The PI3K/AKT pathway promotes gefitinib resistance in mutant KRAS lung adenocarcinoma by a deacetylase‐dependent mechanism

To select the appropriate patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs), it is important to gain a better understanding of the intracellular pathways leading to EGFR‐TKI resistance, which is a common problem in patients with lung cancer. We recently reported that mutant KRAS adenocarcinoma is resistant to gefitinib as a result of amphiregulin and insulin‐like growth factor‐1 receptor overexpression. This resistance leads to inhibition of Ku70 acetylation, thus enhancing the BAX/Ku70 interaction and preventing apoptosis. Here, we determined the intracellular pathways involved in gefitinib resistance in lung cancers and explored the impact of their inhibition. We analyzed the activation of the phosphatidyl inositol‐3‐kinase (PI3K)/AKT pathway and the mitogen‐activated protein kinase/extracellular‐signal regulated kinase (MAPK/ERK) pathway in lung tumors. The activation of AKT was associated with disease progression in tumors with wild‐type EGFR from patients treated with gefitinib (phase II clinical trial IFCT0401). The administration of IGF1R‐TKI or amphiregulin‐directed shRNA decreased AKT signaling and restored gefitinib sensitivity in mutant KRAS cells. The combination of PI3K/AKT inhibition with gefitinib restored apoptosis via Ku70 downregulation and BAX release from Ku70. Deacetylase inhibitors, which decreased the BAX/Ku70 interaction, inhibited AKT signaling and induced gefitinib‐dependent apoptosis. The PI3K/AKT pathway is thus a major pathway contributing to gefitinib resistance in lung tumors with KRAS mutation, through the regulation of the BAX/Ku70 interaction. This finding suggests that combined treatments could improve the outcomes for this subset of lung cancer patients, who have a poor prognosis.     

Carcinogen‐induced tumors in SFN‐transgenic mice harbor a characteristic mutation spectrum of human lung adenocarcinoma

The landscape of genetic alterations in disease models such as transgenic mice or mice with carcinogen‐induced tumors has provided a huge amount of information that has shed light on the process of tumorigenesis in human non‐small‐cell lung cancer (NSCLC). We have previously identified stratifin (SFN) as a potent oncogene, and generated SFN‐transgenic (Tg‐SPC‐SFN^+/?) mice, which express human SFN (hSFN) only in the lung. Here, we have found that carcinogen nicotine‐derived nitrosaminoketone (NNK)‐induced tumors developing in Tg‐SPC‐SFN^+/? mice show a similar histology to human lung adenocarcinoma and exhibit high hSFN expression. In order to compare the genetic characteristics of Tg‐SPC‐SFN^+/? tumors and human lung adenocarcinoma, the former were subjected to whole‐exome sequencing. Interestingly, Tg‐SPC‐SFN^+/? tumors showed the distinct distribution of exonic mutations and high number of mutated genes and transversion. Moreover, Tg‐SPC‐SFN^+/? tumors showed 73 genes that were commonly detected in more than 2 tumors, mutations of which were also found in human lung adenocarcinoma. The expression levels of some of these genes were significantly associated with the clinical outcome of lung adenocarcinoma patients. Additionally, mutated genes in Tg‐SPC‐SFN^+/? tumors were closely associated with key canonical pathways such as PI3K/AKT signaling and apoptosis signaling. These results suggest that SFN overexpression is a universal abnormality in human lung adenocarcinogenesis and Tg‐SPC‐SFN^+/? tumors recapitulate key features of major human lung adenocarcinoma. Therefore, Tg‐SPC‐SFN^+/? mice provide a useful model for clarifying the molecular mechanism underlying lung adenocarcinogenesis.     

Antitumor activity of pimasertib,a selective MEK 1/2 inhibitor,in combination with PI3K/mTOR inhibitors or with multi‐targeted kinase inhibitors in pimasertib‐resistant human lung and colorectal cancer cells

The RAS/RAF/MEK/MAPK and the PTEN/PI3K/AKT/mTOR pathways are key regulators of proliferation and survival in human cancer cells. Selective inhibitors of different transducer molecules in these pathways have been developed as molecular targeted anti‐cancer therapies. The in vitro and in vivo anti‐tumor activity of pimasertib, a selective MEK 1/2 inhibitor, alone or in combination with a PI3K inhibitor (PI3Ki), a mTOR inhibitor (everolimus), or with multi‐targeted kinase inhibitors (sorafenib and regorafenib), that block also BRAF and CRAF, were tested in a panel of eight human lung and colon cancer cell lines. Following pimasertib treatment, cancer cell lines were classified as pimasertib‐sensitive (IC₅₀ for cell growth inhibition of 0.001 µM) or pimasertib‐resistant. Evaluation of basal gene expression profiles by microarrays identified several genes that were up‐regulated in pimasertib‐resistant cancer cells and that were involved in both RAS/RAF/MEK/MAPK and PTEN/PI3K/AKT/mTOR pathways. Therefore, a series of combination experiments with pimasertib and either PI3Ki, everolimus, sorafenib or regorafenib were conducted, demonstrating a synergistic effect in cell growth inhibition and induction of apoptosis with sustained blockade in MAPK‐ and AKT‐dependent signaling pathways in pimasertib‐resistant human colon carcinoma (HCT15) and lung adenocarcinoma (H1975) cells. Finally, in nude mice bearing established HCT15 and H1975 subcutaneous tumor xenografts, the combined treatment with pimasertib and BEZ235 (a dual PI3K/mTOR inhibitor) or with sorafenib caused significant tumor growth delays and increase in mice survival as compared to single agent treatment. These results suggest that dual blockade of MAPK and PI3K pathways could overcome intrinsic resistance to MEK inhibition.     

Higher susceptibility of spontaneous and NNK‐induced lung neoplasms in connexin 43 deficient CD1 × AJ F1 mice: Paradoxical expression of connexin 43 during lung carcinogenesis

Connexins (Cxs) are proteins that form the communicating gap junctions, and reportedly have a role in carcinogenesis. Here, we evaluated the importance of Connexin43 (Cx43) in spontaneous and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK)‐induced lung carcinogenesis. Male wild‐type (Cx43^+/+) and hemizygote (Cx43^+/?) CD1 × AJ F1 mice were injected with NNK or saline. After 60 weeks mice were euthanized; lung nodules were counted, measured, and fixed in formalin or snap frozen. Immunohistochemistry for Cx43 and Beta‐catenin (β‐catenin) was performed and Cx43 mRNA expression was evaluated by real‐time PCR. Cx43 deletion significantly increased the incidence and number of spontaneous nodules in the CD1 × AJ F1 mice and the number of gross lesions and the aggressiveness of lesions in NNK‐treated mice. Cx43 mRNA increased significantly and was correlated with the aggressiveness of tumors, although lesions from Cx43^+/? mice expressed less Cx43 RNAm than their counterparts. Lung parenchyma presented a Cx43 immunostaining pattern with points or plaques between cells. In hyperplasias and adenomas, Cx43 was found in the membrane and in cytoplasm. Malignant lesions presented increased Cx43 in cytoplasm and a few membrane spots of immunostaining. β‐catenin was weakly expressed in lung parenchyma. Though hyperplasias presented some cells with nuclear β‐catenin, NNK‐induced tumors contained a higher number of this staining pattern. Also, no difference in β‐catenin occurred between both genotypes independently of the histological grade. In summary, our results indicate that Cx43 acts as a tumor suppressor gene in early lung tumorigenesis and loses this property in advanced carcinogenesis. Therefore, Cxs are better classified as conditional tumor suppressors. © 2012 Wiley Periodicals, Inc.     

Protease‐activated receptor‐2 accelerates intestinal tumor formation through activation of nuclear factor‐κB signaling and tumor angiogenesis in ApcMin/+ mice

Hepatocyte growth factor activator inhibitor‐1 (HAI‐1), encoded by the SPINT1 gene, is a membrane‐bound protease inhibitor expressed on the surface of epithelial cells. Hepatocyte growth factor activator inhibitor‐1 regulates type II transmembrane serine proteases that activate protease‐activated receptor‐2 (PAR‐2). We previously reported that deletion of Spint1 in Apc^Min/+ mice resulted in accelerated formation of intestinal tumors, possibly through enhanced nuclear factor‐κB signaling. In this study, we examined the role of PAR‐2 in accelerating tumor formation in the Apc^Min/+ model in the presence or absence of Spint1. We observed that knockout of the F2rl1 gene, encoding PAR‐2, not only eliminated the enhanced formation of intestinal tumors caused by Spint1 deletion, but also reduced tumor formation in the presence of Spint1. Exacerbation of anemia and weight loss associated with HAI‐1 deficiency was also normalized by compound deficiency of PAR‐2. Mechanistically, signaling triggered by deregulated protease activities increased nuclear translocation of RelA/p65, vascular endothelial growth factor expression, and vascular density in Apc^Min/+‐induced intestinal tumors. These results suggest that serine proteases promote intestinal carcinogenesis through activation of PAR‐2, and that HAI‐1 plays a critical tumor suppressor role as an inhibitor of matriptase, kallikreins, and other PAR‐2 activating proteases.     

Secondary bile acid‐induced dysbiosis promotes intestinal carcinogenesis

The gut microbiota plays an important role in maintaining intestinal homeostasis. Dysbiosis is associated with intestinal tumorigenesis. Deoxycholic acid (DCA), a secondary bile acid increased by a western diet, correlates with intestinal carcinogenesis. However, evidence relating bile acids, intestinal microbiota and tumorigenesis are limited. In our study, we investigated the effect of DCA on induction of intestinal dysbiosis and its roles in intestinal carcinogenesis. Alteration of the composition of the intestinal microbiota was induced in DCA‐treated APC^min/+ mice, which was accompanied by impaired intestinal barrier, gut low grade inflammation and tumor progression. The transfer of fecal microbiota from DCA‐treated mice to another group of Apc^min/+ mice increased tumor multiplicity, induced inflammation and recruited M2 phenotype tumor‐associated macrophages. Importantly, the fecal microbiota transplantation activated the tumor‐associated Wnt/β‐catenin signaling pathway. Moreover, microbiota depletion by a cocktail of antibiotics was sufficient to block DCA‐induced intestinal carcinogenesis, further suggesting the role of dysbiosis in tumor development. Our study demonstrated that alteration of the microbial community induced by DCA promoted intestinal carcinogenesis.     

A newly synthesized compound, 4′‐geranyloxyferulic acid–N(omega)‐nitro‐l‐arginine methyl ester suppresses inflammation‐associated colorectal carcinogenesis in male mice

We previously reported the cancer chemopreventive activity of 4′‐geranyloxyferulic acid (GOFA, Miyamoto et al., Nutr Cancer 2008; 60:675‐84) and a β‐cyclodextrin inclusion compound of GOFA (Tanaka et al., Int J Cancer 2010; 126:830‐40) in colitis‐related colorectal carcinogenesis. In our study, the chemopreventive effects of a newly synthesized GOFA‐containing compound, GOFA–N(omega)‐nitro‐l ‐arginine methyl ester (L‐NAME), which inhibits inducible nitric oxide (iNOS) and cyclooxygenase‐2 (COX) enzymes, were investigated using a colitis‐associated mouse colorectal carcinogenesis model with azoxymethane (AOM) and dextran sodium sulfate (DSS). The dietary administration of GOFA–L‐NAME after the AOM and DSS treatments significantly reduced the multiplicity of adenocarcinomas (inhibition rates: 100 ppm, 84%, p < 0.001; 500 ppm, 94%, p < 0.001) compared with the AOM + DSS group. Dietary GOFA–L‐NAME significantly decreased the proliferation (p < 0.001) and increased the apoptosis (p < 0.001) of colonic adenocarcinoma cells. A subsequent short‐term experiment revealed that dietary GOFA–L‐NAME decreased the mRNA expression of inflammatory enzymes, such as iNOS and COX‐2, and proinflammatory cytokines, such as tumor necrosis factor‐α, interleukin (IL)?1β, IL‐6 and macrophage inflammatory protein (MIP)?2 in the colonic mucosa of mice that received 1.5% DSS in their drinking water for 7 days. Our findings indicate that GOFA–L‐NAME is able to inhibit colitis‐associated colon carcinogenesis by modulating inflammation, proliferation, apoptosis and the expression of proinflammatory cytokines in mice.     

Up‐regulation of interleukin‐17 expression by human papillomavirus type 16 E6 in nonsmall cell lung cancer

BACKGROUND:

Human papillomavirus (HPV) 16/18 infection is associated with nonsmoking lung cancer. In this study, the authors investigated a putative correlation between interleukin (IL)‐17 expression and HPV infection in clinical nonsmall cell lung cancer (NSCLC) tissues and examined the effects of HPV infection on a human NSCLC cell line.

METHODS:

IL‐17 expression was investigated in 79 NSCLC tumor tissues by immunohistochemistry. Growth rate, IL‐17 mRNA, and secreting protein levels were also examined in HPV 16/18 E6‐transfected H1299 human NSCLC cells.

RESULTS:

Immunohistochemical data showed that 48.1% of lung tumors had IL‐17 staining, which was significantly associated with patients' sex (P = .03), HPV infection (P = .002), and tumor stage (P = .03). Significant correlations of IL‐17 with IL‐6 (P < .001) and IL‐17 with Mcl‐1 (P < .001) expression were also observed. Cell growth rate was increased, and IL‐17/Mcl‐1 expression levels were elevated in HPV 16 E6‐transfected H1299 cells. The transfected E6 oncoproteins can significantly up‐regulate expression levels of IL‐17 and antiapoptotic protein Mcl‐1.

CONCLUSIONS:

The study suggests that HPV infection‐induced IL‐17 levels can stimulate Mcl‐1 expression through the PI3K pathway and promote lung tumor cell progression through a p53‐ and IL‐6‐independent pathway. Cancer 2010. © 2010 American Cancer Society.     

Endogenous conversion of ω‐6 to ω‐3 polyunsaturated fatty acids in fat‐1 mice attenuated intestinal polyposis by either inhibiting COX‐2/β‐catenin signaling or activating 15‐PGDH/IL‐18

Omega‐3 polyunsaturated fatty acids (ω‐3PUFAs) have inhibitory effects in various preclinical cancer models, but their effects in intestinal polyposis have never been examined. As attempts have been made to use nutritional intervention to counteract colon cancer development, in this study we evaluated the effects of ω‐3 PUFAs on intestinal polyposis in the Apc^Min/+ mouse model. The experimental groups included wild‐type C56BL/6 mice, Apc^Min/+ mice, fat‐1 transgenic mice expressing an n‐3 desaturase to enable ω‐3 PUFA synthesis, and Apc^Min/+ × fat‐1 double‐transgenic mice; all mice were 20 weeks of age. Small intestines were collected for gross and pathologic evaluation, including assessment of polyp number and size, followed by immunohistochemical staining and Western blotting. After administration of various concentrations of ω‐3 PUFAs, PUFA levels were measured in small intestine tissue by GC/MS/MS analysis to compare with PUFA synthesis of between C57BL6 and fat‐1mice. As a result, ω‐3 PUFAs significantly attenuated Apc mutation–induced intestinal polyposis accompanied with significant inhibition of Wnt/β‐catenin signaling, COX‐2 and PGE_2, but induced significant levels of 15‐PGDH. In addition, significant induction of the inflammasome‐related substrates as IL‐1β and IL‐18 and activation of caspase‐1 was observed in Apc^Min/+ × fat‐1 mice. Administration of at least 3 g/60 kg ω‐3 PUFAs was equivalent to ω‐3 PUFAs produced in fat‐1 mice and resulted in significant increase in the expression of IL‐1β, caspase‐3 and IL‐18, as seen in Apc^Min/+ × fat‐1 mice. We conclude that ω‐3PUFAs can prevent intestinal polyp formation by inhibition of Wnt/β‐catenin signaling, but increased levels of 15‐PGDH and IL‐18.     

Atorvastatin inhibits pancreatic carcinogenesis and increases survival in LSL‐KrasG12D‐LSL‐Trp53R172H‐Pdx1‐Cre mice

There are several studies supporting the role of HMG‐CoA reductase inhibitors such as atorvastatin against carcinogenesis, in which inhibiting the generation of prenyl intermediates involved in protein prenylation plays the crucial role. Mutation of Kras gene is the most common genetic alteration in pancreatic cancer and the Ras protein requires prenylation for its membrane localization and activity. In the present study, the effectiveness of atorvastatin against pancreatic carcinogenesis and its effect on protein prenylation were determined using the LSL‐Kras^G12D‐LSL‐Trp53^R172H‐Pdx1‐Cre mouse model (called Pan^kras/p53 mice). Five‐week‐old Pan^kras/p53 mice were fed either an AIN93M diet or a diet supplemented with 100 ppm atorvastatin. Kaplan–Meier survival analysis with Log‐Rank test revealed a significant increase in survival in mice fed 100 ppm atorvastatin (171.9 ± 6.2 d) compared to the control mice (144.9 ± 8.4 d, P < 0.05). Histologic and immunohistochemical analysis showed that atorvastatin treatment resulted in a significant reduction in tumor volume and Ki‐67‐labeled cell proliferation. Mechanistic studies on primary pancreatic tumors and the cultured murine pancreatic carcinoma cells revealed that atorvastatin inhibited prenylation in several key proteins, including Kras protein and its activities, and similar effect was observed in pancreatic carcinoma cells treated with farnesyltransferase inhibitor R115777. Microarray assay on the global gene expression profile demonstrated that a total of 132 genes were significantly modulated by atorvastatin; and Waf1p21, cyp51A1, and soluble epoxide hydrolase were crucial atorvastatin‐targeted genes which involve in inflammation and carcinogenesis. This study indicates that atorvastatin has the potential to serve as a chemopreventive agent against pancreatic carcinogenesis. © 2012 Wiley Periodicals, Inc.     

Integrating expression‐related SNPs into genome‐wide gene‐ and pathway‐based analyses identified novel lung cancer susceptibility genes

Traditional pathway analysis map single nucleotide polymorphisms (SNPs) to genes according to physical position, which lacks sufficient biological bases. Here, we incorporated genetics of gene expression into gene‐ and pathway‐based analysis to identify genes and pathways associated with lung cancer risk. We identified expression‐related SNPs (eSNPs) in lung tissues and integrated these eSNPs into three lung cancer genome‐wide association studies (GWASs), including 12,843 lung cancer cases and 12,639 controls. We used SKAT‐C for gene‐based analysis, and conditional analysis to identify independent eSNPs of each gene. ARTP algorithm was used for pathway analysis. A total of 374,382 eSNPs in the GWAS datasets survived quality control, which were mapped to 5,084 genes and 2,752 pathways. In the gene‐based analysis, nine genes showed significant associations with lung cancer risk. Among them, TP63 (3q28), RP11‐650L12.2 (15q25.1) and CHRNA5 (15q25.1) were located in known lung cancer susceptibility loci. We also validated two newly identified susceptibility loci (RNASET2 and AL133458.1 in 6q27, and MPZL3 in 11q23.3). Besides, DVL3 (3q27.1), RP11‐522I20.3 (9q21.32) and CCDC116 (22q11.21) were identified as novel lung cancer susceptibility genes. Pathway analysis showed that pathways involved in protein structure, the Notch signaling pathway and the nicotinic acetylcholine receptor‐related pathways were associated with lung cancer risk. Combing eSNPs, gene‐ and pathway‐based analysis identifies novel lung cancer susceptibility genes, which serves as a powerful approach to decipher biological mechanisms underlying GWAS findings.     

Requisite role of vasohibin‐2 in spontaneous gastric cancer formation and accumulation of cancer‐associated fibroblasts

The vasohibin (VASH) family consists of two genes, VASH1 and VASH2. VASH1 is mainly expressed in vascular endothelial cells and suppresses angiogenesis in an autocrine manner, whereas VASH2 is mainly expressed in cancer cells and exhibits pro‐angiogenic activity. Employing adenomatous polyposis coli gene mutant mice, we recently reported on the role of Vash2 in the spontaneous formation of intestinal tumors. In this study, we used K19‐Wnt1/C2mE (Gan) mice and examined the role of Vash2 in spontaneous gastric cancer formation. Gan mice spontaneously develop gastric tumors by activation of Wnt and prostaglandin E2 signaling pathways in gastric mucosa after 30 weeks of age. Expression of Vash2 mRNA was significantly increased in gastric tumor tissues compared with normal stomach tissues. When Gan mice were crossed with the Vash2‐deficient (Vash2^LacZ/LacZ) strain, gastric cancer formation was significantly suppressed in Vash2^LacZ/LacZ Gan mice. Normal composition of gastric mucosa was partially maintained in Vash2^LacZ/LacZ Gan mice. Knockout of Vash2 caused minimal reduction of tumor angiogenesis but a significant decrease in cancer‐associated fibroblasts (CAF) in tumor stroma. DNA microarray analysis and real‐time RT‐PCR showed that mRNA levels of epiregulin (Ereg) and interleukin‐11 (Il11) were significantly downregulated in gastric tumors of Vash2^LacZ/LacZ Gan mice. Furthermore, conditioned medium of gastric cancer cells stimulated migration of and α‐smooth muscle actin expression in fibroblasts, whereas conditioned medium of VASH2 knockdown cells attenuated these effects in vitro. These results suggest that VASH2 plays an important role in gastric tumor progression via the accumulation of CAF accompanying upregulation of EREG and IL‐11 expression.     

Targeting breast cancer‐initiating/stem cells with melanoma differentiation‐associated gene‐7/interleukin‐24

Melanoma differentiation‐associated gene‐7/interleukin‐24 (mda‐7/IL‐24) displays a broad range of antitumor properties including cancer‐specific induction of apoptosis, inhibition of tumor angiogenesis and modulation of antitumor immune responses. In our study, we elucidated the role of MDA‐7/IL‐24 in inhibiting growth of breast cancer‐initiating/stem cells. Ad.mda‐7 infection decreased proliferation of breast cancer‐initiating/stem cells without affecting normal breast stem cells. Ad.mda‐7 induced apoptosis and endoplasmic reticulum stress in breast cancer‐initiating/stem cells similar to unsorted breast cancer cells and inhibited the self‐renewal property of breast cancer‐initiating/stem cells by suppressing Wnt/β‐catenin signaling. Prevention of inhibition of Wnt signaling by LiCl increased cell survival upon Ad.mda‐7 treatment, suggesting that Wnt signaling inhibition might play a key role in MDA‐7/IL‐24‐mediated death of breast cancer‐initiating/stem cells. In a nude mouse subcutaneous xenograft model, Ad.mda‐7 injection profoundly inhibited growth of tumors generated from breast cancer‐initiating/stem cells and also exerted a potent “bystander” activity inhibiting growth of distant uninjected tumors. Further studies revealed that tumor growth inhibition by Ad.mda‐7 was associated with a decrease in proliferation and angiogenesis, two intrinsic features of MDA‐7/IL‐24, and a reduction in vivo in the percentage of breast cancer‐initiating/stem cells. Our findings demonstrate that MDA‐7/IL‐24 is not only nontoxic to normal cells and normal stem cells but also can kill both unsorted cancer cells and enriched populations of cancer‐initiating/stem cells, providing further documentation that MDA‐7/IL‐24 might be a safe and effective way to eradicate cancers and also potentially establish disease‐free survival.     

Expression of TLR9 in tumor‐infiltrating mononuclear cells enhances angiogenesis and is associated with a worse survival in lung cancer

Toll‐like receptors (TLRs) play a crucial role in the innate and adaptive immune responses against microbial infection, tissue injury and cancer. Ligands of TLR9 have been developed as therapy in non‐small‐cell lung carcinoma (NSCLC). However, phase III clinical trials in metastatic NSCLC were negative. Our objective was to determine whether TLR9 affects tumor growth. We generated a mouse model of lung adenocarcinoma (ADC) mutated for K‐ras (K‐ras^LA1), with and without TLR9 inactivation (K‐ras^LA1TLR9^?/? and K‐ras^LA1TLR9^+/+, respectively). TLR9 was functionally expressed only in mononuclear cells of K‐ras^LA1TLR9^+/+ mice. These mice had significantly worse survival and a higher tumor burden than K‐ras^LA1TLR9^?/? mice. Lung tumors were analyzed for 24 cytokines/growth factors using Bio‐Plex multiplex bead‐based assays. Factor VIII was assessed by immunochemistry. Tumors from K‐ras^LA1TLR9^+/+ mice were characterized by an angiogenic phenotype with higher concentrations of vascular endothelial growth factor (VEGF) and higher microvessel density than from K‐ras^LA1TLR9^?/? mice. LKR13 cells, an ADC cell line derived from K‐ras^LA1 mice, were subcutaneously injected into TLR9^?/? and TLR9^+/+ mice. Syngeneic tumors regressed in TLR9^?/? but not in TLR9^+/+ mice. Peripheral blood mononuclear cells from TLR9^?/? mice released less VEGF than those from TLR9^+/+ mice. In 61 patients with early‐stage NSCLC, TLR9 was expressed in mononuclear cells that infiltrated tumors, as assessed by immunochemistry, and contributed to worse survival. Our results suggest that TLR9 expression in mononuclear cells was associated with an angiogenic phenotype and promoted lung cancer progression. These findings may aid clinical development of TLR9 ligands to treat cancers.