K-rasLA1 Mice Research Articles

Gammaherpesvirus Infection Stimulates Lung Tumor-Promoting Inflammation.

Lung tumor-promoting environmental exposures and γherpesvirus infections are associated with Type 17 inflammation. To test the effect of γherpesvirus infection in promoting lung tumorigenesis, we infected mutant K-Ras-expressing (K-RasLA1) mice with the murine γherpesvirus MHV68 via oropharyngeal aspiration. After 7 weeks, the infected mice displayed a more than 2-fold increase in lung tumors relative to their K-RasLA1 uninfected littermates. Assessment of cytokines in the lung revealed that expression of Type 17 cytokines (Il-6, Cxcl1, Csf3) peaked at day 7 post-infection. These observations correlated with the post-infection appearance of known immune mediators of tumor promotion via IL-17A in the lungs of tumor-bearing mice. Surprisingly, Cd84, an immune cell marker mRNA, did not increase in MHV68-infected wild-type mice lacking lung tumors. Csf3 and Cxcl1 protein levels increased more in the lungs of infected K-RasLA1 mice relative to infected wild-type littermates. Flow cytometric and transcriptomic analyses indicated that the infected K-RasLA1 mice had increased Ly6Gdim/Ly6Chi immune cells in the lung relative to levels seen in uninfected control K-RasLA1 mice. Selective methylation of adenosines (m6A modification) in immune-cell-enriched mRNAs appeared to correlate with inflammatory infiltrates in the lung. These observations implicate γherpesvirus infection in lung tumor promotion and selective accumulation of immune cells in the lung that appears to be associated with m6A modification of mRNAs in those cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Luteolin and its derivative apigenin suppress the inducible PD-L1 expression to improve anti-tumor immunity in KRAS-mutant lung cancer.

Upregulated expression of immune checkpoint molecules correlates with exhausted phenotype and impaired function of cytotoxic T cells to evade host immunity. By disrupting the interaction of PD-L1 and PD1, immune checkpoint inhibitors can restore immune system function against cancer cells. Growing evidence have demonstrated apigenin and luteolin, which are flavonoids abundant in common fruits and vegetables, can suppress growth and induce apoptosis of multiple types of cancer cells with their potent anti-inflammatory, antioxidant and anticancer properties. In this study, the effects and underlying mechanisms of luteolin, apigenin, and anti-PD-1 antibody combined with luteolin or apigenin on the PD-L1 expression and anti-tumorigenesis in KRAS-mutant lung cancer were investigated. Luteolin and apigenin significantly inhibited lung cancer cell growth, induced cell apoptosis, and down-regulated the IFN-γ-induced PD-L1 expression by suppressing the phosphorylation of STAT3. Both luteolin and apigenin showed potent anti-cancer activities in the H358 xenograft and Lewis lung carcinoma model in vivo, and the treatment with monoclonal PD1 antibody enhanced the infiltration of T cells into tumor tissues. Apigenin exhibited anti-tumor activity in Genetically engineered KRASLA2 mice. In conclusion, both apigenin and luteolin significantly suppressed lung cancer with KRAS mutant proliferation, and down-regulated the IFN-γ induced PD-L1 expression. Treatment with the combination of PD-1 blockade and apigenin/luteolin has a synergistic effect and might be a prospective therapeutic strategy for NSCLC with KRAS-mutant.

Whole-Exome Sequencing of Radiation-Induced Thymic Lymphoma in Mouse Models Identifies Notch1 Activation as a Driver of p53 Wild-Type Lymphoma.

Mouse models of radiation-induced thymic lymphoma are widely used to study the development of radiation-induced blood cancers and to gain insights into the biology of human T-cell lymphoblastic leukemia/lymphoma. Here we aimed to identify key oncogenic drivers for the development of radiation-induced thymic lymphoma by performing whole-exome sequencing using tumors and paired normal tissues from mice with and without irradiation. Thymic lymphomas from irradiated wild-type (WT), p53+/-, and KrasLA1 mice were not observed to harbor significantly higher numbers of nonsynonymous somatic mutations compared with thymic lymphomas from unirradiated p53-/- mice. However, distinct patterns of recurrent mutations arose in genes that control the Notch1 signaling pathway based on the mutational status of p53. Preferential activation of Notch1 signaling in p53 WT lymphomas was also observed at the RNA and protein level. Reporter mice for activation of Notch1 signaling revealed that total-body irradiation (TBI) enriched Notch1hi CD44+ thymocytes that could propagate in vivo after thymocyte transplantation. Mechanistically, genetic inhibition of Notch1 signaling in immature thymocytes prevented formation of radiation-induced thymic lymphoma in p53 WT mice. Taken together, these results demonstrate a critical role of activated Notch1 signaling in driving multistep carcinogenesis of thymic lymphoma following TBI in p53 WT mice. SIGNIFICANCE: These findings reveal the mutational landscape and key drivers in murine radiation-induced thymic lymphoma, a classic animal model that has been used to study radiation carcinogenesis for over 70 years.

Reply to Krupenko et al. Comment on "Lee et al. The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer Cancers 2020, 12, 1382".

In the Cancers paper, we observed the increase ALDH1L1 protein expression following oncogenesis, as well as a therapeutic effect, by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC) [...].

Plumbagin suppresses non-small cell lung cancer progression through downregulating ARF1 and by elevating CD8+ T cells

Non-small cell lung cancer (NSCLC) is one of the most frequently diagnosed cancers and the leading causes of cancer death worldwide. Therefore, new therapeutic agents are urgently needed to improve patient outcomes. Plumbagin (PLB), a natural sesquiterpene present in many Chinese herbal medicines, has been reported for its anti-cancer activity in various cancer cells. In this study, the effects and underlying mechanisms of PLB on the tumorigenesis of NSCLC were investigated. PLB dose-dependently inhibited the growth of NSCLC cell lines. PLB promoted ROS production, activated the endoplasmic reticulum (ER) stress pathway, and induced cell apoptosis, accompanied by the decreased expression level of ADP-ribosylation factor 1 (ARF1) in NSCLC cancer cells, and those effects of PLB could be reversed by the pretreatment with N-acetyl-L-cysteine (NAC). More importantly, the calcium chelator (BM) significantly reversed PLB-induced cell apoptosis. Furthermore, PLB significantly inhibited the growth of both H1975 xenograft and LLC1 tumors and exhibited antitumor activity by enhancing the number and the effector function of CD8+ T cells in KRASLA2 mice model and the LLC1 xenograft. Our findings suggest that PLB exerts potent antitumor activity against NSCLC in vitro and in vivo through ARF1 downregulation and induction of antitumor immune response, indicating that PLB is a new novel therapeutic candidate for the treatment of patients with NSCLC.

The Impact of the Deepwater Horizon Oil Spill upon Lung Health-Mouse Model-Based RNA-Seq Analyses.

We used a transcriptomic approach to interrogate the effects of a saline-accommodated fraction from the Macondo 252 well (MC252) oil and Corexit dispersants on lung tissue. Wild-type C57BL/6 male and female mice were exposed on days 0, 7 and 13 by oropharyngeal aspiration to saline accommodated fractions (SAF) of crude oil from the Macondo (MC252) well, Corexit 9500, Corexit 9527, 9500+oil and 9527+oil or a saline solution as the vehicle control. These treatments did not cause overt toxicity, with the exception of the Corexit exposures which caused brief weight loss after the first exposure. On day 14, total RNA was isolated from the left lung for RNA-seq analyses. KEGG-pathway-based differential expression revealed that Corexit 9527 elicited the strongest changes involving the upregulation of 19 KEGG pathways (FDR < 0.10), followed by Corexit 9500 with the upregulation of seven pathways (FDR < 0.10). As an important signature, pathways related to a response to DNA damage (e.g., p53 signaling and mismatch repair) dominate those upregulated by Corexit 9527 and Corexit 9500. In addition, pro-inflammatory pathways (e.g., cytokine-cytokine receptor interaction, IL-17 signaling pathway and TNF signaling pathways) were upregulated selectively in oil-treated male mice. Surprisingly, oil + dispersant combinations caused lesser effects than the individual treatments at the transcriptomic level. Overall, these findings support potential genotoxicity, inflammation and cell death due to dispersant or oil exposures. Similar exposures to lung tumor bearing K-RasLA1 mice provided evidence for tumor promotion by oil and Corexit dispersant treatments. Our mouse RNA-seq analyses may be relevant to the pulmonary health hazards of MC252 oil and dispersants experienced in exposed populations.

The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer.

Lung adenocarcinoma cells express high levels of ALDH1L1, an enzyme of the one-carbon pathway that catalyzes the conversion of 10-formyltetrahydrofolate into tetrahydrofolate and NAD(P)H. In this study, we evaluated the potential of ALDH1L1 as a therapeutic target by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC). Reporter assays revealed KRAS-mediated upregulation of the ALDH1L1 promoter in human NSCLC cells. Aldh1l1−/− mice exhibited a normal phenotype, with a 10% decrease in Kras-driven lung tumorigenesis. By contrast, the inhibition of oxidative phosphorylation inhibition using phenformin in Aldh1l1−/−; KrasLA2 mice dramatically decreased the number of tumor nodules and tumor area by up to 50%. Furthermore, combined treatment with pan-ALDH inhibitor and phenformin showed a decreased number and area of lung tumors by 70% in the KrasLA2 lung cancer model. Consistent with this, previous work showed that the combination of ALDH1L1 knockdown and phenformin treatment decreased ATP production by as much as 70% in NSCLS cell lines. Taken together, these results suggest that the combined inhibition of ALDH activity and oxidative phosphorylation represents a promising therapeutic strategy for NSCLC.

MiR-301a promotes lung tumorigenesis by suppressing Runx3

BackgroundOur previous report demonstrated that genetic ablation of miR-301a reduces Kras-driven lung tumorigenesis in mice. However, the impact of miR-301a on host anti-tumor immunity remains unexplored. Here we assessed the underlying molecular mechanisms of miR-301a in the tumor microenvironment.MethodsThe differentially expressed genes were identified by using deep sequencing. The immune cell counts, and cytokines expression were analyzed by realtime PCR, immunohistochemistry and flow cytometry. The role of miR-301a/Runx3 in lung tumor was evaluated on cell growth, migration and invasion. The function of miR-301a/Runx3 in regulating tumor microenvironment and tumor metastasis were evaluated in Kras transgenic mice and B16/LLC1 syngeneic xenografts tumor models.ResultsIn this work, we identified 1166 up-regulated and 475 down-regulated differentially expressed genes in lung tumor tissues between KrasLA2 and miR-301a−/−; KrasLA2 mice. Immune response and cell cycle were major pathways involved in the protective role of miR-301a deletion in lung tumorigenesis. Overexpression of the miR-301a target, Runx3, was an early event identified in miR-301a−/−; KrasLA2 mice compared to WT-KrasLA2 mice. We found that miR-301a deletion enhanced CD8+ T cell accumulation and IFN-γ production in the tumor microenvironment and mediated antitumor immunity. Further studies revealed that miR-301a deficiency in the tumor microenvironment effectively reduced tumor metastasis by elevating Runx3 and recruiting CD8+ T cells, whereas miR-301a knockdown in tumor cells themselves restrained cell migration by elevating Runx3 expression.ConclusionsOur findings further underscore that miR-301a facilitates tumor microenvironment antitumor immunity by Runx3 suppression in lung tumorigenesis.

Proton radiation-induced cancer progression

There are considerable health risks related to ionizing and proton radiation exposure. While there is a long history of health risks associated with ionizing (photon) radiation exposure, there is a limited understanding of the long-term health risks associated with proton radiation exposure. Since proton radiation is becoming more common in cancer therapy, the long-term biological effects of proton radiation remain less well characterized in terms of radiotherapy and well as for astronauts during deep space explorations. In this study, we compared the long-term side effects of proton radiation to equivalent doses of X-rays in the initiation and progression of premalignant lesions in a lung cancer susceptible mouse model (K-rasLA1). We show proton irradiation causes more complex DNA damage that is not completely repaired resulting in increased oxidative stress in the lungs both acutely and persistently. We further observed K-rasLA1 mice irradiated with protons had an increased number and size of initiated and premalignant lesions and adenomas that were often infiltrated with inflammatory cells. Proton irradiated mice had a lower median survival and increased carcinoma incidence as compared to unirradiated controls and X-rays exposed mice. Our conclusion is that exposure to proton irradiation enhances the progression of premalignant lesions to invasive carcinomas through persistent DNA damage, chronic oxidative stress, and immunosuppression.

Thy-1+ Cancer-associated Fibroblasts Adversely Impact Lung Cancer Prognosis

Cancer-associated fibroblasts (CAFs) regulate diverse intratumoral biological programs and can promote or inhibit tumorigenesis, but those CAF populations that negatively impact the clinical outcome of lung cancer patients have not been fully elucidated. Because Thy-1 (CD90) marks CAFs that promote tumor cell invasion in a murine model of KrasG12D–driven lung adenocarcinoma (KrasLA1), here we postulated that human lung adenocarcinomas containing Thy-1+ CAFs have a worse prognosis. We first examined the location of Thy-1+ CAFs within human lung adenocarcinomas. Cells that co-express Thy-1 and α-smooth muscle actin (αSMA), a CAF marker, were located on the tumor periphery surrounding collectively invading tumor cells and in perivascular regions. To interrogate a human lung cancer database for the presence of Thy-1+ CAFs, we isolated Thy-1+ CAFs and normal lung fibroblasts (LFs) from the lungs of KrasLA1 mice and wild-type littermates, respectively, and performed global proteomic analysis on the murine CAFs and LFs, which identified 425 proteins that were differentially expressed. Used as a probe to identify Thy-1+ CAF-enriched tumors in a compendium of 1,586 lung adenocarcinomas, the presence of the 425-gene signature predicted a significantly shorter survival. Thus, Thy-1 marks a CAF population that adversely impacts clinical outcome in human lung cancer.

Inflammasome Independent Leukotriene B4 Production Drives Crystalline Silica Induced Sterile Inflammation in Lungs

Abstract Occupational exposure to Crystalline Silica (CS) is a major public health concern with millions of workers continuously exposed to CS. Deposition of fine silica particles in the lungs can lead to severe lung inflammation and cancer. Lipid chemokine Leukotriene B4 (LTB4) and IL-1b that mediate leukocyte recruitment to the site of injury, are the key mediators of CS-induced sterile inflammation. Our previous studies have shown that CS exposure increases lung tumor burden in K-rasLA1 mice and that is abrogated in the LTB4 receptor, BLT1 deficient mice. Here, we show that phagocytosis of CS sequentially activates a series of pathways leading to LTB4, IL-1b and CXC chemokine production. CS uptake induces rapid formation of lipid bodies termed “lipidosome” independent of inflammasome activation. Studies with inhibitors suggested that CS-induced lipidosome formation and LTB4 production is linked to the phagocytosis pathway, an observation confirmed by confocal microscopy. We further demonstrated the SiRNA knockdown of cPla2, JNK or Rab40c prevents the formation of lipidosome and production of LTB4. Lastly, we demonstrate using an air pouch model in mice that JNK inhibitor, BI-78D3 is effective in reducing CS induced sterile-inflammation in-vivo. In summary, our studies highlight a novel inflammasome independent and JNK activation dependent lipidosome pathway leading to the production LTB4 as a major regulator of CS-induced sterile inflammation.

Acute DNA damage activates the tumour suppressor p53 to promote radiation-induced lymphoma.

Genotoxic cancer therapies, such as chemoradiation, cause haematological toxicity primarily by activating the tumour suppressor p53. While inhibiting p53-mediated cell death during cancer therapy ameliorates haematologic toxicity, whether it also impacts carcinogenesis remains unclear. Here we utilize a mouse model of inducible p53 short hairpin RNA (shRNA) to show that temporarily blocking p53 during total-body irradiation (TBI) not only ameliorates acute toxicity, but also improves long-term survival by preventing lymphoma development. Using KrasLA1 mice, we show that TBI promotes the expansion of a rare population of thymocytes that express oncogenic KrasG12D. However, blocking p53 during TBI significantly suppresses the expansion of KrasG12D-expressing thymocytes. Mechanistically, bone marrow transplant experiments demonstrate that TBI activates p53 to decrease the ability of bone marrow cells to suppress lymphoma development through a non-cell-autonomous mechanism. Together, our results demonstrate that the p53 response to acute DNA damage promotes the development of radiation-induced lymphoma.

High Inorganic Phosphate Intake Promotes Tumorigenesis at Early Stages in a Mouse Model of Lung Cancer

Inorganic phosphate (Pi) is required by all living organisms for the development of organs such as bone, muscle, brain, and lungs, regulating the expression of several critical genes as well as signal transduction. However, little is known about the effects of prolonged dietary Pi consumption on lung cancer progression. This study investigated the effects of a high-phosphate diet (HPD) in a mouse model of adenocarcinoma. K-rasLA1 mice were fed a normal diet (0.3% Pi) or an HPD (1% Pi) for 1, 2, or 4 months. Mice were then sacrificed and subjected to inductively coupled plasma mass/optical emission spectrometry and laser ablation inductively coupled plasma mass-spectrometry analyses, western blot analysis, histopathological, immunohistochemical, and immunocytochemical analyses to evaluate tumor formation and progression (including cell proliferation, angiogenesis, and apoptosis), changes in ion levels and metabolism, autophagy, epithelial-to-mesenchymal transition, and protein translation in the lungs. An HPD accelerated tumorigenesis, as evidenced by increased adenoma and adenocarcinoma rates as well as tumor size. However, after 4 months of the HPD, cell proliferation was arrested, and marked increases in liver and lung ion levels and in energy production via the tricarboxylic acid cycle in the liver were observed, which were accompanied by increased autophagy and decreased angiogenesis and apoptosis. These results indicate that an HPD initially promotes but later inhibits lung cancer progression because of metabolic adaptation leading to tumor cell quiescence. Moreover, the results suggest that carefully regulated Pi consumption are effective in lung cancer prevention.

Abstract 2330: Activation of cancer stem cells by oncogenic K-Ras requires activated Wnt/β-catenin signaling

Abstract Adenomatous polyposis coli (APC) loss-of-function mutations and K-Ras gain-of-function mutations are common abnormalities that occur during the initiation and intermediate adenoma stages of colorectal tumorigenesis, respectively. However, little is known about the role of these mutations in the regulation of cancer stem cells (CSCs) associated with colorectal cancer (CRC) tumorigenesis. We analyzed tissue from CRC patients (n = 49) and observed an association between K-Ras mutations and CSC activation during colorectal tumorigenesis. The oncogenic K-Ras mutations enhanced stemness of CRC cells carrying an APC mutation, as shown by comparisons of sphere formation, transforming potential, and differentiation in vitro and tumor-forming capacity in vivo between isogenic APC mutated CRC cells harboring either wild-type or mutant K-Ras. Moreover, the activation of CSC by K-Ras mutations in CRC required an additional APC mutation, as revealed by the induction of CD44, CD133, and CD166 in intestinal tumor tissues of APCMin/+/K-RasLA2 double-mutant mice, but not in K-RasLA2 mice. Overall, the progression and metastasis of CRC induced by K-Ras mutation occurs by initial activation of CSC by APC loss and further activation of Wnt/β-catenin signaling and subsequent activation of Ras-ERK signaling. Citation Format: Byoung-San Moon, Kug Hwa Lee, Kyounghwa Koo, Kang-Yell Choi. Activation of cancer stem cells by oncogenic K-Ras requires activated Wnt/β-catenin signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2330. doi:10.1158/1538-7445.AM2015-2330

Abstract 895: Genetic deletion of cyclooxygenase-2 suppresses K-ras induced lung tumorigenesis

Abstract Lung cancer is the leading cause of cancer-related death worldwide. Proto-oncogene K-ras is mutated in 30-50% of lung adenocarcinomas, the most common histological subtype of the non-small cell lung cancer. Cyclooxygenases are the key enzymes in the conversion of arachidonic acid to prostaglandins (PGs) which are thought to promote tumor growth, angiogenesis and metastasis in various tumors including lung cancer. However, the role of cyclooxygenase 2 (COX-2) in lung tumorigenesis in mice carrying the K-ras oncogene remains unclear. We evaluated the contribution of COX-2 in lung tumorigenesis in K-rasLA1(G12D) mice and its relevant molecular mechanisms. We used a genetic knockout approach by crossing COX-2 homozygous mice (COX-2-/-) with a mouse strain which develops lung tumorigenesis driven by mutant K-ras (K-rasLA1) to obtain COX-2 deficient mice with K-ras expression (K-ras/COX-2-/-) and COX-2 wild type mice with K-ras expression (K-ras). Loss of COX-2 was associated with a significant reduction in both multiplicity and tumor volume of lung adenocarcinoma examined by ex-vivo MRI. The average number of lung nodules in K-ras/COX-2-/- mice was 6.8 per mouse which was significantly less than that of K-ras mice (19.8) (p&amp;lt;0.05). Similarly, tumor size in K-ras mice (65.6 ± 31.1 mm3) was larger than that of K-ras/COX-2-/- mice (25.5 ± 8.7 mm3). Histological examination of mouse lung demonstrated that there was a significantly higher incidence of bronchioalveolar hyperplasia in lung tissues of the K-ras group (9.5±3.2) than in the K-ras/COX-2-/- group (5.3±2.5) (p&amp;lt;0.05). In line with this, studying tumor cell proliferation by Ki67 staining suggested that COX-2 deletion in K-ras mice was associated with less lung tumor cell proliferative potential evidenced by Ki67 positive cells being significantly decreased from 6.2% in K-ras mice to 2.9% in K-ras/COX-2-/- mice. The level of PGE2 was notably lower in lung tumor tissue derived from K-ras/COX-2-/- mice than that of K-ras mice. Similarly, the urinary metabolite of PGE2, PGEM, was also significantly reduced by 3-fold in K-ras/COX-2-/- mice in comparison with K-ras mice. In contrast, level of 13,14-dihydro-15-keto-PGE2, a metabolite of PGE2 in K-ras/COX-2-/- mouse lung tumor tissue was significantly increased by almost 3-fold. Furthermore, MEK and p-ErK1/2 expressions significantly decreased in lung tissues of K-ras/COX-2-/- mice compare to that of K-ras mice. Together, our data suggest that COX-2 deletion contributes to repression of K-ras-induced lung tumorigenesis through reducing tumor cell proliferation rate, decreasing production of PGE2, and increasing 13,14-dihydro-15-keto-PGE2 in the tumors, which potentially involves the MAPK pathway. These observations suggested the importance of COX-2 in lung tumorigenesis, especially under K-ras mutation and further identified COX-2 as a potential target in lung cancer prevention. The study is supported by NIH Grant R01CA144053. Citation Format: Yong Pan, Yan Jiang, Lin Tan, Murali K. Ravoori, Mihai Gagea-Iurascu, Mihai Gagea-Iurascu, Vikus Kundra, Susan M. Fischer, Peiying Yang. Genetic deletion of cyclooxygenase-2 suppresses K-ras induced lung tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 895. doi:10.1158/1538-7445.AM2015-895

Abstract 4416: IKBKE is a key mediator of Ras activation of NF-κB and Ras oncogenic function

Abstract Accumulating evidence indicates that the NF-κB transcription factor pathway is important for oncogenic RAS to transform cells and to drive tumorigenesis in animal models. IKBKE has been shown to activate NF-κB through both canonical and non-canonical pathways. Here we show that activating mutations of Ras significantly induce IKBKE kinase activity and that depletion of KRas results in decrease of IKBKE activity only in KRAS mutant but wild-type cells. KRas-induced NF-κB DNA-binding and transcription activity was abrogated by knockdown of IKBKE. In addition, we demonstrated significant decrease of lung tumor development in Ikbke-/-;KRasLA1 mice when compared to Ikbke+/-;KRasLA1 and Ikbke+/+;KRasLA1 animals. Furthermore, depletion of IKBKE selectively reduces cell survival and tumor growth in K-Ras mutant non-small cell lung cancer cells. These data identify IKBKE as a downstream effector of Ras which mediates RAS activation of NF-κB and Ras tumorigenic activity. Citation Format: Donghwa Kim, Jianping Guo, Sridevi Challa, Domenico Coppola, Jin Q. Cheng. IKBKE is a key mediator of Ras activation of NF-κB and Ras oncogenic function. [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 4416. doi:10.1158/1538-7445.AM2014-4416

Radiation-Enhanced Lung Cancer Progression in a Transgenic Mouse Model of Lung Cancer Is Predictive of Outcomes in Human Lung and Breast Cancer

Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment, including the modification of inflammatory responses from antitumorigenic to protumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis. K-ras(LA1) mice were irradiated with various doses and dose regimens and then monitored until death. Microarray analyses were performed using Illumina BeadChips on whole lung tissue 70 days after irradiation with a fractionated or acute dose of radiation and compared with age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets. Radiation exposure accelerates lung cancer progression in the K-ras(LA1) lung cancer mouse model with dose fractionation being more permissive for cancer progression. A nonrandom inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature. These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by affecting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis.

Role of Oncogenic K-Ras in Cancer Stem Cell Activation by Aberrant Wnt/β-Catenin Signaling

Adenomatous polyposis coli (APC) loss-of-function mutations and K-Ras gain-of-function mutations are common abnormalities that occur during the initiation and intermediate adenoma stages of colorectal tumorigenesis, respectively. However, little is known about the role these mutations play in cancer stem cells (CSCs) associated with colorectal cancer (CRC) tumorigenesis. We analyzed tissue from CRC patients (n = 49) to determine whether K-Ras mutations contributed to CSC activation during colorectal tumorigenesis. DLD-1-K-Ras-WT and DLD-1-K-Ras-MT cells were cultured and evaluated for their ability to differentiate, form spheroids in vitro, and form tumors in vivo. Interaction between APC and K-Ras mutations in colorectal tumorigenesis was evaluated using APC (Min/+)/K-Ras (LA2) mice and DLD-1-K-Ras-WT and DLD-1-K-Ras-MT cell xenografts. (n = 4) Group differences were determined by Student t test. All statistical tests were two-sided. The sphere-forming capability of DLD-1-K-Ras-MT cells was statistically significantly higher than that of DLD-1-K-Ras-WT cells (DLD-1-K-Ras-MT mean = 86.661 pixel, 95% confidence interval [CI] = 81.701 to 91.621 pixel; DLD-1-K-Ras-WT mean = 42.367 pixel, 95% CI = 36.467 to 48.267 pixel; P = .003). Moreover, both the size and weight of tumors from DLD-1-K-Ras-MT xenografts were markedly increased compared with tumors from DLD-1-K-Ras-WT cells. Expression of the CSC markers CD44, CD133, and CD166 was induced in intestinal tumors from APC (Min/+)/K-Ras (LA2)mice, but not K-Ras (LA2) mice, indicating that APC mutation is required for CSC activation by oncogenic K-Ras mutation. K-Ras mutation activates CSCs, contributing to colorectal tumorigenesis and metastasis in CRC cells harboring APC mutations. Initial activation of β-catenin by APC loss and further enhancement through K-Ras mutation induces CD44, CD133, and CD166 expression.

The effect of RNAi silencing of p62 using an osmotic polysorbitol transporter on autophagy and tumorigenesis in lungs of K-rasLA1 mice

Treating cancer patients by conventional chemotherapy to achieve prolonged survival still remains complicated. Autophagy is a topic of considerable interest in recent times, as it may contribute greatly to tumor suppression. Recent studies indicate that autophagy-deficient cells accumulate high levels of p62, an ubiquitin-binding scaffold protein, involved greatly in tumorigenesis. Here, we synthesized an osmotically active polysorbitol-mediated transporter (PSMT) to downregulate p62 using an RNAi strategy and described the mechanism of how p62 silencing using PSMT/siRNA p62 system activates autophagy and contributes to tumor suppression in the lungs of K-rasLA1 mice. Downregulation of p62 by PSMT/siRNA p62 activated autophagy confirmed by the formation of autophagosomes and swelling of Golgi apparatus with a decreasing level of GM130, a cis-Golgi matrix protein. Activation of osmotic PSMT-mediated autophagy remarkably reduced the size and number of tumors by suppressing proliferative cell nuclear antigen, cluster of differentiation 31, and vascular endothelial growth factor levels. Furthermore, an increase in apoptosis was observed in the lungs of PSMT/siRNA p62-delivered K-rasLA1 mice.