-infiltrating natural killer and natural killer T cells. This was accompanied by a reduction in effector T cells and an increase of CD4+ regulatory T cells (Treg) in subsolid nodules. An independent set of resected pulmonary nodules consisting of both adenocarcinomas and associated premalignant lesions corroborated the early increment of Tregs in premalignant lesions compared with the associated normal lung tissues by MIF. Gene expression analysis indicated that cancer-associated alveolar type 2 cells and fibroblasts may contribute to the deregulation of the extracellular matrix, potentially affecting immune infiltration in subsolid nodules through ligand-receptor interactions. These findings suggest that there is a suppression of immune surveillance across the spectrum of early-stage lung adenocarcinoma.SignificanceAnalysis of a spectrum of subsolid pulmonary nodules by single-cell RNA sequencing provides insights into the immune regulation and cell-cell interactions in the tumor microenvironment during early lung tumor development.

BackgroundDespite recent advances in immunotherapy, many patients with non-small cell lung cancer (NSCLC) do not respond to immune checkpoint inhibitors (ICI). Resistance to ICI may be driven by suboptimal priming of antitumor T lymphocytes due to poor antigen presentation as well as their exclusion and impairment by the immunosuppressive tumor microenvironment (TME). In a recent phase I trial in patients with NSCLC, in situ vaccination (ISV) with dendritic cells engineered to secrete CCL21 (CCL21-DC), a chemokine that facilitates the recruitment of T cells and DC, promoted T lymphocyte tumor infiltration and PD-L1 upregulation.MethodsMurine models of NSCLC with distinct driver mutations (KrasG12D/P53+/-/Lkb1-/- (KPL); KrasG12D/P53+/- (KP); and KrasG12D (K)) and varying tumor mutational burden were used to evaluate the efficacy of combination therapy with CCL21-DC ISV plus ICI. Comprehensive analyses of longitudinal preclinical samples by flow cytometry, single cell RNA-sequencing (scRNA-seq) and whole-exome sequencing were performed to assess mechanisms of combination therapy.ResultsISV with CCL21-DC sensitized immune-resistant murine NSCLCs to ICI and led to the establishment of tumor-specific immune memory. Immunophenotyping revealed that CCL21-DC obliterated tumor-promoting neutrophils, promoted sustained infiltration of CD8 cytolytic and CD4 Th1 lymphocytes and enriched progenitor T cells in the TME. Addition of ICI to CCL21-DC further enhanced the expansion and effector function of T cells both locally and systemically. Longitudinal evaluation of tumor mutation profiles revealed that CCL21-DC plus ICI induced immunoediting of tumor subclones, consistent with the broadening of tumor-specific T cell responses.ConclusionsCCL21-DC ISV synergizes with anti-PD-1 to eradicate murine NSCLC. Our data support the clinical application of CCL21-DC ISV in combination with checkpoint inhibition for patients with NSCLC.

Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

PurposeCell-free DNA (cfDNA) offers a noninvasive approach to monitor cancer. Here we develop a method using whole-exome sequencing (WES) of cfDNA for simultaneously monitoring the full spectrum of cancer treatment outcomes, including minimal residual disease (MRD), recurrence, evolution, and second primary cancers.Experimental designThree simulation datasets were generated from 26 patients with cancer to benchmark the detection performance of MRD/recurrence and second primary cancers. For further validation, cfDNA samples (n = 76) from patients with cancer (n = 35) with six different cancer types were used for performance validation during various treatments.ResultsWe present a cfDNA-based cancer monitoring method, named cfTrack. Taking advantage of the broad genome coverage of WES data, cfTrack can sensitively detect MRD and cancer recurrence by integrating signals across known clonal tumor mutations of a patient. In addition, cfTrack detects tumor evolution and second primary cancers by de novo identifying emerging tumor mutations. A series of machine learning and statistical denoising techniques are applied to enhance the detection power. On the simulation data, cfTrack achieved an average AUC of 99% on the validation dataset and 100% on the independent dataset in detecting recurrence in samples with tumor fractions ≥0.05%. In addition, cfTrack yielded an average AUC of 88% in detecting second primary cancers in samples with tumor fractions ≥0.2%. On real data, cfTrack accurately monitors tumor evolution during treatment, which cannot be accomplished by previous methods.ConclusionsOur results demonstrated that cfTrack can sensitively and specifically monitor the full spectrum of cancer treatment outcomes using exome-wide mutation analysis of cfDNA.

Early cancer detection by cell-free DNA faces multiple challenges: low fraction of tumor cell-free DNA, molecular heterogeneity of cancer, and sample sizes that are not sufficient to reflect diverse patient populations. Here, we develop a cancer detection approach to address these challenges. It consists of an assay, cfMethyl-Seq, for cost-effective sequencing of the cell-free DNA methylome (with > 12-fold enrichment over whole genome bisulfite sequencing in CpG islands), and a computational method to extract methylation information and diagnose patients. Applying our approach to 408 colon, liver, lung, and stomach cancer patients and controls, at 97.9% specificity we achieve 80.7% and 74.5% sensitivity in detecting all-stage and early-stage cancer, and 89.1% and 85.0% accuracy for locating tissue-of-origin of all-stage and early-stage cancer, respectively. Our approach cost-effectively retains methylome profiles of cancer abnormalities, allowing us to learn new features and expand to other cancer types as training cohorts grow.

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand-receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. SIGNIFICANCE: The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception.This article is highlighted in the In This Issue feature, p. 2355.

Cancer interception refers to actively blocking the cancer development process by preventing progression of premalignancy to invasive disease. The rate-limiting steps for effective lung cancer interception are the incomplete understanding of the earliest molecular events associated with lung carcinogenesis, the lack of preclinical models of pulmonary premalignancy, and the challenge of developing highly sensitive and specific methods for early detection. Recent advances in cancer interception are facilitated by developments in next-generation sequencing, computational methodologies, as well as the renewed emphasis in precision medicine and immuno-oncology. This review summarizes the current state of knowledge in the areas of molecular abnormalities in lung cancer continuum, preclinical human models of lung cancer pathogenesis, and the advances in early lung cancer diagnostics.

Conditional genetically engineered mouse models (GEMMs) of non-small cell lung cancer (NSCLC) harbor common oncogenic driver mutations of the disease, but in contrast to human NSCLC these models possess low tumor mutational burden (TMB). As a result, these models often lack tumor antigens that can elicit host adaptive immune responses, which limits their utility in immunotherapy studies. Here, we establish Kras-mutant murine models of NSCLC bearing the common driver mutations associated with the disease and increased TMB, by in vitro exposure of cell lines derived from GEMMs of NSCLC [KrasG12D (K), KrasG12DTp53-/-(KP), KrasG12DTp53+/-Lkb1-/- (KPL)] to the alkylating agent N-methyl-N-nitrosourea (MNU). Increasing the TMB enhanced host anti-tumor T cell responses and improved anti-PD-1 efficacy in syngeneic models across all genetic backgrounds. However, limited anti-PD-1 efficacy was observed in the KPL cell lines with increased TMB, which possessed a distinct immunosuppressed tumor microenvironment (TME) primarily composed of granulocytic myeloid-derived suppressor cells (G-MDSCs). This KPL phenotype is consistent with findings in human KRAS-mutant NSCLC where LKB1 loss is a driver of primary resistance to PD-1 blockade. In summary, these novel Kras-mutant NSCLC murine models with known driver mutations and increased TMB have distinct TMEs and recapitulate the therapeutic vulnerabilities of human NSCLC. We anticipate that these immunogenic models will facilitate the development of innovative immunotherapies in NSCLC.

LKB1 inactivating mutations are commonly observed in patients with KRAS-mutant non-small cell lung cancer (NSCLC). Although treatment of NSCLC with immune checkpoint inhibitors (ICI) has resulted in improved overall survival in a subset of patients, studies have revealed that co-occurring KRAS/LKB1 mutations drive primary resistance to ICIs in NSCLC. Effective therapeutic options that overcome ICI resistance in LKB1-mutant NSCLC are limited. Here, we report that loss of LKB1 results in increased secretion of the C-X-C motif (CXC) chemokines with an NH2-terminal Glu-Leu-Arg (ELR) motif in premalignant and cancerous cells, as well as in genetically engineered murine models (GEMM) of NSCLC. Heightened levels of ELR+ CXC chemokines in LKB1-deficient murine models of NSCLC positively correlated with increased abundance of granulocytic myeloid-derived suppressor cells (G-MDSC) locally within the tumor microenvironment and systemically in peripheral blood and spleen. Depletion of G-MDSCs with antibody or functional inhibition via all-trans-retinoic acid (ATRA) led to enhanced antitumor T-cell responses and sensitized LKB1-deficent murine tumors to PD-1 blockade. Combination therapy with anti-PD-1 and ATRA improved local and systemic T-cell proliferation and generated tumor-specific immunity. Our findings implicate ELR+ CXC chemokine-mediated enrichment of G-MDSCs as a potential mediator of immunosuppression in LKB1-deficient NSCLC and provide a rationale for using ATRA in combination with anti-PD-1 therapy in patients with LKB1-deficient NSCLC refractory to ICIs. SIGNIFICANCE: These findings show that accumulation of myeloid-derived suppressor cells in LKB1-deficient non-small cell lung cancer can be overcome via treatment with all-trans-retinoic acid, sensitizing tumors to immunotherapy.

IntroductionEstrogen receptors (ER) (ERα, ERβ) and aromatase (key enzyme for estrogen synthesis) are expressed in most human NSCLCs. High intratumoral estrogen levels and elevated aromatase expression in NSCLC predict poor outcome. This open-label, phase 1b, single-center study evaluated the safety and tolerability of escalating doses of the aromatase inhibitor, exemestane, in combination with carboplatin and pemetrexed in postmenopausal women with stage IV nonsquamous NSCLC.MethodsPatients received exemestane (starting 1-wk before chemotherapy) at 25 mg orally (PO) daily (cohort 1) or 50 mg PO daily (cohort 2) combined with carboplatin (area under the curve 6 mg × min/mL) and pemetrexed (500 mg/m2) intravenously every 3 weeks for four cycles. Thereafter, patients were eligible for continued therapy with exemestane and pemetrexed or pemetrexed alone.ResultsA total of 10 patients consented for therapy, and two patients failed in the screening. Four patients completed the therapy in cohort 1 and four patients in cohort 2. The median number of cycles administered was 15 (range: 1-54). Maximum tolerated dose was exemestane 50 mg PO daily with combination chemotherapy. Intention-to-treat analysis revealed an objective response rate (ORR) of 62.5% (five of eight patients with partial response) and a clinical benefit rate of 87.5% (seven of eight patients with either stable disease or partial response). ORR was associated with aromatase expression (p = 0.02). Circulating estrogen levels decreased with exemestane use, and quality of life measurements did not significantly change during the treatment. There were no adverse events.ConclusionsThe combination of carboplatin, pemetrexed, and exemestane in postmenopausal women with metastatic NSCLC is safe and well tolerated. Biomarker studies revealed that ORR correlates with tumor aromatase expression. These findings support future clinical trials to confirm the antitumor efficacy with this combination therapy.

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAF non-V600, and BRAF V600 mutations. Tumor cell-intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8+ T cells, and durable tumor regression elicited by this combination requires CD8+ T cells, which can be reinvigorated by anti-PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy. SIGNIFICANCE: Type I RAFi + MEKi are indicated only in certain BRAF V600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8+ T cells are mechanisms that may be further exploited.This article is highlighted in the In This Issue feature, p. 521.

An in-depth understanding of lung cancer biology and mechanisms of tumor progression has facilitated significant advances in the treatment of lung cancer. There remains a pressing need for the development of innovative approaches to detect and intercept lung cancer at its earliest stage of development. Recent advances in genomics, computational biology, and innovative technologies offer unique opportunities to identify the immune landscape in the tumor microenvironment associated with early-stage lung carcinogenesis, and provide further insight in the mechanism of lung cancer evolution. This review will highlight the concept of immunoediting and focus on recent studies assessing immune changes and biomarkers in pulmonary premalignancy and early-stage non-small cell lung cancer. A protumor immune response hallmarked by an increase in checkpoint inhibition and inhibitory immune cells and a simultaneous reduction in antitumor immune response have been correlated with tumor progression. The potential systemic biomarkers associated with early lung cancer will be highlighted along with current clinical efforts for lung cancer interception. Research focusing on the development of novel strategies for cancer interception prior to the progression to advanced stages will potentially lead to a paradigm shift in the treatment of lung cancer and have a major impact on clinical outcomes.See all articles in this CEBP Focus section, "NCI Early Detection Research Network: Making Cancer Detection Possible."

In this issue of Cancer Discovery, Pennycuick and colleagues comprehensively evaluate the immune contexture of progressive and regressive lesions in squamous pulmonary premalignancy. The authors dissect the molecular features of these lesions and the potential pathways of immune escape operative in progression to invasive cancer.See related article by Pennycuick et al., p. 1489.

Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.

Oncogenic KRAS mutations are frequently found in non-small cell lung carcinoma (NSCLC) and cause constitutive activation of the MEK-ERK pathway. Many cancer types have been shown to overexpress PD-L1 to escape immune surveillance. FRA1 is a MEK/ERK-dependent oncogenic transcription factor and a member of the AP-1 transcriptional factor superfamily. This study assesses the hypothesis that KRAS mutation directly regulates PD-L1 expression through the MEK-ERK pathway mediated by FRA1. Premalignant human bronchial epithelial cell (HBEC) lines harboring the KRAS mutationV12, EGFR mutation, p53 knock-down, or both KRAS mutation and p53 knock-down were tested for levels of PD-L1, FRA1, and ERK activation (pERK). Our results showed that KRAS mutation alone, but not other genetic alterations, induced significantly higher expression of PD-L1 compared to its vector counterparts. The increased PD-L1 expression in the KRAS mutated cells was dramatically reduced by inhibition of ERK activation. Furthermore, the MEK-ERK pathway-dependent PD-L1 expression was markedly reduced by FRA1 silencing. Interestingly, FRA1 silencing led to inhibition of ERK activation, indicating that FRA1 plays a role in PD-L1 regulation via positive feedback of ERK activation. Correlation of PD-L1 and FRA1 mRNA expression was validated using human lung cancer specimens from The Cancer Genome Atlas (TCGA) and established NSCLC cell lines from Cancer Cell Line Encyclopedia (CCLE). FRA1 expression was significantly associated with PD-L1 expression, and high FRA1 expression was correlated with poor overall survival. Our findings suggest that oncogenic KRAS-driven PD-L1 expression is dependent on MEK-ERK and FRA1 in high risk, premalignant HBEC.

Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer.

Tumor cells are hypothesized to use proteolytic enzymes to facilitate invasion. Whether circulating tumor cells (CTCs) secrete these enzymes to aid metastasis is unknown. A quantitative and high-throughput approach to assay CTC secretion is needed to address this question. We developed an integrated microfluidic system that concentrates rare cancer cells >100,000-fold from 1 mL of whole blood into ∼50,000 2-nL drops composed of assay reagents within 15 min. The system isolates CTCs by size, exchanges fluid around CTCs to remove contaminants, introduces a matrix metalloprotease (MMP) substrate, and encapsulates CTCs into microdroplets. We found CTCs from prostate cancer patients possessed above baseline levels of MMP activity (1.7- to 200-fold). Activity of CTCs was generally higher than leukocytes from the same patient (average CTC/leukocyte MMP activity ratio, 2.6 ± 1.5). Higher MMP activity of CTCs suggests active proteolytic processes that may facilitate invasion or immune evasion and be relevant phenotypic biomarkers enabling companion diagnostics for anti-MMP therapies.

ObjectivesThis open-label, randomized phase II trial evaluated antitumor efficacy of an antiestrogen, fulvestrant, in combination with human epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in advanced non-small cell lung cancer (NSCLC) patients.Materials and methodsPatients with advanced or metastatic NSCLC, ECOG 0-2, previous chemotherapy unless patient refusal, and no prior EGFR-directed therapy were randomized 2:1 to erlotinib 150 mg oral daily plus 500 mg intramuscular fulvestrant on day 1, 15, 29 and every 28 days thereafter or erlotinib alone 150 mg oral daily. The primary end point was objective response rate (ORR); secondary endpoints included progression free survival (PFS) and overall survival (OS).ResultsAmong 106 randomized patients, 100 received at least one dose of study drug. ORR was 16.4% (11 of 67 patients) for the combination versus 12.1% (4 of 33 patients) for erlotinib (p = 0.77). PFS median 3.5 versus 1.9 months [HR = 0.86, 95% CI (0.52-1.43), p = 0.29] and OS median 9.5 versus 5.8 months [HR = 0.92, 95% CI (0.57-1.48), p = 0.74] numerically favored the combination. In an unplanned subset analysis, among EGFR wild type patients (n = 51), but not EGFR mutant patients (n = 17), median PFS was 3.5 versus 1.7 months [HR = 0.35, 95% CI (0.14-0.86), p = 0.02] and OS was 6.2 versus 5.2 months [HR = 0.72, 95% CI (0.35-1.48), p = 0.37] for combined therapy versus erlotinib, respectively. Notably, EGFR WT patients were more likely to be hormone receptor-positive (either estrogen receptor α- and/or progesterone receptor-positive) compared to EGFR mutant patients (50% versus 9.1%, respectively) (p = 0.03). Treatment was well tolerated with predominant grade 1-2 dermatologic and gastrointestinal adverse effects.ConclusionAddition of fulvestrant to erlotinib was well tolerated, with increased activity noted among EGFR wild type patients compared to erlotinib alone, albeit in an unplanned subset analysis.

Altered metabolism is a hallmark of cancer growth, forming the conceptual basis for development of metabolic therapies as cancer treatments. We performed in vivo metabolic profiling and molecular analysis of lung squamous cell carcinoma (SCC) to identify metabolic nodes for therapeutic targeting. Lung SCCs adapt to chronic mTOR inhibition and suppression of glycolysis through the GSK3α/β signaling pathway, which upregulates glutaminolysis. Phospho-GSK3α/β protein levels are predictive of response to single-therapy mTOR inhibition while combinatorial treatment with the glutaminase inhibitor CB-839 effectively overcomes therapy resistance. In addition, we identified a conserved metabolic signature in a broad spectrum of hypermetabolic human tumors that may be predictive of patient outcome and response to combined metabolic therapies targeting mTOR and glutaminase.

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.

Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines. This enhanced migratory capacity was found to be Rac1-dependent and heritable, as evidenced by maintenance of the phenotype through multiple cell divisions continuing more than 8 weeks after selection. The morphology of this lung epithelial subpopulation was characterized by increased cell protrusion intensity. In a murine model of micrometastatic seeding and pulmonary colonization, the motility-selected premalignant cells exhibit both enhanced survival in short-term assays and enhanced outgrowth of premalignant lesions in longer-term assays, thus overcoming important aspects of "metastatic inefficiency." Overall, our findings indicate that among immortalized premalignant airway epithelial cell lines, subpopulations with heritable motility-related biophysical properties exist, and these may explain micrometastatic seeding occurring early in the pathogenesis of lung cancer. Understanding, targeting, and preventing these critical biophysical traits and their underlying molecular mechanisms may provide a new approach to prevent metastatic behavior. Cancer Prev Res; 10(9); 514-24. ©2017 AACRSee related editorial by Hynds and Janes, p. 491.

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 × 106, 5 × 106, 1 × 107, or 3 × 107 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 mm2). 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.

Inactivation of the LKB1 tumor suppressor is a frequent event in non-small cell lung carcinoma (NSCLC) leading to the activation of mTOR complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing comutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas and squamous cell carcinomas (SCC). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1-mutant human cell lines and genetically engineered mouse models of NSCLC that develop both adenocarcinomas and SCCs. Specifically, we found that KRAS/LKB1-mutant lung adenocarcinomas responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors, thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1-mutant adenocarcinomas and squamous cell tumors.

BackgroundCyclooxygenase 2 (COX-2)-dependent signaling represents a potential mechanism of resistance to therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. This is mediated in part through an EGFR-independent activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) by prostaglandin E2 (PGE2). PGE2 promotes downregulation of E cadherin and epithelial to mesenchymal transition. The current study investigated EGFR and COX-2 inhibition in patients with non-small cell lung cancer (NSCLC) and elevated baseline urinary metabolite of PGE2 (PGEM).MethodsPatients with stage IIIB/IV (AJCC 6th edition) NSCLC who progressed after at least 1 line of therapy or refused standard chemotherapy were randomized to receive erlotinib and celecoxib versus erlotinib and placebo. The primary endpoint was progression-free survival (PFS) with 80% power to detect a 50% improvement with a 1-sided significance level of .2 in the intent-to-treat and elevated baseline PGEM populations. Secondary endpoints included response rate, overall survival, and evaluation of molecular markers to assess targeting COX-2-related pathways and evaluate EGFR tyrosine kinase inhibitor resistance.ResultsA total of 107 patients were enrolled with comparable baseline characteristics. Among the patients treated with celecoxib, those with wild-type EGFR were found to have an increased PFS (3.2 months vs 1.8 months; P = .03). PFS was numerically improved among patients in the intent-to-treat group who received erlotinib and celecoxib compared with those treated with erlotinib and placebo (5.4 months vs 3.5 months; P = .33) and was increased in patients in the erlotinib and celecoxib arm with elevated baseline PGEM (5.4 months vs 2.2 months; P = .15). Adverse events were similar in both treatment arms.ConclusionsThe combination of erlotinib and celecoxib did not appear to improve outcomes in an unselected population, but selection by elevated baseline PGEM led to an increase in PFS with this combination. Patients with EGFR wild-type status may benefit from the combination of erlotinib and celecoxib.

Metastatic disease is responsible for 90% of death from solid tumors. However, only a minority of metastasis-specific targets has been exploited therapeutically, and effective prevention and suppression of metastatic disease is still an elusive goal. In this review, we will first summarize the current state of knowledge about the molecular features of the disease, with particular focus on steps and targets potentially amenable to therapeutic intervention. We will then discuss the reasons underlying the paucity of metastatic drugs in the current oncological arsenal and potential ways to overcome this therapeutic gap. We reason that the discovery of novel promising targets, an increased understanding of the molecular features of the disease, the effect of disruptive technologies, and a shift in the current preclinical and clinical settings have the potential to create more successful drug development endeavors.

BackgroundThe 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.ObjectiveThe 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 resultsWestern 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.ConclusionCombined 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.

ObjectiveThis 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 designIn vivo study.SettingAcademic research laboratory.Subjects and methodsMice 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).ResultsThe 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).ConclusionHerein, 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.

Understanding the molecular pathogenesis of lung cancer is necessary to identify biomarkers/targets specific to individual airway molecular profiles and to identify options for targeted chemoprevention. Herein, we identify mechanisms by which loss of microRNA (miRNA)125a-3p (miR125a) contributes to the malignant potential of human bronchial epithelial cells (HBEC) harboring an activating point mutation of the K-ras proto-oncogene (HBEC K-ras). Among other miRNAs, we identified significant miR125a loss in HBEC K-ras lines and determined that miR125a is regulated by the PEA3 transcription factor. PEA3 is upregulated in HBEC K-ras cells, and genetic knockdown of PEA3 restores miR125a expression. From a panel of inflammatory/angiogenic factors, we identified increased CXCL1 and vascular endothelial growth factor (VEGF) production by HBEC K-ras cells and determined that miR125a overexpression significantly reduces K-ras-mediated production of these tumorigenic factors. miR125a overexpression also abrogates increased proliferation of HBEC K-ras cells and suppresses anchorage-independent growth (AIG) of HBEC K-ras/P53 cells, the latter of which is CXCL1-dependent. Finally, pioglitazone increases levels of miR125a in HBEC K-ras cells via PEA3 downregulation. In addition, pioglitazone and miR125a overexpression elicit similar phenotypic responses, including suppression of both proliferation and VEGF production. Our findings implicate miR125a loss in lung carcinogenesis and lay the groundwork for future studies to determine whether miR125a is a possible biomarker for lung carcinogenesis and/or a chemoprevention target. Moreover, our studies illustrate that pharmacologic augmentation of miR125a in K-ras-mutated pulmonary epithelium effectively abrogates several deleterious downstream events associated with the mutation.

Serum mass profiling can discern physiological changes associated with specific disease states and their progression. Sera (86 total) from control individuals and patients with stage I nonsmall cell lung cancer or benign small pulmonary nodules were discriminated retrospectively by serum changes discerned by mass profiling. Control individuals were distinguished from patients with Stage I lung cancer or benign nodules with test sensitivities of 89% and 83%. Lung cancer patients versus those with benign nodules were distinguished with 80% sensitivity. This study exhibits progress toward a minimally-invasive aid in early detection of lung cancer and monitoring small pulmonary nodules for malignancy.

Lung squamous cell carcinoma (SCC) is thought to arise from premalignant lesions in the airway epithelium; therefore, studying these lesions is critical for understanding lung carcinogenesis. Previous microarray and sequencing studies designed to discover early biomarkers and therapeutic targets for lung SCC had limited success identifying key driver events in lung carcinogenesis, mostly due to the cellular heterogeneity of patient samples examined and the interindividual variability associated with difficult to obtain airway premalignant lesions and appropriate normal control samples within the same patient. We performed RNA sequencing on laser-microdissected representative cell populations along the SCC pathologic continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. This is the first gene expression profiling study of airway premalignant lesions with patient-matched SCC tumor samples. Our results provide much needed information about the biology of premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of SCC, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients.

UnlabelledAberrant 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.ImplicationsThis study describes a novel mechanism, involving c-Myc and miR-17-92, which integrates cell proliferation and apoptosis resistance.

ObjectivesDichloroacetate (DCA) is a highly bioavailable small molecule that inhibits pyruvate dehydrogenase kinase, promoting glucose oxidation and reversing the glycolytic phenotype in preclinical cancer studies. We designed this open-label phase II trial to determine the response rate, safety, and tolerability of oral DCA in patients with metastatic breast cancer and advanced stage non-small cell lung cancer (NSCLC).Materials and methodsThis trial was conducted with DCA 6.25 mg/kg orally twice daily in previously treated stage IIIB/IV NSCLC or stage IV breast cancer. Growth inhibition by DCA was also evaluated in a panel of 54 NSCLC cell lines with and without cytotoxic chemotherapeutics (cisplatin and docetaxel) in normoxic and hypoxic conditions.Results and conclusionsUnder normoxic conditions in vitro, single-agent IC50 was >2 mM for all evaluated cell lines. Synergy with cisplatin was seen in some cell lines under hypoxic conditions. In the clinical trial, after seven patients were enrolled, the study was closed based on safety concerns. The only breast cancer patient had stable disease after 8 weeks, quickly followed by progression in the brain. Two patients withdrew consent within a week of enrollment. Two patients had disease progression prior to the first scheduled scans. Within 1 week of initiating DCA, one patient died suddenly of unknown cause and one experienced a fatal pulmonary embolism. We conclude that patients with previously treated advanced NSCLC did not benefit from oral DCA. In the absence of a larger controlled trial, firm conclusions regarding the association between these adverse events and DCA are unclear. Further development of DCA should be in patients with longer life expectancy, in whom sustained therapeutic levels can be achieved, and potentially in combination with cisplatin.

Definition of the molecular pathogenesis of lung cancer allows investigators an enhanced understanding of the natural history of the disease, thus fostering development of new prevention strategies. In addition to regulating epithelial-to-mesenchymal transition (EMT), the transcription factor Snail exerts global effects on gene expression. Our recent studies reveal that Snail is upregulated in non-small cell lung cancer (NSCLC), is associated with poor prognosis, and promotes tumor progression in vivo. Herein, we demonstrate that overexpression of Snail leads to the upregulation of secreted protein, acidic and rich in cysteine (SPARC) in models of premalignancy and established disease, as well as in lung carcinoma tissues in situ. Snail overexpression leads to increased SPARC-dependent invasion in vitro, indicating that SPARC may play a role in lung cancer progression. Bioinformatic analysis implicates transforming growth factor beta (TGF-β), extracellular signal-regulated kinase (ERK)1/2, and miR-29b as potential intermediaries in Snail-mediated upregulation of SPARC. Both the TGF-β1 ligand and TGF-β receptor 2 (TGF-βR2) are upregulated following Snail overexpression. Treatment of human bronchial epithelial cell (HBEC) lines with TGF-β1 and inhibition of TGF-β1 mRNA expression modulates SPARC expression. Inhibition of MAP-ERK kinase (MEK) phosphorylation downregulates SPARC. MiR-29b is downregulated in Snail-overexpressing cell lines, whereas overexpression of miR-29b inhibits SPARC expression. In addition, miR-29b is upregulated following ERK inhibition, suggesting a Snail-dependent pathway by which Snail activation of TGF-β and ERK signaling results in downregulation of miR-29b and subsequent upregulation of SPARC. Our discovery of pathways responsible for Snail-induced SPARC expression contributes to the definition of NSCLC pathogenesis.

Health disparities are an immense challenge to American society. Clinical and Translational Science Awards (CTSAs) housed within the National Center for Advancing Translational Science (NCATS) are designed to accelerate the translation of experimental findings into clinically meaningful practices and bring new therapies to the doorsteps of all patients. Research Centers at Minority Institutions (RCMI) program at the National Institute on Minority Health and Health Disparities (NIMHD) are designed to build capacity for biomedical research and training at minority serving institutions. The CTSA created a mechanism fostering formal collaborations between research intensive universities and minority serving institutions (MSI) supported by the RCMI program. These consortium-level collaborations activate unique translational research approaches to reduce health disparities with credence to each academic institutions history and unique characteristics. Five formal partnerships between research intensive universities and MSI have formed as a result of the CTSA and RCMI programs. These partnerships present a multifocal approach; shifting cultural change and consciousness toward addressing health disparities, and training the next generation of minority scientists. This collaborative model is based on the respective strengths and contributions of the partnering institutions, allowing bidirectional interchange and leveraging NIH and institutional investments providing measurable benchmarks toward the elimination of health disparities.

Abstract: LKB1/STK11 is a tumor suppressor gene that was originally identified in an autosomal-dominant inherited disorder, Peutz-Jeghers Syndrome (PJS). Patients with PJS develop an overgrowth of differentiated tissues in the gastrointestinal tract with 100% penetration and demonstrate an increased predisposition toward a number of additional malignancies, including breast, ovarian, pancreatic and lung cancers. Loss-of-function in LKB1/STK11 characterizes up to 20-30% of non-small cell lung cancers (NSCLC). In a murine model of lung carcinogenesis with inducible Kras activation and biallelic deletion of LKB1(KrasG12D Lkb1-/-), loss of LKB1 yielded a high frequency of NSCLC tumors and metastasis. However, few mechanisms have been proposed to explain how LKB1 loss contributes to pulmonary tumorigenesis, especially during early stage of tumor development. In this study, we use human bronchial epithelial cells (HBECs) that were immortalized in the absence of viral onco-proteins and therefore represent an accurate model for studying early events during lung carcinogenesis. We knocked down LKB1 expression in HBECs (LKB1-KD HBECs) and observed numerous cancer-associated characteristics, including preference for glycolytic metabolism and induction of epithelial mesenchymal transition. Intriguingly, we also found dysregulation of several inflammatory factors in addition to these anticipated phenotypes. We have focused on the pro-angiogenic chemokine CXCL8, which is also associated with chemotaxis, tumorigenicity and metastasis via binding to its membrane receptors CXCR1/2. Knockdown of LKB1 leads to transcriptional upregulation and significantly elevated secretion of CXCL8, while re-introduction of wild type LKB1 decreases CXCL8 production in LKB1-null NSCLC tumor cell lines. Interestingly, the phosphorylation levels of GSK3β and β-catenin (S552) are elevated in LKB1-KD HBECs, and a TCF/β-catenin binding inhibitor abolishes the induction of CXCL8 upon LKB1 loss. This suggests that the WNT pathway may mediate these effects. Together, these findings suggest that loss of LKB1 promotes tumorigenesis and metastasis through a CXCL8 mediated-pro-inflammatory tumor microenvironment. Therefore, blocking CXCL8 function via neutralizing peptide or receptor antagonist may represent a novel therapeutic strategy for NSCLC prevention and treatment.These studies were supported by the DOD #LC120407 (SMD, DS, TCW), China Scholarship Council (CSC) #2010632164 (RL) and NCI SPORE P50CA70907 (JDM,JEL). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C296. Citation Format: Rui Li, Tonya C. Walser, Kostyantyn Krysan, David Shackelford, Jill E. Larsen, John D. Minna, Steven M. Dubinett. The contribution of LKB1/STK11-mediated CXCL8 dysregulation to lung carcinogenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C296.

BackgroundThe CXC chemokine, CXCL12, and its receptor, CXCR4 promote metastases of a variety of solid tumors, including non-small cell lung cancer (NSCLC). The expression of CXCR4 on tumor cells may represent a critical biomarker for their propensity to metastasize. This study was performed to evaluate the hypothesis that co-expression of pan-cytokeratin and CXCR4 may be a prognostic marker for patients with advanced NSCLC.MethodsWe evaluated CXCR4 levels on circulating pan-cytokeratin positive cells from patients with NSCLC. NSCLC tumor and metastases were also assessed for the presence of CXCR4.ResultsPan-cytokeratin positive cells were increased in the circulation of patients with NSCLC, as compared to normal control subjects. Patients with pan-cytokeratin +/CXCR4+ = 2,500 cells/ml had a significant improvement in median survival when compared with patients with pan-cytokeratin +/CXCR4+ >2,500 cells/ml (not achieved versus 14 weeks). CXCR4 expression was found on NSCLC tumors and at sites of tumor metastasis.ConclusionThis study suggests that CXCR4 may be a prognostic marker in NSCLC, and provides hypothesis-generating results, which may be important in determining metastatic potential. In future studies, we will prospectively evaluate the prognostic significance of pan-cytokeratin/CXCR4+ cells, and determine the mechanisms involved in the regulation of CXCR4 expression on tumor cells in a larger patient population.

BackgroundOur previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability. In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity. These results have provided the rationale to generate a clinical grade adenoviral vector encoding CCL-21 for ex vivo transduction of human DC in order to assess intratumoral administration in late stage human lung cancer.MethodsIn the current study, human monocyte-derived DC were differentiated by exposure to GM-CSF and IL-4 from cryopreserved mononuclear cells obtained from healthy volunteers. Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein.ResultsCCL21 protein production from transduced DC was detected in supernatants (24-72 hours, 3.5-6.7 ng/4-5 x 10(6) cells). DC transduced with the clinical grade adenoviral vector were > 88% viable (n = 16), conserved their phenotype and maintained integral biological activities including dextran uptake, production of immunostimulatory cytokines/chemokines and antigen presentation. Furthermore, supernatant from CCL21-DC induced the chemotaxis of T2 cells in vitro.ConclusionViable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.

This article reviews progress in chemopreventive drug development, especially data and concepts that are new since the 2002 AACR report on treatment and prevention of intraepithelial neoplasia. Molecular biomarker expressions involved in mechanisms of carcinogenesis and genetic progression models of intraepithelial neoplasia are discussed and analyzed for how they can inform mechanism-based, molecularly targeted drug development as well as risk stratification, cohort selection, and end-point selection for clinical trials. We outline the concept of augmenting the risk, mechanistic, and disease data from histopathologic intraepithelial neoplasia assessments with molecular biomarker data. Updates of work in 10 clinical target organ sites include new data on molecular progression, significant completed trials, new agents of interest, and promising directions for future clinical studies. This overview concludes with strategies for accelerating chemopreventive drug development, such as integrating the best science into chemopreventive strategies and regulatory policy, providing incentives for industry to accelerate preventive drugs, fostering multisector cooperation in sharing clinical samples and data, and creating public-private partnerships to foster new regulatory policies and public education.

BACKGROUND:SLC/CCL21, normally expressed in high endothelial venules and in T cell zones of spleen and lymph nodes, strongly attracts T cells and dendritic cells (DC). We have previously shown that SLC/CCL21-mediated anti-tumor responses are accompanied by significant induction of IFNgamma and the CXC chemokines, monokine induced by IFNgamma (MIG/CXCL9) and IFNgamma-inducible protein-10 (IP-10/CXCL10). RESULTS:We assessed the importance of IFNgamma, IP-10/CXCL10 and MIG/CXCL9 in SLC/CCL21 therapy. In vivo depletion of IP-10/CXCL10, MIG/CXCL9 or IFNgamma significantly reduced the anti-tumor efficacy of SLC/CCL21. Assessment of cytokine production at the tumor site showed an interdependence of IFNgamma, MIG/CXCL9 and IP-10/CXCL10; neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines. Similarly, neutralization of any one of these cytokines led to a decrease in the frequency of CXCR3+ve T cells and CD11c+ve DC at the tumor site. CONCLUSION:These findings indicate that the full potency of SLC/CCL21-mediated anti-tumor responses require in part the induction of IFNgamma, MIG/CXCL9 and IP-10/CXCL10.