Primary Human Lung Adenocarcinoma Research Articles

Abstract 7528: Coordinated regulation of critical immune checkpoint proteins by the integrated stress response (ISR) pathway in lung cancer

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Despite recent advances, high rates of recurrence highlight the need for novel treatment options. Groundbreaking discoveries in identifying and exploiting the PD-1/PD-L1 (Programmed Death 1/Programmed Death Ligand 1) immune checkpoint have resulted in the approval of monoclonal antibodies that disrupt this interaction as a first-line therapy for lung cancer patients. However, only ~20% of NSCLC patients benefit from checkpoint blockade. A critical question remains as to what mechanisms facilitate resistance to PD-1/PD-L1 therapy and whether other immune checkpoints or pathways can be pursued clinically in combination with this therapy. The integrated stress response (ISR) pathway represents an emerging therapeutic vulnerability. The ISR is a generalized response to maintain cellular homeostasis, and is known to be activated by hypoxia, heme deprivation, amino acid starvation, and oncogenic activation. We previously demonstrated that ISR activation potently induces PD-L1 in NSCLC and suppression of anti-tumor immunity (Suresh et al, Nature Cancer, 2020). We discovered that ISR pathway activation enhances PD-L1 translation through the bypass of inhibitory upstream open reading frames (uORFs) in the PD-L1 5' UTR and a mechanistic link to the translation initiation factor eIF5B. Our latest studies demonstrate that the immune checkpoint protein, CD155 (Cluster of Differentiation 155), is induced by ISR activation. We find that both PD-L1 and CD155 are induced by multiple arms of the ISR pathway, and CD155 harbors inhibitory uORFs in its 5' UTR. Importantly, we observed a significant correlation between PD-L1 and CD155 expression in a panel of primary human lung adenocarcinomas. We further demonstrated that ISR activation inhibits T cell function in vitro and promotes tumor growth in the CMT167 syngeneic mouse model. Analysis of immune cell infiltration using mass cytometry in this in vivo model is ongoing. Currently, we are determining the extent to which ISR inhibition suppresses tumorigenesis by promoting anti-tumor immunity and whether this may synergize with existing immune checkpoint therapies. Overall, these studies will set the stage for determining whether inhibition of the ISR pathway alone or in combination with immune checkpoint blockade will benefit lung cancer patients. Our studies may also lead to new therapeutic approaches to trigger anti-cancer immune responses and improve current strategies. Citation Format: Shayna Elizabeth Thomas-Jardin, Shruthy Suresh, Cheryl Lewis, Chul Ahn, Bret M. Evers, John D. Minna, Kathryn A. O'Donnell. Coordinated regulation of critical immune checkpoint proteins by the integrated stress response (ISR) pathway in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7528.

COL11A1 regulates PI3K/Akt/GSK-3β pathway and promotes human lung adenocarcinoma primary cell migration and invasion

Objective: To investigate the role and mechanism of COL11A1 in lung adenocarcinoma migration and invasion. Methods: Surgical pathological tissues of 4 patients with lung adenocarcinoma admitted to the Affiliated Hospital of Guizhou Medical University from September to November 2020 were used. Immunohistochemical methods were used to identify lung adenocarcinoma tissues, para-cancerous tissues and parallel transcriptome sequencing. Genetic prognostic analysis was conducted by TCGA and GTEx databases.The expression level of COL11A1 gene in lung adenocarcinoma and adjacent tissues was detected by Western blotting.The primary human lung adenocarcinoma cells cultured. The COL11A1 siRNA was transfected into primary human lung adenocarcinoma cells, then the transcriptome sequencing of differential genes was performed,and KEGG enrichment analysis of differential gene enrichment pathway was conducted. Protein expression and phosphorylation were detected by Western blot method. Cell migration was detected by scratch healing test. Cell proliferation was detected by CCK8 method and invasion ability was detected by Transwell method. Results: Ten differentially expressed genes were screened by transcription sequencing in lung adenocarcinoma. Prognostic analysis of single gene showed that COL11A1 gene expression level was correlated with survival rate (P<0.001). The expression of COL11A1 in lung adenocarcinoma was higher than that in adjacent tissues by Western blot (P<0.001). Transcriptome sequencing of COL11A1 siRNA transfection into primary human lung adenocarcinoma cells showed that differential genes were concentrated in PI3K-akt pathway. The expression of tumor suppressor gene PTEN in siRNA transfection group was significantly higher than that in control group and negative transfection group by Western blot. The expression of Aktp-Akt 473 p-Akt 308 p-PTENp-PDK1p-c-Rafp-GSK-3 β was down-regulated (all P<0.05).Compared with the negative control group, the ability of migration, proliferation and invasion of primary human lung adenocarcinoma cells in siRNA transfection group decreased (all P<0.05). COL11A1 regulates PI3K/Akt/GSK-3 β pathway to promote migration and invasion of primary human lung adenocarcinoma cells. Conclusion: COL11A1 regulates PI3K/Akt/GSK-3 β pathway to promote migration and invasion of primary human lung adenocarcinoma cells.

FARP1, ARHGEF39, and TIAM2 are essential receptor tyrosine kinase effectors for Rac1-dependent cell motility in human lung adenocarcinoma.

Despite the undisputable role of the small GTPase Rac1 in the regulation of actin cytoskeleton reorganization, the Rac guanine-nucleotide exchange factors (Rac-GEFs) involved in Rac1-mediated motility and invasion in human lung adenocarcinoma cells remain largely unknown. Here, we identify FARP1, ARHGEF39, and TIAM2 as essential Rac-GEFs responsible for Rac1-mediated lung cancer cell migration upon EGFR and c-Met activation. Noteworthily, these Rac-GEFs operate in a non-redundant manner by controlling distinctive aspects of ruffle dynamics formation. Mechanistic analysis reveals a leading role of the AXL-Gab1-PI3K axis in conferring pro-motility traits downstream of EGFR. Along with the positive association between the overexpression of Rac-GEFs and poor lung adenocarcinoma patient survival, we show that FARP1 and ARHGEF39 are upregulated in EpCam+ cells sorted from primary human lung adenocarcinomas. Overall, our study reveals fundamental insights into the complex intricacies underlying Rac-GEF-mediated cancer cell motility signaling, hence underscoring promising targets for metastatic lung cancer therapy.

Outgrowth of erlotinib-resistant subpopulations recapitulated in patient-derived lung tumor spheroids and organoids.

A model that recapitulates development of acquired therapeutic resistance is needed to improve oncology drug development and patient outcomes. To achieve this end, we established methods for the preparation and growth of spheroids from primary human lung adenocarcinomas, including methods to culture, passage, monitor growth, and evaluate changes in mutational profile over time. Primary lung tumor spheroids were cultured in Matrigel® with varying concentrations of erlotinib, a small molecule kinase inhibitor of epidermal growth factor receptor (EGFR) that is ineffective against KRAS mutant cells. Subtle changes in spheroid size and number were observed within the first two weeks of culture. Spheroids were cultured for up to 24 weeks, during which time interactions between different cell types, movement, and assembly into heterogeneous organoid structures were documented. Allele-specific competitive blocker PCR (ACB-PCR) was used to quantify low frequency BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA H1047R mutant subpopulations in tumor tissue residue (TR) samples and cultured spheroids. Mutant subpopulations, including multiple mutant subpopulations, were quite prevalent. Twelve examples of mutant enrichment were found in eight of the 14 tumors analyzed, based on the criteria that a statistically-significant increase in mutant fraction was observed relative to both the TR and the no-erlotinib control. Of the mutants quantified in erlotinib-treated cultures, PIK3CA H1047 mutant subpopulations increased most often (5/14 tumors), which is consistent with clinical observations. Thus, this ex vivo lung tumor spheroid model replicates the cellular and mutational tumor heterogeneity of human lung adenocarcinomas and can be used to assess the outgrowth of mutant subpopulations. Spheroid cultures with characterized mutant subpopulations could be used to investigate the efficacy of lung cancer combination therapies.

Reduced Protumorigenic Tumor-Associated Macrophages With Statin Use in Premalignant Human Lung Adenocarcinoma.

BackgroundStatins have anticancer properties by acting as competitive inhibitors of the mevalonate pathway. They also have anti-inflammatory activity, but their role in suppressing inflammation in a cancer context has not been investigated to date.MethodsWe have analyzed the relationship between statin use and tumor-associated macrophages (TAMs) in a cohort of 262 surgically resected primary human lung adenocarcinomas. TAMs were evaluated by multiplex immunostaining for the CD68 pan-TAM marker and the CD163 protumorigenic TAM marker followed by digital slide scanning and partially automated quantitation. Links between statin use and tumor stage, virulence, and cancer-specific survival were also investigated in a wider cohort of 958 lung adenocarcinoma cases. All statistical tests were two-sided.ResultsWe found a statin dose-dependent reduction in protumorigenic TAMs (CD68+CD163+) in both stromal (P = .021) and parenchymal (P = .003) compartments within regions of in situ tumor growth, but this association was lost in invasive regions. No statistically significant relationship between statin use and tumor stage was observed, but there was a statin dose-dependent shift towards lower histological grade as assessed by growth pattern (P = .028). However, statin use was a predictor of slightly worse cancer-specific survival (P = .032), even after accounting for prognostic variables in a multivariable Cox proportional hazards survival model (hazard ratio = 1.38, 95% confidence interval = 1.04 to 1.84).ConclusionsStatin use is associated with reduced numbers of protumorigenic TAMs within preinvasive lung adenocarcinoma and is related to reduced tumor invasiveness, suggesting a chemo-preventive effect in early tumor development. However, invasive disease is resistant to these effects, and no beneficial relationship between statin use and patient outcome is observed.

Comprehensive nucleosome mapping of the human genome in cancer progression.

Altered chromatin structure is a hallmark of cancer, and inappropriate regulation of chromatin structure may represent the origin of transformation. Important studies have mapped human nucleosome distributions genome wide, but the role of chromatin structure in cancer progression has not been addressed. We developed a MNase-Transcription Start Site Sequence Capture method (mTSS-seq) to map the nucleosome distribution at human transcription start sites genome-wide in primary human lung and colon adenocarcinoma tissue. Here, we confirm that nucleosome redistribution is an early, widespread event in lung (LAC) and colon (CRC) adenocarcinoma. These altered nucleosome architectures are consistent between LAC and CRC patient samples indicating that they may serve as important early adenocarcinoma markers. We demonstrate that the nucleosome alterations are driven by the underlying DNA sequence and potentiate transcription factor binding. We conclude that DNA-directed nucleosome redistributions are widespread early in cancer progression. We have proposed an entirely new hierarchical model for chromatin-mediated genome regulation.

Abstract 1949: P38MAPK plays a tumor suppressive role through differentiation of ALDHhigh/CD44high tumor iniating cells in primary human lung adenocarcinoma

Abstract The p38 MAPK cascade regulates body responses to a wide range of stress stimuli in a context-dependent and tissue-specific manner. The isoform p38alpha (p38) mediates both tumor suppressive and tumor-supportive functions in different cancer models. However, its role in lung cancer is not clear. Since EGFR is frequently mutated in lung adenocarcinomas in non-smoking women and p38 is a downstream target of EGFR signaling, it is possible that p38 plays a tumor-supportive role. Using immunohistochemistry to analyze nuclear activated p38 expression in 90 lung adenocarcinomas, we observed p38 expression was significantly associated with well differentiated compared to poorly-differentiated carcinomas (p&amp;lt;0.001) and better patient outcome (p=0.019, disease-free survival; p=0.03, overall survival). In vitro p38 suppression in lung cancer cell lines did not lead to enhanced cell death. On the other hand, p38 suppression alone or in combination with EGFR inhibition led to increased CD44high/ALDHhigh lung tumor initiating cells (TIC); isolated lung TIC also showed higher p38 expression. Together, the data implicate p38 plays a tumor suppressive role in lung cancer by driving tumor cell differentiation. Disrupting pathways that inhibit p38 expression or enhancement of p38 expression might be useful for lung cancer treatment. Citation Format: Jing Liu, Lai-han Leung, Zhi-jie Xiao, Vicky PC Tin, Maria Pik Wong. P38MAPK plays a tumor suppressive role through differentiation of ALDHhigh/CD44high tumor iniating cells in primary human lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1949. doi:10.1158/1538-7445.AM2014-1949

Abstract 266: Notch3 is a marker of tumor-propagating cells in non-small cell lung cancer and is required for their self-renewal.

Abstract We investigated tumor cell heterogeneity in mouse models of non-small cell lung cancer (NSCLC) that recapitulate key genetic events in this disease. In tumors driven by simultaneous expression of an oncogenic Kras allele and loss of the tumor suppressor Trp53, only a relatively rare subset of tumor cells is capable of propagating tumor growth in both a clonogenic in vitro assay and an in vivo orthotopic transplantation assay. The tumor-propagating cell (TPC) population was enriched by sorting for the combination of the cell surface markers CD24, ITGB4 and Notch(1-4). We demonstrate a critical and specific role for Notch3 signaling in the maintenance of TPCs in the KrasG12D; Trp53fl/fl mouse model. Knock-down of Notch3, but not Notch 1, 2 or 4, decreased self-renewal of TPCs in vitro. In addition, knock-down of Notch3 was critical for tumor propagation in vivo. The relevance of these studies to human NSCLC was confirmed by demonstrating a significant effect of Notch inhibition on the self-renewal of primary human lung adenocarcinoma cells in ex vivo cultures established directly from patients. These findings identify a novel subpopulation of tumor cells with distinct functional capabilities, underscoring the importance of interrogating heterogeneity in NSCLC. The unique role of Notch3 in tumor propagation may provide a novel therapeutic target for the treatment of non-small cell lung cancer. Citation Format: Yanyan Zheng, Cecile de la Cruz, Leanne Sayles, Chris Alleyne-Chin, Yue Xu, Chuong D. Hoang, Joseph B. Shrager, Erica Jackson, Alejandro Sweet-Cordero. Notch3 is a marker of tumor-propagating cells in non-small cell lung cancer and is required for their self-renewal. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 266. doi:10.1158/1538-7445.AM2013-266

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

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

Interplay between Menin and K-Ras in Regulating Lung Adenocarcinoma

MEN1, which encodes the nuclear protein menin, acts as a tumor suppressor in lung cancer and is often inactivated in human primary lung adenocarcinoma. Here, we show that the inactivation of MEN1 is associated with increased DNA methylation at the MEN1 promoter by K-Ras. On one hand, the activated K-Ras up-regulates the expression of DNA methyltransferases and enhances the binding of DNA methyltransferase 1 to the MEN1 promoter, leading to increased DNA methylation at the MEN1 gene in lung cancer cells; on the other hand, menin reduces the level of active Ras-GTP at least partly by preventing GRB2 and SOS1 from binding to Ras, without affecting the expression of GRB2 and SOS1. In human lung adenocarcinoma samples, we further demonstrate that reduced menin expression is associated with the enhanced expression of Ras (p < 0.05). Finally, excision of the Men1 gene markedly accelerates the K-Ras(G12D)-induced tumor formation in the Men1(f/f);K-Ras(G12D/+);Cre ER mouse model. Together, these findings uncover a previously unknown link between activated K-Ras and menin, an important interplay governing tumor activation and suppression in the development of lung cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Vascular endothelial growth factor and soluble intercellular adhesion molecule-1 in lung adenocarcinoma with malignant pleural effusion: Correlations with patient survival and pleural effusion control

The mechanisms by which vascular endothelial growth factor (VEGF) and soluble intercellular adhesion molecule-1 (sICAM-1) contribute to lung cancer growth have not been fully elucidated. This study aimed to assess the role of VEGF and sICAM-1 in control of pleural effusions (PE) and survival in patients with primary human lung adenocarcinoma. Using enzyme-linked immunoadsorbent assay, the concentrations of VEGF and sICAM-1 were measured in pleural effusions and serum from a total of 79 lung adenocarcinoma patients with malignant pleural effusions (MPE) and 24 patients with tuberculosis. Data were correlated with the efficacy of MPE control and survival. Compared to patients with tuberculosis, the levels of VEGF and sICAM-1 in both PE and serum were significantly higher in patients with lung adenocarcinoma. Statistically significant correlation was observed between PE VEGF levels and MPE control. PE VEGF≥2760 pg/ml was used as a cut-off point for failure to MPE control (odds ratio=7.06; 95% confidence interval (CI), 2.40-20.78; P<0.001). The median progression-free survival (PFS) from response assessment was 3 months. In a multivariate analysis, PE VEGF (hazard ratio [HR], 1.16; 95% CI, 1.02-1.32), serum sICAM-1 (HR, 1.90; 95% CI, 1.17-3.07) were confirmed as independent prognostic factors for PFS. The levels of VEGF in PE can be used to predict the therapeutic efficacy in the control of MPE and this, together with serum level of sICAM-1 is potential survival factors in lung adenocarcinoma patients with MPE.

Abstract 2899: Restoring sensitivity to anti-EGFR-based therapy in lung adenocarcinoma by downstream modulation of the FOXO1/KLF6 transcriptional network

Abstract Lung cancer is currently the leading cause of cancer-related death. This high mortality rate has prompted numerous exhaustive efforts to identify novel therapeutic targets and treatment modalities for this deadly disease. The characterization of specific signaling cascades involved in the development and progression of lung cancer has been instrumental in developing novel therapeutic strategies. This paradigm is best exemplified by recent studies demonstrating that the epidermal growth factor receptor (EGFR) is a critical diagnostic and therapeutic target in metastatic lung adenocarcinoma. The clinical utility of anti-EGFR-based strategies is, however, ultimately limited by primary or acquired drug resistance, that can develop through several distinct molecular mechanisms. Thus, a more complete molecular characterization of downstream mediators of both cancer progression and treatment resistance will allow for the development of rationally designed combination based therapies to overcome the specific molecular lesions driving the reistant phenotype. Though much is known about the specific molecular lesions conferring resistance to anti-EGFR-based therapies, additional molecular characterization of the downstream mediators of EGFR signaling may lead to the development of new classes of targeted molecular therapies to treat resistant disease. Here we identify a transcriptional network involving the KLF6 and FOXO1 tumor suppressor genes that regulates response to anti-EGFR-based therapies in both cell culture and in vivo models of the disease. Specifically, inhibition of AKT signaling promotes FOXO1 stabilization resulting in transactivation of the KLF6 tumor suppressor gene and induction of apoptosis in lung adenocarcinoma cell lines. Furthermore, the use of the FDA-approved drug Trifluoperazine Hydrochloride (TFP), which has been shown to inhibit FOXO1 nuclear export, restores sensitivity to erlotinib-resistant cell lines through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft lung adenocarcinoma models. Further validation of the requirement for KLF6 activation in erlotinib response was demonstrated by loss-of-function experiments, in which targeted reduction of KLF6 using sequence specific siRNAs abrogated the utility of erlotinib on lung tumors in vivo. In addition, a significant correlation between activated oncogenic EGFR signaling and downregulation of the FOXO1 and KLF6 tumor suppressor gene network in both primary human lung adenocarcinoma patient samples and a transgenic mouse model of the disease was observed. Combined, these studies define a novel transcriptional network regulating oncogenic EGFR signaling and identify a class of FDA-approved drugs to restore chemosensitivity to anti-EGFR-based therapy for the treatment of metastatic lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2899. doi:10.1158/1538-7445.AM2011-2899

Abstract B41: Restoring sensitivity to anti-EGFR-based therapy in lung adenocarcinoma by downstream modulation of the FOXO1/KLF6 transcriptional network

Abstract Anti-EGFR-based strategies have shown clinical efficacy in the treatment of metastatic lung adenocarcinoma. Nevertheless, treatment resistance invariably develops through either de novo activation of downstream RAS/AKT signaling or acquired mutations in the Epidermal growth factor receptor (EGFR). A more thorough molecular characterization of the downstream mediators of EGFR signaling may lead to the development of newer classes of targeted molecular therapies to treat resistant disease. Here we identify a transcriptional network involving the KLF6 and FOXO1 tumor suppressor genes that regulate response to anti-EGFR-based therapies in both cell culture and in vivo models of the disease. Specifically, inhibition of AKT signaling promotes FOXO1 stabilization resulting in transactivation of the KLF6 tumor suppressor gene and induction of apoptosis in lung adenocarcinoma cell lines. Furthermore, the use of FDA-approved drug trifluoperazine hydrochloride, a FOXO1 nuclear export inhibitor restores sensitivity to erlotinib-resistant cell lines through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft lung adenocarcinoma models. In addition, quantitative real-time PCR-based analysis and Western blotting confirm a significant correlation between activated oncognic EGFR signaling and downregulation of the FOXO1 and KLF6 tumor suppressor gene network in both primary human lung adenocarcinoma patient samples and a transgenic mouse model of the disease. Combined, these studies define a novel transcriptional network regulating oncogenic EGFR signaling and identify targeted molecular agents to restore chemosensitivity to anti-EGFR-based therapy in culture and in vivo. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B41.

RUNX3 Modulates DNA Damage-mediated Phosphorylation of Tumor Suppressor p53 at Ser-15 and Acts as a Co-activator for p53

Although it has been shown that the gastric tumor suppressor RUNX3 has a growth inhibitory activity, the precise molecular mechanisms behind RUNX3-mediated tumor suppression remained unclear. In this study, we found that RUNX3 is closely involved in DNA damage-dependent phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The small interference RNA-mediated knockdown of RUNX3 inhibited adriamycin (ADR)-dependent apoptosis in p53-proficient cells but not in p53-deficient cells in association with a significant reduction of p53-target gene expression as well as phosphorylation of p53 at Ser-15. In response to ADR, RUNX3 was induced to accumulate in the cell nucleus and co-localized with p53. Immunoprecipitation experiments demonstrated that RUNX3 forms a complex with p53 in cells. In vitro pulldown assays revealed that the COOH-terminal portion of p53 is required for the interaction with RUNX3. Forced expression of RUNX3 enhanced p53-mediated transcriptional activation. Additionally, RUNX3 had an ability to induce the phosphorylation of p53 at Ser-15, thereby promoting p53-dependent apoptosis. Intriguingly, RUNX3 interacted with phosphorylated forms of ataxia telangiectasia-mutated in response to ADR; however, it did not affect the extent of DNA damage. From the clinical point of view, coordinated p53 mutation and decreased expression of RUNX3 in 105 human lung adenocarcinomas were significantly associated with the poor outcome of patients (p = 0.0203). Thus, our present results strongly suggest that RUNX3 acts as a novel co-activator for p53 through regulating its DNA damage-induced phosphorylation at Ser-15 and also provide a clue to understanding the molecular mechanisms underlying RUNX3-mediated tumor suppression.

Abstract LB-250: Smoking reprograms human airway basal cells in vivo toward an embryonic stem cell-like molecular phenotype similar to that of lung adenocarcinoma

Abstract Activation of the human embryonic stem cell (hESC)-like transcriptional program has been observed in various epithelial cancers, including lung adenocarcinomas, in association with tumor aggressiveness. We hypothesized that initial changes related to the acquisition of this “cancerous” hESC-like phenotype occur in the stem/progenitor cell compartment of healthy epithelia under the influence of a chronic carcinogenic stress as a step toward malignant tissue derangement. Given that cigarette smoking is capable of inducing molecular changes in the airway epithelium leading to lung cancer, we analyzed expression of hESC-specific genes in the freshly isolated large airway epithelium of healthy nonsmokers (LAE-NS; n=21) and healthy smokers (LAE-S; n=31) and in the pure basal cell (BC) populations obtained from the LAE-NS (BC-NS; n=4) and LAE-S (BC-S; n=4). Expression patterns were then compared to those in primary human lung adenocarcinoma samples (n=193) and pure human lung adenocarcinoma cells propagated as xenografts in the NOD/SCID/IL2Rg-null immunodeficient mice (n=4) using a list of 40 hESC-specific genes based on published meta-analysis of the hESC transcriptome. The microarray analysis revealed low-level constitutive expression of a subset of hESC-specific genes in the LAE-NS, with higher expression in the BC population. Smoking dramatically and selectively up-regulated hESC-specific gene expression in BC, an observation confirmed by massive parallel sequencing (RNA-Seq). Remarkably, 35% of known hESC-specific genes were found up-regulated in BC-S by both microarrays and RNA-Seq. The majority of these genes (71%) were not detected in nonsmokers’ BC indicative of their de novo induction by smoking. Strikingly, 82% of hESC-specific genes induced in BC-S were also significantly up-regulated in both primary human lung adenocarcinomas and xenograft-derived human lung adenocarcinoma cells. Clustering analysis identified a subgroup of lung adenocarcinoma samples (n=32; 6%) among 193 primary lung adenocarcinoma samples based on the high expression of hESC-specific signature found up-regulated in BC of healthy smokers. This subset of adenocarcinoma subjects exhibited distinct clinical/pathological characteristics, such as longer smoking history (p&amp;lt;0.0002), lower lung function parameters such as FEV1 and DLCO (both p&amp;lt;0.001), larger tumor size (p&amp;lt;0.0002), more advanced tumor stage, poorer differentiation grade, and higher frequency of TP53 gene mutations (50% vs 23% in other adenocarcinoma subjects). Thus, prior to any clinical manifestations of lung cancer, cigarette smoking reprograms airway basal stem/progenitor cell population in vivo toward a lung cancer-associated hESC-like molecular state. The subset of lung adenocarcinomas highly expressing smoking-induced basal cell hESC-like gene signature have a distinct, aggressive clinical phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-250.

Suppression of lung adenocarcinoma through menin and polycomb gene-mediated repression of growth factor pleiotrophin

Menin upregulates transcription of cell-cycle inhibitors to suppress endocrine tumors, but it is poorly understood how menin suppresses non-endocrine tumors such as lung cancer. Here, we show that menin inhibits proliferation of human lung cancer cells and growth of lung cancer in mice. The menin-mediated tumor suppression requires repression of growth factor pleiotrophin (PTN), which binds to its cell surface receptor, anaplastic lymphoma kinase (ALK) that is activated in certain lung adenocarcinomas. Menin represses PTN transcription and PTN-induced proliferation of human lung cancer cells, and menin expression is substantially reduced in primary human lung adenocarcinomas. Notably, menin binds the PTN locus and enhances Polycomb gene Enhancer of Zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation (H3K27m3), a negative mark for gene transcription but does not affect histone H3K4 methylation that is usually upregulated by menin in endocrine cells. Together, our findings indicate that menin suppresses lung cancer partly through increasing Polycomb gene-mediated H3K27 methylation and repressing PTN transcription, unraveling a novel, epigenetically regulated PTN-ALK signaling pathway in suppressing lung cancer.

Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas

Persistently activated or tyrosine-phosphorylated STAT3 (pSTAT3) is found in 50% of lung adenocarcinomas. pSTAT3 is found in primary adenocarcinomas and cell lines harboring somatic-activating mutations in the tyrosine kinase domain of EGFR. Treatment of cell lines with either an EGFR inhibitor or an src kinase inhibitor had no effect on pSTAT3 levels, whereas a pan-JAK inhibitor (P6) blocked activation of STAT3 and inhibited tumorigenesis. Cell lines expressing these persistently activated mutant EGFRs also produced high IL-6 levels, and blockade of the IL-6/gp130/JAK pathway led to a decrease in pSTAT3 levels. In addition, reduction of IL-6 levels by RNA interference led to a decrease in tumorigenesis. Introduction of persistently activated EGFR into immortalized breast epithelial cells led to tumorigenesis, IL-6 expression, and STAT3 activation, all of which could be inhibited with P6 or gp130 blockade. Furthermore, inhibition of EGFR activity in multiple cell lines partially blocked transcription of IL-6 and concurrently decreased production and release of IL-6. Finally, immunohistochemical analysis revealed a positive correlation between pSTAT3 and IL-6 positivity in primary lung adenocarcinomas. Therefore, mutant EGFR could activate the gp130/JAK/STAT3 pathway by means of IL-6 upregulation in primary human lung adenocarcinomas, making this pathway a potential target for cancer treatment.

Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma

BackgroundLung cancer is the number one cancer killer of both men and women in the United States. Three quarters of lung cancer patients are diagnosed with regionally or distantly disseminated disease; their 5-year survival is only 15%. DNA hypermethylation at promoter CpG islands shows great promise as a cancer-specific marker that would complement visual lung cancer screening tools such as spiral CT, improving early detection. In lung cancer patients, such hypermethylation is detectable in a variety of samples ranging from tumor material to blood and sputum. To date the penetrance of DNA methylation at any single locus has been too low to provide great clinical sensitivity. We used the real-time PCR-based method MethyLight to examine DNA methylation quantitatively at twenty-eight loci in 51 primary human lung adenocarcinomas, 38 adjacent non-tumor lung samples, and 11 lung samples from non-lung cancer patients.ResultsWe identified thirteen loci showing significant differential DNA methylation levels between tumor and non-tumor lung; eight of these show highly significant hypermethylation in adenocarcinoma: CDH13, CDKN2A EX2, CDX2, HOXA1, OPCML, RASSF1, SFPR1, and TWIST1 (p-value << 0.0001). Using the current tissue collection and 5-fold cross validation, the four most significant loci (CDKN2A EX2, CDX2, HOXA1 and OPCML) individually distinguish lung adenocarcinoma from non-cancer lung with a sensitivity of 67–86% and specificity of 74–82%. DNA methylation of these loci did not differ significantly based on gender, race, age or tumor stage, indicating their wide applicability as potential lung adenocarcinoma markers. We applied random forests to determine a good classifier based on a subset of our loci and determined that combined use of the same four top markers allows identification of lung cancer tissue from non-lung cancer tissue with 94% sensitivity and 90% specificity.ConclusionThe identification of eight CpG island loci showing highly significant hypermethylation in lung adenocarcinoma provides strong candidates for evaluation in patient remote media such as plasma and sputum. The four most highly ranked loci, CDKN2A EX2, CDX2, HOXA1 and OPCML, which show significant DNA methylation even in stage IA tumor samples, merit further investigation as some of the most promising lung adenocarcinoma markers identified to date.

LKB1 modulates lung cancer differentiation and metastasis

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz-Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.