Carcinogen-induced Lung Cancer Research Articles

157 (PB145): Dietary sugar potentiates tobacco carcinogen-induced lung cancer development and progression by inducing the recruitment and protumoral polarization of macrophages

157 (PB145): Dietary sugar potentiates tobacco carcinogen-induced lung cancer development and progression by inducing the recruitment and protumoral polarization of macrophages

Diallyl trisulfide inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung cancer via modulating gut microbiota and the PPARγ/NF-κB pathway.

Smoking is the primary risk factor for developing lung cancer. Chemoprevention could be a promising strategy to reduce the incidence and mortality rates of lung cancer. Recently, we reported that A/J mice exposed to tobacco smoke carcinogens displayed the reshaping of gut microbiota. Additionally, garlic oil was found to effectively inhibit the carcinogenic effects of tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in lung tumorigenesis. Diallyl trisulfide (DATS), which is the predominant compound in garlic oil, exhibits various biological activities. To further explore the chemopreventive action and potential mechanism of DATS on lung tumorigenesis, we established a lung adenocarcinoma model in A/J mice stimulated by NNK. Subsequently, we employed multi-omics combined molecular biology technologies to clarify the mechanism. The results indicated that DATS significantly decreased the number of lung tumors in NNK induced A/J mice. Interestingly, we discovered that DATS could modulate gut microbiota, particularly increasing the abundance of F. rodentium, which has inhibitory effects on tumor growth. Mechanistically, DATS could activate the PPARγ pathway, leading to the negative regulation of the NF-κB signaling pathway and subsequent suppression of NF-κB-mediated inflammatory factors. Collectively, these findings provide support for DATS as a potential novel chemopreventive agent for tobacco carcinogen-induced lung cancer.

A novel agonist of 4-1BB costimulatory receptor shows therapeutic efficacy against a tobacco carcinogen-induced lung cancer.

Immunotherapy utilizing checkpoint inhibitors has shown remarkable success in the treatment of cancers. In addition to immune checkpoint inhibitors, immune co-stimulation has the potential to enhance immune activation and destabilize the immunosuppressive tumor microenvironment. CD137, also known as 4-1BB, is one of the potent immune costimulatory receptors that could be targeted for effective immune co-stimulation. The interaction of the 4-1BB receptor with its natural ligand (4-1BBL) generates a strong costimulatory signal for T cell proliferation and survival. 4-1BBL lacks costimulatory activity in soluble form. To obtain co-stimulatory activity in soluble form, a recombinant 4-1BBL protein was generated by fusing the extracellular domains of murine 4-1BBL to a modified version of streptavidin (SA-4-1BBL). Treatment with SA-4-1BBL inhibited the development of lung tumors in A/J mice induced by weekly injections of the tobacco carcinogen NNK for eight weeks. The inhibition was dependent on the presence of T cells and NK cells; depletion of these cells diminished the SA-4-1BBL antitumor protective effect. The number of lung tumor nodules was significantly reduced by the administration of SA-4-1BBL to mice during ongoing exposure to NNK. The data presented in this paper suggest that utilizing an immune checkpoint stimulator as a single agent generate a protective immune response against lung cancer in the presence of a carcinogen. More broadly, this study suggests that immune checkpoint stimulation can be extended to a number of other cancer types, including breast and prostate cancers, for which improved diagnostics can detect disease at the preneoplastic stage.

Abstract A007: Assessing immunoprevention efficacy of an agonist of CD137 pathway in a tobacco carcinogen-induced lung cancer preclinical model

Abstract Purpose: Lung cancer is the leading cause of cancer-related deaths worldwide. Cigarette smoking is the primary risk factor for lung cancer and is linked to 80-90% of lung cancer deaths according to the Centers for Disease Control (CDC). The most carcinogenic component of tobacco smoke is NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). SA-4-1BBL is a novel CD137 receptor agonist that has shown cancer immunoprevention efficacy in transplantable preclinical tumor models by invoking an innate immune surveillance mechanism involving CD4+ CD44+ T and NK cells. As a preclinical model for high-risk populations, we evaluated the immunoprevention efficacy of SA-4-1BBL in lung tumor formation induced by NNK. Experimental Procedures: A/J female mice were used as a preclinical model for lung cancer immunoprevention due to their susceptibility to tobacco-carcinogen-induced lung cancer. A/J mice were i.p. injected with NNK weekly for eight consecutive treatments. The mice were also treated s.c. with 100 µg SA-4-1BBL twice, two weeks apart on weeks 6 and 8 of NNK treatment. Animals were monitored for 21 weeks and euthanized to harvest the lung and lung-draining lymph nodes for histology (H&E staining and PCNA immunofluorescence staining). In separate groups, mice were treated with a depleting antibody to CD4 twice two weeks apart on weeks 6 and 8 of NNK treatment to investigate the role of CD4+ immune cells in cancer immunoprevention. Results: NNK treatment resulted in lung tumor formation as assessed by PCNA immunofluorescence staining in all saline-injected control mice by the 21-week experimental end-point. Treatment with SA-4-1BBL resulted in significantly fewer macroscopic and microscopic tumor numbers than the control group (p < 0.01). Animals treated with SA-4-1BBL and subjected to CD4+ cell depletion showed similar numbers of macroscopic and microscopic lung tumors to the vehicle group. Conclusions: Prophylactic treatment with SA-4-1BBL significantly prevents NNK-induced lung tumor formation and CD4+ immune cells play a critical role in the observed immunoprevention efficacy of SA-4-1BBL. Supported by a grant from DOD LC190524 to Esma S. Yolcu. Citation Format: Ayse Ece Gulen, Mohammad Tarique, Vahap Ulker, Evrim Agca, Haval Shirwan, Esma S. Yolcu. Assessing immunoprevention efficacy of an agonist of CD137 pathway in a tobacco carcinogen-induced lung cancer preclinical model [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A007.

Hypermethylation involved in DNA profiles of lung cancer specific tumour suppressor genes and epigenetic modification caused by an ultra-highly diluted homeopathic drug, Condurango 30C, in vitro and in vivo

Background and objectives: DNA hyper-methylation is an important aspect involved in carcinogenesis and cancer progression, which affects mainly CpG islands of DNA and causes inactivation of tumour suppressor genes. Therefore DNA hypermethylation status of the genomic DNA in both the transformed cancerous cell lines and in carcinogen-induced lung cancer was ascertained by analysis of expressions of certain major lung cancer specific tumour suppressor genes. The other objective was to examine if ultra highly diluted homeopathic drug, Condurango 30C, had ability to modulate DNA methylation. Methods: DNA methylation activity, if any, has been ascertained in H460-NSCLC cells in vitro and in BaP-induced lung cancer of rats in vivo, in respect of tumour suppressor genes like p15, p16, p18 and p53 by using PCR-SSCP analyses. The ability of modulation of DNA methylation, if any, by Condurango 30C was also verified against placebo control in a blinded manner. Results: Condurango 30C-treated DNA showed significant decrease in band-intensity of p15 and p53 genes especially in methylated condition, in vitro, at the IC50 dose (2.43µl/100µl). SSCP analysis of p15 and p53 genes in Condurango 30C-treated DNA also supported ability of Condurango 30C to modulate methylation state, in vitro. Inhibition of p15 hypermethylation was observed after post cancer treatment of rat with Condurango 30C. SSCP results gave a better indication of differences in band-position and single strand separation of p15 and p53 in Condurango 30C treated samples. Conclusion: Condurango 30C could trigger epigenetic modification in lung cancer via modulation of DNA hypermethylation but placebos could not.

ATF3 inhibits arsenic-induced malignant transformation of human bronchial epithelial cells by attenuating inflammation

Arsenic is a naturally occurring metalloid strongly associated with the incidence of lung cancer. Understanding the mechanisms of arsenic-induced carcinogenesis favors the development of effective interventions to reduce the incidence and mortality of lung cancer. In this study, we investigated the role of activating transcription factor 3 (ATF3) in arsenic-induced transformation of human bronchial epithelial cells. ATF3 was upregulated during chronic exposure to 0.25 μM arsenic, and loss of ATF3 promoted arsenic-induced transformation. Moreover, arsenic-transformed ATF3 knockout (ATF3 KO-AsT) cells exhibited more aggressive characteristics, including acceleration in proliferation, resistance to chemotherapy and increase in migratory capacity. RNA-seq revealed that pathways involved in inflammation, cell cycle, EMT and oncogenesis were affected due to ATF3 deficiency during chronic arsenic exposure. Further experiments confirmed the overproduction of IL-6, IL-8 and TNFα as well as enhanced phosphorylation of AKT and STAT3 in ATF3 KO-AsT cells. Our results demonstrate that ATF3 upregulated by chronic low-dose arsenic exposure represses cell transformation and acquisition of malignant characteristics through inhibiting the production of proinflammatory cytokines and activation of downstream proteins AKT and STAT3, providing a new strategy for the prevention of carcinogen-induced lung cancer.

Loss of Parkinson\u2019s susceptibility gene LRRK2 promotes carcinogen-induced lung tumorigenesis

Pathological links between neurodegenerative disease and cancer are emerging. LRRK2 overactivity contributes to Parkinson’s disease, whereas our previous analyses of public cancer patient data revealed that decreased LRRK2 expression is associated with lung adenocarcinoma (LUAD). The clinical and functional relevance of LRRK2 repression in LUAD is unknown. Here, we investigated associations between LRRK2 expression and clinicopathological variables in LUAD patient data and asked whether LRRK2 knockout promotes murine lung tumorigenesis. In patients, reduced LRRK2 was significantly associated with ongoing smoking and worse survival, as well as signatures of less differentiated LUAD, altered surfactant metabolism and immunosuppression. We identified shared transcriptional signals between LRRK2-low LUAD and postnatal alveolarization in mice, suggesting aberrant activation of a developmental program of alveolar growth and differentiation in these tumors. In a carcinogen-induced murine lung cancer model, multiplex IHC confirmed that LRRK2 was expressed in alveolar type II (AT2) cells, a main LUAD cell-of-origin, while its loss perturbed AT2 cell morphology. LRRK2 knockout in this model significantly increased tumor initiation and size, demonstrating that loss of LRRK2, a key Parkinson’s gene, promotes lung tumorigenesis.

Lipopolysaccharide-Mediated Chronic Inflammation Promotes Tobacco Carcinogen-Induced Lung Cancer and Determines the Efficacy of Immunotherapy.

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease that is associated with increased risk of lung cancer. Pseudomonas aeruginosa (PA) infections are frequent in patients with COPD, which increase lung inflammation and acute exacerbations. However, the influences of PA-induced inflammation on lung tumorigenesis and the efficacy of immune checkpoint blockade remain unknown. In this study, we initiated a murine model of lung cancer by treating FVB/NJ female mice with tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alone or in combination with PA-lipopolysaccharide (LPS). LPS-mediated chronic inflammation induced T-cell exhaustion, increased the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis, and enhanced NNK-induced lung tumorigenesis through an immunosuppressive microenvironment characterized by accumulation of myeloid-derived suppressive cells (MDSC) and regulatory T cells. Anti-PD-1 antibody treatment reduced tumors in NNK/LPS-treated mice with a 10-week LPS treatment but failed to inhibit tumor growth when LPS exposure was prolonged to 16 weeks. Anti-Ly6G antibody treatment coupled with depletion of MDSC alone reduced tumor growth; when combined with anti-PD-1 antibody, this treatment further enhanced antitumor activity in 16-week NNK/LPS-treated mice. Immune gene signatures from a human lung cancer dataset of PD-1 blockade were identified, which predicted treatment responses and survival outcome and overlapped with those from the mouse model. This study demonstrated that LPS-mediated chronic inflammation creates a favorable immunosuppressive microenvironment for tumor progression and correlates with the efficacy of anti-PD-1 treatment in mice. Immune gene signatures overlap with human and mouse lung tumors, providing potentially predictive markers for patients undergoing immunotherapy. SIGNIFICANCE: This study identifies an immune gene signature that predicts treatment responses and survival in patients with tobacco carcinogen-induced lung cancer receiving immune checkpoint blockade therapy.

Electrospray drying of docetaxel nanosuspension: A study on particle formation and evaluation of nanocrystals thereof

Nanocrystallization has emerged as a potential solubility enhancement approach for many active pharmaceutical ingredients. Electrospraying has generated a lot of curiosity with its tremendously unexplored potential in the field of pharmaceutical sciences. The present work aims to improve the solubility of Docetaxel by nanocrystallization approach, exploring electrospraying as a novel solidification technique. Electrosprayed nanocrystals were evaluated for wettability, the physical state of drug present, size after re-dispersion, and the % drug release. In vivo studies were also performed in the carcinogen-induced lung cancer model to compare the efficacy of nanocrystals versus unprocessed drug. The results revealed that the drug to stabilizer (Pluronic® F-127) ratio of 1:5, a solvent to the anti-solvent ratio (Ethanol: Water) of 1:10 v/v, and a stirring speed of 600 rpm, led to the formation of drug nanosuspension having the size of 271 ± 19 nm, and PDI of 0.287 ± 0.25 respectively. Upon electrospraying, the size was further increased to 458 ± 57 nm. Results revealed that the drug was present in the crystalline form, even after electrospraying. In-vivo studies conducted in carcinogen-induced lung cancer model showed no significant difference in the number of tumorous nodules formed. However, the developed formulation was more effective in reducing tumor load. Thus, the study concluded that electrospraying could be utilized as one of the potential drying techniques for nano-formulations.

The oncogenic potential of a mutant TP53 gene explored in two spontaneous lung cancer mice models

BackgroundLung cancer is the number one cancer killer worldwide. A major drawback in the lung cancer treatment field is the lack of realistic mouse models that replicate the complexity of human malignancy and immune contexture within the tumor microenvironment. Such models are urgently needed. Mutations of the tumor protein p53 are among the most common alterations in human lung cancers.MethodsPreviously, we developed a line of lung cancer mouse model where mutant human TP53-273H is expressed in a lung specific manner in FVB/N background. To investigate whether the human TP53 mutant has a similar oncogenic potential when it is expressed in another strain of mouse, we crossed the FVB/N-SPC-TP53-273H mice to A/J strain and created A/J-SPC-TP53-273H transgenic mice. We then compared lung tumor formation between A/J-SPC-TP53-273H and FVB/N-SPC-TP53-273H.ResultsWe found the TP53-273H mutant gene has a similar oncogenic potential in lung tumor formation in both mice strains, although A/J strain mice have been found to be a highly susceptible strain in terms of carcinogen-induced lung cancer. Both transgenic lines survived more than 18 months and developed age related lung adenocarcinomas. With micro CT imaging, we found the FVB-SPC-TP53-273H mice survived more than 8 weeks after initial detection of lung cancer, providing a sufficient window for evaluating new anti-cancer agents.ConclusionsOncogenic potential of the most common genetic mutation, TP53-273H, in human lung cancer is unique when it is expressed in different strains of mice. Our mouse models are useful tools for testing novel immune checkpoint inhibitors or other therapeutic strategies in the treatment of lung cancer.

The Effect of Low Carbohydrate Diets on Preventing and Treating Carcinogen-Induced Lung Cancer in Mice

Abstract Objectives Since cancer cells typically rely more on glycolysis than normal cells, we hypothesized that lowering carbohydrate intake may reduce cancer risk. We aimed to investigate the efficacy of low-carbohydrate (CHO) diets in preventing and treating a tobacco-specific carcinogen-induced lung cancer in female A/J mice. Methods We evaluated the role of different types of CHO (easily digestible vs resistant), protein (casein vs. soy) and fat (fish vs. coconut vs. a mixture of oils) in modulating 4-(N-methyl-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK)-induced lung nodule formation in these mice. To assess the efficacy of these diets in preventing NNK-induced lung nodule formation, we put these mice in the different diets for 2 weeks, intraperitoneally-injected NNK once a week for two weeks to initiate lung nodule formation. After 5 months, the lung nodules in these mice were counted. Results The lowering of easily digestible CHO significantly reduced constitutive blood glucose levels and lung nodule formation in the mice. Interestingly, diets low in easily digestible starch, high in fish oil (FO) and soy protein (15%Amylose/Soy/FO) were the most effective at preventing the formation of NNK-induced lung nodules. To determine if this 15%Amylose/Soy/FO is also effective at slowing tumor progression, we fed NNK-injected A/J mice a Western diet until tumors were established (5 months post NNK) and then either switched them to the 15%Amylose/Soy/FO or kept them on the Western diet for 5 additional months. The 15%Amylose/Soy/FO diet prevented the formation of additional lung tumor nodules and reduced the size of the tumors, although no significant difference was observed in tumor stage. The reduction in size of the lung tumors on the 15%Amylose/Soy/FO diet was not due to a lower tumor proliferation (Ki67 index) but an increase in apoptosis, as determined by TUNEL assays. Conclusions We conclude that a diet change that lowers glucose intake, incorporates FO and soy protein may be effective not only in preventing lung cancer formation but also in slowing the growth of established lung tumors. Funding Sources Lotte & John Hecht Memorial Foundation.

Antineoplastic potential of eupatilin against benzo[a]pyrene-induced lung carcinogenesis

Background: Most of the conservative therapies used to treat lung cancer show serious side effects. In addition, the prevalence and death rates due to lung cancer have been increasing alarmingly across the globe. Eupatilin (EUP) is a naturally occurring flavone which is primarily the active ingredient of the traditional Chinese medicine Artemisia asiatica. Materials and Methods: In this study, we evaluated the antineoplastic effect of EUP against benzo(a)pyrene-induced lung cancer in Swiss albino mice. We analyzed the level of xenobiotics, liver dysfunction enzymes (LDEs), pro-inflammatory cytokines, and histology of the liver. Furthermore, we conducted in vitro experiments (A549 cells) to elucidate the amount of cell proliferation, apoptosis, and their markers (caspases 3 and 9). Results: The EUP (30 mg/kg bw) treatment of tumor-bearing mice with EUP revealed the normal levels of xenobiotic, LDEs, antioxidant enzymes, lipid peroxidation in the liver and further carcinoembryonic antigen, pro-inflammatory marker, and histology in lung tissues. EUP inhibited the proliferation of A549 cells and induced the formation of reactive oxygen species and apoptosis by upregulating the expression of caspases 3 and 9. Conclusion: Overall, these results substantiate the anti-neoplastic effects of EUP against carcinogen-induced lung cancer in in vitro and in vivo models.

XPC protects against smoking- and carcinogen-induced lung adenocarcinoma.

Cigarette smoke (CS) contains hundreds of carcinogens and is a potent inducer of oxidative and bulky DNA damage, which when insufficiently repaired leads to activation of DNA damage response and possibly mutations. The DNA repair protein xeroderma pigmentosum group C (XPC) is primed to play an important role in CS-induced DNA damage because of its function in initiating repair of both bulky oxidative DNA damage. We hypothesized that loss of XPC function will increase susceptibility to developing CS- and carcinogen-induced lung cancer through impaired repair of oxidative DNA damage. Mice deficient in XPC (XPC-/-) exposed to chronic CS developed lung tumors whereas their wild-type littermates (XPC+/+) did not. XPC-/- mice treated with the CS-carcinogen urethane developed lung adenocarcinomas representing progressive stages of tumor development, with lung tumor number increased 17-fold compared with XPC+/+ mice. Mice heterozygous for XPC (XPC+/-) demonstrated a gene-dose effect, developing an intermediate number of lung tumors with urethane treatment. Treatment of XPC-/- mice with the carcinogen 3-methylcholanthrene followed by the proliferative agent butylated hydroxytoluene resulted in a 2-fold increase in lung adenocarcinoma development. Finally, tumor number decreased 7-fold in the lungs of XPC-/- mice by concurrent treatment with the antioxidant, N-acetylcysteine. Altogether, this supports a mechanism by which decreased XPC expression promotes lung adenocarcinoma development in response to CS-carcinogen exposure, due in part to impaired oxidative DNA damage repair.

The effect of diet and exercise on tobacco carcinogen-induced lung cancer.

In previous studies, we found that low-carbohydrate (CHO) diets reduced the incidence of tumors in mice genetically predisposed to cancer. However, because >90% of human cancers arise via carcinogen-induced somatic mutations, we investigated, herein, the role that different types and levels of CHO, protein and lipid play in lung cancer induced by the tobacco-specific carcinogen, nicotine-derived nitrosamine ketone (NNK) in A/J mice. We found lowering CHO levels significantly reduced lung nodules and blood glucose levels. We also found that soy protein was superior to casein and that coconut oil was ineffective at reducing lung nodules. Diets containing amylose or inulin (at 15% of total calories), soy protein (at 35%) and fat (at 50%, 30% being fish oil) were the most effective at reducing lung nodules. These fish oil-containing diets increased plasma levels of the ketone body, β-hydroxybutyrate, while reducing both insulin and 8-isoprostane in plasma and bronchoalveolar interleukin-12 and lung PGE2 levels. After only 2 weeks on this diet, the levels of γ-H2AX were significantly reduced, 24 hours after NNK treatment. Housing these mice in two-tiered rat cages with exercise wheels led to similar mouse weights on the different diets, whereas keeping mice in standard mouse cages led to both significant weight differences between the low-CHO, soy protein, fish oil diet and Western diet and substantially more lung nodules than in the two-tiered cages. Our results suggest that low-CHO, soy protein, fish oil-containing diets, together with exercise, may reduce the incidence of lung cancer.

MA16.01 Targeted Gene Therapy for Tobacco Carcinogen-Induced Lung Cancer

Rab25, a member of Rab family of small GTPases, is associated with progression of various types of human cancer including lung cancer that is the leading cause of cancer-associated deaths around the globe. In this study, we report the gene therapeutic effect of short hairpin Rab25 (shRab25) on 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorgenesis in female A/J mice. Initially, Mice (six-week-old) were injected with single dose of NNK (2 mg/0.1 ml saline/mouse) by intraperitoneal injection to induce the tumor. 8 weeks later, shRab25 was delivered with GPT-SPE (glycerol propoxylate triacrylate (GPT) and spermine) complex into tobacco-induced lung cancer models through a nose-only inhalation system twice a week for 2 month. Remarkably, aerosol-delivered shRab25 significantly decreased the expression level of Rab25 and other prominent apoptosis related proteins in female A/J mice. The apoptosis in these mice were determined by detecting the expression level of Bcl-2, PCNA, Bax and further confirmed by TUNEL assay. Our results strongly confirm the tumorigenic role of Rab25 in tobacco carcinogen induced-lung cancer and hence demonstrate aerosol delivery of shRab25 as a therapeutic target for lung cancer treatment.

Suppression of Tobacco Carcinogen-Induced Lung Tumorigenesis by Aerosol-Delivered Glycerol Propoxylate Triacrylate-Spermine Copolymer/Short Hairpin Rab25 RNA Complexes in Female A/J Mice.

Rab25, a member of Rab family of small guanosine triphosphatase, is associated with progression of various types of human cancers, including lung cancer, the leading cause of cancer-associated deaths around the globe. In this study, we report the gene therapeutic effect of short hairpin Rab25 RNA (shRab25) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in female A/J mice. Initially, mice (6 weeks old) were injected with single dose of NNK (2 mg/0.1 mL saline/mouse) by intraperitoneal injection to induce the tumor. Eight weeks later, shRab25 was complexed with glycerol propoxylate triacrylate-spermine (GPT-SPE) copolymer and delivered into tobacco-induced lung cancer models through a nose-only inhalation system twice a week for 2 months. GPT-SPE/shRab25 largely decreased the tobacco-induced tumor numbers and tumor volume in the lungs compared to GPT-SPE- or GPT-SPE/shScr-delivered groups. Remarkably, aerosol-delivered GPT-SPE/shRab25 significantly decreased the expression level of Rab25 and other prominent apoptosis-related proteins in female A/J mice. The apoptosis in these mice was determined by detecting the expression level of Bcl-2, proliferating cell nuclear antigen, Bax, and further confirmed by TUNEL assay. Our results strongly confirm the tumorigenic role of Rab25 in tobacco carcinogen-induced lung cancer and hence demonstrate aerosol delivery of shRab25 as a therapeutic target for lung cancer treatment.

Oral kanglaite injection (KLTI) attenuates the lung cancer-promoting effect of high-fat diet (HFD)-induced obesity.

Obesity is a risk factor for cancer and cancer-related mortality, however, its role in lung cancer progression remains controversial. This study aimed to assess whether high-fat diet (HFD)-induced obesity promotes lung cancer progression and whether the promotion can be decreased by Kanglaite injection (KLTI). In vivo, HFD-induced overweight or obesity increases the lung carcinoma incidence and multiplicity in a urethane-induced lung carcinogenic model and cancer-related mortality in a LLC allograft model by increasing oxidative stress and cellular signaling molecules including JAK, STAT3, Akt, mTOR, NF-κB and cyclin D1. These changes resulted in increases in vascular disruption and the lung water content, thereby promoting lung epithelial proliferation and the epithelial-mesenchymal transition (EMT) during carcinogenesis. Chronic KLTI treatment substantially prevented the weight gain resulting from HFD consumption, thereby reversing the metabolic dysfunction-related physiological changes and reducing susceptibility to lung carcinogenesis. In vitro, KLTI significantly suppressed the proliferation and induced apoptosis and differentiation in 3T3-L1 preadipocyte cells and attenuated endothelial cell permeability in HUVECs. Our study indicates that there is a potential relationship between obesity and lung cancer. This is the first study to show that obesity can directly accelerate carcinogen-induced lung cancer progression and that KLTI can decrease the lung cancer-promoting effect of HFD-induced obesity.

Roles of Palmitoylation and the KIKK Membrane-Targeting Motif in Leukemogenesis By Oncogenic KRAS4A

The RAS family includes three RAS genes, which encode four highly homologous proteins: H-, N-, and KRAS4A and 4B, the latter two being alternatively spliced forms differing only at the carboxyl terminus. Hyperactivation of RAS is common in human cancer, including hematological malignancies. Since RAS proteins are difficult to target, identification of alternative means to block RAS oncogenic signaling is critical for developing therapies against RAS-driven cancers. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Among RAS PTMs, palmitoylation is required for the high affinity plasma membrane binding of NRAS, HRAS and KRAS4A. We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for NRAS-related cancers. For KRAS-driven cancer, although much research has been focused on the KRAS4B splice variant, which does not undergo palmitoylation, KRAS4A has recently been shown to play an essential role in the development of carcinogen-induced lung cancer in mice and to be widely expressed in human cancers. However, the role of palmitoylation in KRAS4A tumorigenesis is not clear. In this study we show that KRAS4A is expressed in human leukemia cell lines with KRAS mutations and that mutation at the palmitoylation site of oncogenic KRAS4A significantly abrogates its leukemogenic potential. However, unlike NRAS, palmitoylation defective KRAS4A still induces leukemia in mice, albeit with a much longer latency. Consistently, palmitoylation defection has less impact on signaling of KRAS4A comparing that of NRAS. Using NRAS/KRAS4A chimeric constructs, we found that the KIKK motif of KRAS4A contributes to the transforming activity of KRAS4A. Mutations at both palmitoylation site and the KIKK motif abolish the ability of oncogenic KRAS4A to induce leukemia in mice. These studies suggest that therapies targeting RAS palmitoylation may also be effective in treating KRAS4A associated malignancies and that interfering the KIKK membrane-targeting motif would enhance the therapeutic effectiveness. DisclosuresNo relevant conflicts of interest to declare.

Roles of palmitoylation and the KIKK membrane-targeting motif in leukemogenesis by oncogenic KRAS4A.

BackgroundWe have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for NRAS-related cancers. For KRAS-driven cancer, although much research has been focused on the KRAS4B splice variant, which does not undergo palmitoylation, KRAS4A has recently been shown to play an essential role in the development of carcinogen-induced lung cancer in mice and to be widely expressed in human cancers. However, the role of palmitoylation in KRAS4A tumorigenesis is not clear.MethodsThe expression of KRAS4A in KRAS-mutated leukemia cell lines and acute myeloid leukemia (AML) cells were checked using western blotting and reverse transcriptions-quantitative polymerase chain reaction (RT-qPCR) analysis, respectively. The leukemogenic potentials of oncogenic KRAS4A and its palmitoylation-defective mutants were examined by a mouse bone marrow transduction and transplantation model and the in vitro transformation assays. The activation of the RAS downstream signaling pathways and the membrane localizations of the KRAS4A and its mutants were analyzed via western blot analysis and confocal microscopy, respectively.ResultsWe show here that KRAS4A is expressed in human leukemia cell lines and in AML cells harboring KRAS mutations and that mutation at the palmitoylation site of oncogenic KRAS4A significantly abrogates its leukemogenic potential. However, unlike NRAS, palmitoylation-defective KRAS4A still induces leukemia in mice, albeit with a much longer latency. Using NRAS/KRAS4A chimeric constructs, we found that the KIKK motif of KRAS4A contributes to the transforming activity of KRAS4A. Mutations at both palmitoylation site and the KIKK motif abolish the ability of oncogenic KRAS4A to induce leukemia in mice.ConclusionsOur studies suggest that therapies targeting RAS palmitoylation may also be effective in treating KRAS4A associated malignancies and that interfering the KIKK membrane-targeting motif would enhance the therapeutic effectiveness.

Pharmacological inhibition of caspase-8 limits lung tumour outgrowth.

Lung cancer is one of the leading causes of cancer death worldwide. Despite advances in therapy, conventional therapy is still the main treatment and has a high risk of chemotherapy resistance. Caspase-8 is involved in cell death and is a recognized marker for poor patient prognosis. To elucidate the role of caspase-8 in lung carcinoma, we used human samples of non-small cell lung cancer (NSCLC) and a mouse model of carcinogen-induced lung cancer. Healthy and cancerous NSCLC samples had similar levels of the active form of caspase-8. Similarly, lung tumour-bearing mice had high levels of the active form of caspase-8. Pharmacological inhibition of caspase-8 by z-IETD-FMK robustly reduced tumour outgrowth and this was closely associated with a reduction in the release of pro-inflammatory cytokines, IL-6, TNF-α, IL-18, IL-1α, IL-33, but not IL-1β. Furthermore, inhibition of caspase-8 reduced the recruitment of innate suppressive cells, such as myeloid-derived suppressor cells, but not of regulatory T cells to lungs of tumour-bearing mice. However, despite the well-known role of caspase-8 in cell death, the apoptotic cascade (caspase-3, caspase-9 and Bcl-2 dependent) was not active in lungs of z-IETD-treated tumour-bearing mice, but instead higher levels of the short segment of c-FLIP (c-FLIPs) were detected. Similarly, human healthy lung samples had higher levels of c-FLIPs than cancerous samples. Our data suggest that caspase-8 is an important orchestrator of cancer-associated inflammation and the presence of short segment of c-FLIP determines whether caspase-8 induces tumour proliferation or tumour arrest/regression in the lung.