CC-LR Mice Research Articles

Abstract LB359: Lung epithelial specific IL-1R promotes K-ras mutant lung cancer by inducing angiogenesis that might be medicated via myeloid tumor microenvironment

Abstract As the leading cause of cancer-related deaths worldwide, the development of targeted therapeutics and immunotherapies as a means for treating lung cancer remains crucial. Non-small cell lung cancer (NSCLC), the most common histological subtype accounting for 85%, predominantly arises from lung adenocarcinoma with driver mutations in the K-ras oncogene (KM-LUAD). Studies have shown that KM-LUAD progression partly occurs through activation of the NF-κB pathway initiating an inflammatory response and creating a pro-tumor microenvironment. Notably, the pro-inflammatory cytokine IL-1β a potent activator and product of the NF-κB pathway is elevated in the lungs and sera of KM-LUAD patients. Our preliminary studies have shown that IL-1β blockade reprograms the lung immune microenvironment to that of an anti-tumor immune phenotype in a mouse model of KM-LUAD driven by lung epithelial cell-specific expression of KRASG12D (CCSPCre/LSL-KRASG12D, CC-LR mouse), suggesting that indeed this cytokine mediates tumor-promoting inflammation in KM-LUAD pathogenesis. Yet, cell-specific mechanisms that underlie tumor-promoting roles of IL-1β signaling are still poorly understood. Thus, we sought to elucidate the role of IL-1β signaling via its ability to bind to its receptor, IL-1R, in the context of KM-LUAD by conditionally knocking out the IL-1R in KRAS-mutant lung epithelial cells in CC-LR mice. Tumor development as well as immune microenvironment in 14 and 18-week-old CC-LR mice with epithelial specific conditional knockout of IL-1R (LR/IL-1RΔ/Δ) in comparison to age- and sex-matched control CC-LR littermates were studied. Notably reduced tumor burden in LR/IL-1RΔ/Δ mice with an interesting shift toward atypical adenomatous hyperplasia (AHH) lesion formation was evident when compared to their CC-LR counterpart. Having previously shown that targeting IL-1R leads to a reduction in the myeloid cell compartment also known for the regulation and production of angiogenic markers, an immunohistochemical evaluation of the angiogenic marker ERG was performed, showing a significantly diminished expression in tissue collected from LR/IL-1RΔ/Δ mice. Further studies assessing the role of angiogenic modulation by IL-1R in the context of KM-LUAD progression and its regulation by myeloid cells are currently under investigation. Overall, these findings provide insight into cell-specific mechanisms underlying the tumor-promoting effects of IL-1β signaling and support the role of tumor cell-intrinsic factors in tumor progression via shaping the tumor microenvironment. Citation Format: Avantika Krishna, Michael J. Clowers, Bo Yuan, Vivian Ha, Katherine Larsen, Nastaran Karimi, Arnav Gaitonde, Jocelynn Colunga, Maria T. Grimaldo, Maria Jose Arredondo Sancristobal, Valentina Villalba-Sabat, Angelica Baca De Anda, Yasmina Rezai, Jyotika Sharma, Humam Kadara, Seyed Javad Moghaddam. Lung epithelial specific IL-1R promotes K-ras mutant lung cancer by inducing angiogenesis that might be medicated via myeloid tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB359.

Abstract 5326: Selective deletion of estrogen receptor β in lung epithelium suppresses K-ras mutant lung tumorigenesis

Abstract Although lung cancer is the most prolific cause of global cancer-related deaths, outcomes by sex have gained traction as a means to stratify patients and tailor therapies accordingly. In recent years, females have begun to shoulder a greater burden of lung cancer diagnoses, specifically within the lung adenocarcinoma (LUAD) subtype; conversely, mortality is worsened for males. However, other sex-dependent effects in lung cancer are harder to predict. For example, hormone replacement therapy for post-menopausal women has been shown to predict better survival in some cohorts while auguring worse outcomes in others. Similar controversy exists regarding menopausal status, gender affirming care, and castration/oophorectomy status. However, there is consensus that these differential effects relate to a patient’s sex hormone composition. Estrogen, a sex hormone abounding in females, boasts well-described pro-tumor signaling in breast cancer. Unfortunately, its role in lung cancer remains elusive. Estrogen is reported to exert inflammatory properties in the lung environment, to drive immune function, and to induce proliferation of lung cancer cells. This functional antagonism has led to questions about how these compartment-specific effects are balanced. In the lung, estrogen signaling occurs mainly via estrogen receptor beta (ERβ), a nuclear hormone receptor highly expressed in lung tissue and lung tumors. ERβ is understudied relative to its sibling ERα, a well-known oncogene in breast cancer. While it is tempting to analogize ERα oncogene function to ERβ, the literature lacks consensus on the role of ERβ in lung cancer, with some authors classifying it as an oncogene and others as a tumor suppressor. In LUAD, Kras mutations encompass a quarter of driver mutations, and interaction of Kras with ERβ is understudied. Here, we tested the effects of genetic ablation of ERβ in a murine model of Kras mutant LUAD (KM-LUAD) termed CCSPCre/LSL-KrasG12D (CC-LR). Esr2 floxed mice were crossed with CC-LR mice to create a lung epithelial-specific conditional knockout of ERβ (LR/Esr2Δ/Δ). LR/Esr2Δ/Δ mice show significant decrease in tumor burden and total lung immune infiltration, indicating reduced tumor development and tumor-promoting inflammation. Of note, this effect manifests regardless of sex. RNA and protein-level analysis further suggest that ERβ deletion alters the balance of inflammatory transcription factors, resulting in skewing from STAT3- to NF-κB-driven inflammation and a net anti-tumor response. These data suggest that ERβ offers a promising early target in nascent KM-LUAD tumors with therapeutic intervention possible regardless of sex-specific effects. Acknowledgement: supported by NIH/NCI grant awarded to Seyed Javad Moghaddam (R01 CA225977); Michael J Clowers partly supported by NIH/NCI (F31 CA261121); Annamarie L Thompson supported by CPRIT Research Training Award (RP210028). Citation Format: Michael Joseph Clowers, Annamarie L. Thompson, Carlos Ignacio Rodriguez Reyna, Shanshan Deng, Seyed Javad Moghaddam. Selective deletion of estrogen receptor β in lung epithelium suppresses K-ras mutant lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5326.

Abstract 652: Tumor cell-specific IL-1/IL-1R signaling promotes KRAS mutant lung tumorigenesis

Abstract Despite improved diagnosis and treatment strategies such as immunotherapy, lung cancer is still the leading cause of cancer-related deaths worldwide in both men and women. Lung adenocarcinoma (LUAD) with driver mutations in the KRAS oncogene is the most prevalent molecular subtype of lung cancer. KRAS mutant LUAD exhibits aggressive biology in part due to enhanced pro-tumor inflammation mediated by activation of the nuclear factor-κB (NF-κB) and, consequently, elevated expression of various cytokines. While the pro-inflammatory cytokine IL-1β is a product of the NF-κB pathway it also acts as a potent activator, further amplifying the production of protumor cytokines via a positive feedback loop. We have shown that IL-1β blockade enhances anti-tumor immune responses while inhibiting immunosuppression in a mouse model of KRAS mutant LUAD driven by lung epithelial cell-specific expression of KRASG12D (CCSPCre/LSL-KRASG12D, CC-LR mouse). Cell lineage-specific mechanisms that underlie these anti-tumor effects following inhibition of IL-1β are still poorly understood. To fill this void, we here explored the effects of targeting the IL-1R receptor in KRAS mutant lung epithelial cells in the CC-LR mouse model. We studied tumor development and host immune response in 14-week-old CC-LR mice with conditional knockout of IL-1R in KRAS mutant lung epithelial cells (LR/IL-1RΔ/Δ) in comparison to age- and sex-matched control CC-LR littermates. LR/IL-1RΔ/Δ mice displayed markedly reduced tumor multiplicity (~50%) when compared to control CC-LR mice concomitant with decreased cell proliferation and angiogenesis evidenced by attenuated immunohistochemical expression of Ki-67 and ERG. Flow cytometry analysis showed an elevated inflammatory response, most evidently seen through the significant increase in infiltrating monocytes. Interestingly, a shift in type-1 conventional dendritic cells, commonly involved in antigen-cross presentation in tumorigenesis, to type-2 conventional dendritic cells was seen in LR/IL-1RΔ/Δ mice, suggesting a different method of cross-presentation resulting from the conditional knockout of IL-1R that could potentially lead to a protective T-cell response. These results were further confirmed via gene expression analysis of respective markers. Our findings provide further support for the role of the IL-1 cytokine family in the development and progression of KRAS mutant LUAD as well as warrant further studies targeted towards understanding the mechanistic effects IL-1β has on the tumor microenvironment. Citation Format: Avantika Krishna, Michael J. Clowers, Bo Yuan, Milind Mutala, Maria Jose Arredondo Sancristobal, Jocelynn Colunga, Ryan De Maleki, Linda Phan, Humam Kadara, Seyed Javad Moghaddam. Tumor cell-specific IL-1/IL-1R signaling promotes KRAS mutant lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 652.

Abstract 649: Comparative effects of combustible cigarette versus electronic cigarette exposures on K-ras mutant lung cancer

Abstract Combustible cigarette smoking (CCS) is causally related to ~90% of all lung cancers and induces various tumor-initiating effects, some of which (e.g., inflammation) are not reversible even after smoking cessation. More recently, use of non-combustible smoking vectors, such as electronic cigarette vapors (ECV), have rapidly increased, especially among youth. Despite this emerging public health concern, the long-term impact of ECV exposure is poorly understood. Our lab sought to assess the safety of ECV and determine their influence on immune dysfunction and lung cancer progression by comparing the effects of CCS and ECV in an airway epithelial specific K-ras mutant mouse model of lung adenocarcinoma (CC-LR). Three cohorts of 6-week-old CC-LR mice were exposed to either room air, CCS, or ECV 5 days per week for 8 weeks. For CCS exposure, mice were exposed to CCS from 3R4F research cigarettes at a rate of 3 puffs/min for 2 hours each day using the SCIREQ integrated Cigarette Smoking Robot. For ECV exposure, mice were exposed to 72 mg/ml of liquid nicotine in 50% propylene glycol/vegetable glycerin solution at a rate of 3 puffs/min for 2 hours using the SCIREQ Electronic Nicotine Delivery System. While CCS led to a significant increase in tumor burden (25%), ECV showed no significant changes compared to room air-exposed control. Bronchoalveolar lavage fluid had significantly increased total lung immune cell infiltrates in both CCS (30%) and ECV (25%), particularly in macrophage and lymphocyte populations. Immunophenotyping of CCS and ECV exposed lungs displayed pronounced pro-tumor immunosuppressive phenotypes, characterized by significantly decreased CD4+ IFNγ+ and CD8+ GZMB+ cytotoxic T cells along with a significantly elevated CD4+ FOXP3+ regulatory T cells. Furthermore, the cytotoxic cytokine IFNγ was significantly reduced in lungs of CCS- (7-fold) and ECV-exposed (2-fold) mice compared to room air-exposed controls. We also found increased levels of the immunosuppressive cytokine IL-10 in lungs of CCS- and ECV-exposed mice. Previous studies have shown IL-10 to play an important role in microbiome-associated immune modulation, hence we performed a preliminary gut and lung microbiome study to assess microbiome dissimilarity in these cohorts. Taxonomic profiling via 16S rRNA gene sequencing of matched stool and lung samples showed differences in the relative abundance of several lung Proteobacteria spp. while gut Firmicutes, particularly Turicibacter and Ileibacterium, were increased by CCS and ECV. In conclusion, while both CCS and ECV increased immune suppression, ECV did not significantly promote tumorigenesis during this timeframe of intervention. Future studies probing differences in microbiome-modulated CCS and ECV immune dysfunction can help pave way for identification of new targets that foster new interception and early treatment strategies for K-ras mutant lung cancer. Citation Format: Walter V. Velasco Torrez, Maria T. Grimaldo, Michael J. Clowers, Bo Yuan, Segundo del Aguila Soto, Iman Bouchelkia, Javier Eduardo Moreno Barragan, Umesh C. Karandikar, Joseph F. Petrosino, Florencia McAllister, Humam Kadara, Kristi Louise Hoffman, Seyed Javad Moghaddam. Comparative effects of combustible cigarette versus electronic cigarette exposures on K-ras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 649.

Abstract A012: Repurposing of the macrolide antibiotic clarithromycin for the prevention of lung cancer

Abstract Lung adenocarcinoma (LUAD), particularly K-ras mutant LUAD, is a leading cause of cancer mortality. Therefore, strategies to prevent K-ras-mutant LUAD in its earliest stages in high-risk individuals (e.g., smokers) are urgently needed to reduce the public burden of this fatal disease. We and others have shown that K-ras driven tumorigenesis in lung is intimately linked to chronic inflammation and ultimately, tumor cells immune-escape. The antibiotic clarithromycin (CAM) was identified as one of the most promising candidates for repurposing with demonstrated immunomodulatory and anticancer properties. CAM is widely used and belongs to the macrolide class of antibiotics which are among the safest broad spectrum antimicrobials available. Abundant preclinical and clinical evidence exists demonstrating the in vitro and in vivo anticancer effects of CAM. It has been shown that macrolide antibiotics exert suppression of inflammation without overt immunosuppressive effects mostly through the inhibition of proinflammatory cytokines in vitro and in vivo. In these studies, we tested the lung cancer prevention efficacy of CAM using a Kras mutant lung cancer model. In the CCSPCre; LSL-Kras- G12 D (CC-LR) model, activation of the KrasG12 D mutation takes place in club cells by means of removal of the lox-stop-lox genomic sequence via expression of Cre recombinase under the control of the CCSP promoter. This model is excellent for reproducing the various premalignant to malignant progression steps in the lung. CC-LR mice of both genders were randomly enrolled to four experimental arms comparing three CAM doses: 10mg/kg/day, 50mg/kg/day, and 100mg/kg/day, to vehicle control (H2O). Treatment was administered by oral gavage, 5 times per week for 10 wks., starting at 4 wks. of age. At 14 wks. of age mice were euthanized, lung surface tumors were counted, bronchial lavage fluid, as well as lung samples, were obtained for histological, immunohistochemical (IHC) and qRT-PCR analyses. Clarithromycin treatment led to significant lung cancer prevention efficacy, as determined by lung surface tumor counts. A clear dose response with CAM was observed with a mean lung surface tumor count of 30.2 tumors per mouse for vehicle (n=12 mice), 23.5 for CAM 10mg/kg (n=8), 18.8 for CAM 50mg/kg (n=8) and 13.5 tumors per mouse for the CAM 100mg/kg treatment group (n=12, p=0.0014). A significant decrease in the incidence and multiplicity of premalignant and malignant lesions was also observed in histological analyses. For profiling of the lung immune microenvironment, we analyzed by qRT-PCR the expression of 18 genes identifying various cytokines, cell surface markers and proteins characteristic of specific activation states on the various cell subtypes in the tumor microenvironment. We found significant reduction in the expression of pro-inflammatory cytokines, IL-6, TNF, and IL-1β that are known to have a pro-tumor function in lung tumorigenesis, shown previously by us and other groups. We also found a reduction in the expression of myeloid cell-specific immunosuppressive markers, Arg1 and Fizz 1, that could be due to the reduction in the Gr1+ myeloid cell population, which was detected by means of IHC analyses on the same samples, or due to reprograming of pro-tumor M2 type macrophages toward an anti-tumor M1 phenotype. We also see a trend toward reduction of IL-17 cytokine, which we have previously shown to have an essential role in the promotion of K-ras mutant lung tumors. This was associated with an increase, although not significant, in the expression of anti-tumor Th1-specific transcription factor. However, we see an increase in the expression of PD-1, which could be due to the potential induction of an exhausted T cell phenotype. Taken together, we see a reprograming of the lung microenvironment from a tumor-promoting immunosuppressive phenotype toward an anti-tumor phenotype. This work was supported by NCI PREVENT TORFP75N91019F00131 (TORFP 2019 E-05). Citation Format: Seyed Javad Moghaddam, Tabish Hussain, Melody Zarghooni, Walter V. Velasco, Linda Phan, Michelle I. Savage, Jennifer T. Fox, Shizuko Sei, Powel H. Brown, C. Marcelo Aldaz. Repurposing of the macrolide antibiotic clarithromycin for the prevention of lung cancer [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 A012.

Abstract 2095: Selective inhibition of the STAT3 pathway suppresses K-ras mutant lung tumorigenesis

Abstract K-ras mutant lung adenocarcinoma (KM-LUAD) is a difficult-to-drug cancer subtype characterized by a chronic inflammatory tumor microenvironment (TME). Resistance to therapies, including immune checkpoint blockade (ICB), necessitates therapies that target this inflammatory TME. A major transcription factor that mediates chronic inflammation in KM-LUAD is signal transducer and activator of transcription 3 (STAT3). Inhibiting STAT3 may attenuate pro-tumor inflammation. Moreover, STAT3 modulates PD-L1 transcription, so STAT3 inhibitors in conjunction with ICB may increase ICB response rates. Here, we tested the anti-tumor ability of TTI-101, a selective STAT3 inhibitor, in a STAT3 addicted lung cancer cell line (MDA-F471) and in a transgenic mouse model of KM-LUAD (CCSPCre/LSL-KrasG12D, CC-LR). For in vivo experiments, CC-LR mice were treated daily with 50 mg/kg TTI-101 by oral gavage from 10 to 14 weeks of age to model a preventative regimen or from 14 to 18 weeks of age to survey the treatment effect on established tumors. TTI-101 was compared to anti-PD-1 ICB, with 200 μg injected intraperitoneally 3 times per week. In MDA-F471 cells, TTI-101 treatment decreased cell viability, with an IC50 of ~ 20 μM. In mice treated from 10 to 14 weeks of age, TTI-101 therapy significantly reduced the tumor burden compared to ICB. TTI-101 also reduced the number of proliferating cells within tumors. Mice in the 14-18-week group displayed similar trends, but these experiments are ongoing, as are combination TTI-101 and ICB treatment regimens. Our studies show that TTI-101 can reduce K-ras driven tumor cell proliferation in vitro and in vivo, suggesting STAT3 inhibition as an alternative preventive and therapeutic modality for KM-LUAD. The ongoing combination treatments and the 14-18-week cohorts will elucidate the timing of treatments as well as reveal if by targeting the inflammatory TME we are able to improve response to ICB. Funded by: R01 grant from NIH/NCI (R01CA225977) Citation Format: Michael J. Clowers, Cody Chou, Bo Yuan, Walter V. Velasco, Melody Zarghooni, Stephen Peng, T Kris Eckols, Humam Kadara, David J. Tweardy, Seyed Javad Moghaddam. Selective inhibition of the STAT3 pathway suppresses K-ras mutant lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2095.

Targeting IL-1β as an immunopreventive and therapeutic modality for K-ras-mutant lung cancer.

K-ras–mutant lung adenocarcinoma (KM-LUAD) is associated with abysmal prognosis and is tightly linked to tumor-promoting inflammation. A human mAb, canakinumab, targeting the proinflammatory cytokine IL-1β, significantly decreased the risk of lung cancer in the Canakinumab Anti-inflammatory Thrombosis Outcomes Study. Interestingly, we found high levels of IL-1β in the lungs of mice with K-rasG12D–mutant tumors (CC-LR mice). Here, we blocked IL-1β using an anti–IL-1β mAb in cohorts of 6- or 14-week-old CC-LR mice to explore its preventive and therapeutic effect, respectively. IL-1β blockade significantly reduced lung tumor burden, which was associated with reprogramming of the lung microenvironment toward an antitumor phenotype characterized by increased infiltration of cytotoxic CD8+ T cells (with high IFN-γ and granzyme B expression but low programmed cell death 1 [PD-1] expression) while suppressing neutrophils and polymorphonuclear (PMN) myeloid-derived suppressor cells. When querying the Cancer Genome Atlas data set, we found positive correlations between IL1B expression and infiltration of immunosuppressive PMNs and expression of their chemoattractant, CXCL1, and PDCD1 expressions in patients with KM-LUAD. Our data provide evidence that IL-1β blockade may be a preventive strategy for high-risk individuals and an alternative therapeutic approach in combination with currently available treatments for KM-LUAD.

COPD-Type lung inflammation promotes K-ras mutant lung cancer through epithelial HIF-1α mediated tumor angiogenesis and proliferation.

Chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, is an independent risk factor for lung cancer. Lung tissues obtained from human smokers with COPD and lung cancer demonstrate hypoxia and up-regulated hypoxia inducible factor-1 (HIF-1). HIF-1 activation is the central mechanism for controlling the cellular response to hypoxia during inflammation and tumor development. These facts suggest a link between COPD-related airway inflammation, HIF-1, and lung cancer. We have previously established a mouse model of COPD-like airway inflammation that promotes lung cancer in a K-ras mutant mouse model (CC-LR). Here we show that tumors in the CC-LR model have significantly elevated levels of HIF-1α and HIF-1 activity. To determine the tumor-promoting functions of HIF-1 in CC-LR mice, the gene Hif1a which encodes HIF-1α and is required for HIF-1 activity, was disrupted in the lung epithelium of CC-LR animals. Airway epithelial specific HIF-1α deficient mice demonstrated significant reductions in lung surface tumor numbers, tumor angiogenesis, and tumor cell proliferation in the absence or presence of COPD-like airway inflammation. In addition, when CC-LR mice were bred with transgenic animals that overexpress a constitutively active mutant form of human HIF-1α in the airway epithelium, both COPD- and adenocarcinoma-like phenotypes were observed. HIF-1α overexpressing CC-LR mice had significant emphysema, and they also showed potentiated tumorigenesis, angiogenesis, and cell proliferation accompanied by an invasive metastatic phenotype. Our gain and loss of function studies support a key role for HIF-1α in the promotion of lung cancer by COPD-like inflammation.

IL22 Promotes Kras-Mutant Lung Cancer by Induction of a Protumor Immune Response and Protection of Stemness Properties.

Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras-induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4+ T-helper cell response. IL22 is an effector molecule secreted by CD4+ and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on nonhematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS-mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of protumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8+ T-cell responses, while suppressing the protumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC+CCSP+ stem cells. Thus, we conclude that IL22 promotes Kras-mutant lung tumorigenesis by driving a protumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells. Cancer Immunol Res; 6(7); 788-97. ©2018 AACR.

Abstract 2679: A promoting role for the epithelial MyD88/IRAK4/NF-kB signaling in K-ras mutant lung tumorigenesis

Abstract K-ras mutation is the most common oncogenic alterations associated with lung cancer development. Unfortunately, all attempts to develop therapies directly targeting K-ras have been failed thus far, clearly stating the need for new strategies targeting downstream effectors and/or cooperating pathways of K-ras to overcome lung cancer displaying such a molecular profile. Using a conditional K-ras mutated lung cancer mouse model, CC-LR (CCSPcre/LSL-K-rasG12D) we previously showed that K-ras mutated lung tumorigenesis is associated with lung inflammation due to activation of NF-κB pathway and increased expression of its downstream targets in the lung. Here we have shown that lack of NF-κB activity in the airway epithelium by selectively targeting IKKβ, which is required for NF-kB activation, significantly reduces lung tumor burden (3.4-fold) and changes the inflammatory cells and mediators in the bronchoalveolar lavage fluid (BALF) of CC-LR mice. Immunohistochemically staining of lung tissues with specific markers, Ki-67 and CD-31, demonstrated significantly lower tumor cell proliferation and angiogenesis in CC-LR mice with lack of epithelial NF-kB activity. To further dissect the role of NF-κB pathway in this process, CC-LR mice were crossed with MyD88f/f mice to develop a mouse with lack of MyD88 (an adaptor protein upstream to IKKβ) in the airway epithelial cells (CC-LR/MyD88Δ/Δ mice). As we had hypothesized, the resulting tumor numbers in the lungs were significantly lower (1.9-fold) in CC-LR mice with lack of MyD88 in the airway epithelial cells compared to control CC-LR mice. Tumor reduction in CC-LR-MyD88Δ/Δ mice was also associated with decreased tumor cell proliferation and angiogenesis compared to control CC-LR mice. Surprisingly, unlike to lack of epithelial NF-kB activity, absence of MyD88 in the airway epithelium did not change the BALF inflammatory cell component of CC-LR mice. We then targeted another upstream signaling molecule to NF-kB, IRAK4, which is down stream of MyD88 by crossing CC-LR mice to IRAK4 knock out mice. Similar to lack of MyD88, we found a significant reduction in lung tumor number (1.8-fold) with no changes in BALF inflammatory cell component in CC-LR mice with lack of IRAK4 compared to the control CC-LR mice. Taken these together, we conclude that there is an essential role for MyD88/IRAK4/NF-kB pathway activation in promotion of K-ras mutant lung cancer. Citation Format: Susana Castro, Soudabeh Daliri, Maria Miguelina De La Garza, Amber M. Cumpian, Misha Umer, Diana Del Bosque, Sabah Akbani, Scott E. Evans, Seyed Javad Moghaddam. A promoting role for the epithelial MyD88/IRAK4/NF-kB signaling in K-ras mutant lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2679. doi:10.1158/1538-7445.AM2017-2679

Abstract 3714: Muc5ac plays an essential role in promotion of k-ras mutant lung cancer

Abstract Worldwide, lung cancer is the leading cause of cancer mortality, and cigarette smoking (CS) is its principal cause. However, several studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer (3 to 10-fold) compared to smokers with comparable cigarette exposure but without COPD. Importantly, among smokers with COPD, even following withdrawal of cigarette smoke, inflammation persists and lung function continues to deteriorate as does the increased risk of lung cancer. These facts suggest a strong link between COPD-related lung inflammation and lung cancer, however, the precise mechanistic link is not known. Mucociliary dysfunction and mucin hyperproduction are important features of COPD with inflammation being the major trigger. Interestingly, lung cancer with mucin overexpression has higher malignancy potential and poor prognosis, which around 76% having mutations in K-ras oncogene, the most frequent oncogenic mutation in lung adenocarcinoma. Taken these together, we hypothesized that mucins contribute to promotion of K-ras mutant lung cancer by inflammation. Here we first investigated whether Muc5ac, predominant airway mucin that plays a primary role in inflammatory lung diseases, is predictive of clinical outcome in KRAS-mutant human lung adenocarcinomas. We determined Muc5ac mRNA expression by array analysis of 150 lung adenocarcinomas from patients that did not received neoadjuvant therapy and we found that, Muc5ac mRNA level was a significant predictor of poor disease-free survival in KRAS-mutant lung adenocarcinomas. We have further found increased mucin and high expression of Muc5ac in lung tumor tissues of the mice with airway specific expression of a mutant form of K-ras (CC-LR mice). Then, we crossed previously developed Muc5ac knockout (KO) mice to CC-LR mice in order to develop a K-ras mutant lung cancer mouse model with lack of Muc5ac (CC-LR/Muc5ac KO mice). This resulted in a significant tumor reduction by ~54% (2.2-fold) in lung of CC-LR/Muc5ac KO mice compared to age and sex matched control CC-LR mice. Lung inflammation was evaluated by analysis of bronchoalveolar lavage fluid and revealed a significant reduction (3-fold) in number of macrophages, and levels of IL-6 and IL-17 in CC-LR/Muc5ac KO mice compared to CC-LR control mice. Immunohistopathological analysis of lung sections confirmed lower inflammation, decreased tumor number and size, less adenomatous lesions, and reduced tumor cell proliferation and angiogenesis in CC-LR mice with lack of Muc5ac compared to control CC-LR mice. Our experimental results suggest that Muc5ac has an essential role in promotion of K-ras mutant lung cancer through autocrine cell intrinsic and paracrine immune cell mediated mechanisms. Citation Format: Misha Umer, Amber M. Cumpian, Nasim Khosravi, Zoulikha Azzegagh, Maede Mohebnasab, Mauricio S. Caetano, Edwin J. Ostrin, Ignacio I. Wistuba, Burton Dickey, Humam Kadara, Christopher M. Evans, Seyed J. Moghaddam. Muc5ac plays an essential role in promotion of k-ras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3714. doi:10.1158/1538-7445.AM2017-3714

Abstract 2687: Toll like receptors mediated inflammatory signals mediate promotion of K-ras mutant lung cancer by chronic obstructive pulmonary disease

Abstract Lung cancer is the leading cause of cancer death worldwide, and cigarette smoking is its main cause. However, smoker with chronic obstructive pulmonary disorder (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer (3 to 10 fold) compared to smokers without COPD. Importantly, lung inflammation persists and lung function continues to deteriorate as does the increased risk of lung cancer even after cessation of cigarette smoking among former smokers. These facts suggest a strong link between COPD-related airway inflammation and lung cancer promotion, however, the precise mechanistic link is not known. We have previously developed a COPD-like mouse model of airway inflammation through repeated aerosol challenge to a lysate of nontypeable (i.e., unencapsulated) Haemophilus influenzae (NTHi). NTHi is the most common colonizing bacteria in the lower respiratory tract of patients with COPD and could be a potential cause of perpetuating and promoting airway injury and inflammation in these patients. We then showed that this type of airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR), which was associated with the activation of MyD88/NF-κB pathway and increased expression of its downstream targets in the lung. We have further shown that lack of NF-kB or MyD88 (an adaptor protein upstream to NF-κB) in the airway epithelium of CC-LR mice changes the bronchoalveolar lavage fluid cellular component of CC-LR mice and inhibits the promoting effect of COPD-like airway inflammation on lung tumorigensis. Upstream to MyD88 and NF-κB and downstream to bacterial stimuli is the toll-like receptors family (TLRs), which play critical role in the innate immune response. Among TLRs, TLR-2, TLR4, and TLR9 play critical roles in mediating inflammatory responses in lung and are required for primary epithelial response to inflammatory stimuli and activation of MyD88/NF-kB pathway in the airway epithelium. Accordingly, we further hypothesized that TLR-2, 4, and 9 mediate promoting effect of inflammtion on lung tumorigenesis in an MyD88/NF-kB dependent manner. Therefore, CC-LR mice were separately crossed to TLR-2, TLR-4 and TLR-9 knock out mice in order to test this hypothesis. We found that genetic ablation of these TLRs in CC-LR mice, causes significant reduction in lung surface tumor numbers compared to age and sex matched control CC-LR mice in the presence of COPD-like airway inflammation. This tumor reduction was associated with significant reduction in the numbers of inflammatory cells in bronchoalveolar lavage fluid of mice with lack of these TLRs. Taken these together, we conclude that promoting effect of COPD on lung cancer is mediated through TLR2, 4, 9-mediated activation of epithelial MyD88/NF-kB pathway. Citation Format: Nasim Khosravi, Nelly Torres-Garza, Soudabeh Daliri, Maria Miguelina De La Garza, Amber Cumpian, Evelyn Beltran, Misha Umer, Diana Del Bosque, Saba Akbani, Scott Evans, Seyed Javad Moghaddam. Toll like receptors mediated inflammatory signals mediate promotion of K-ras mutant lung cancer by chronic obstructive pulmonary disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2687. doi:10.1158/1538-7445.AM2017-2687

Abstract 3974: Gender specific function of epithelial IL-6-STAT3 pathway in K-ras mutant lung cancer

Abstract Activating mutations of K-ras are the most common oncogenic alterations found in lung cancer, and are heavily associated with tobacco exposure and poor prognosis. Using a conditional lung cancer mouse model (CC-LR), we showed that K-ras mutant lung tumors have inflammatory characteristics with activation of NF-kB pathway, release of IL-6 and activation of its downstream target, STAT3. We further demonstrated that IL-6 blockade results in significant tumor reduction as well as decreased pSTAT3 expression, tumor cell proliferation, angiogenesis and a reduction in tumor-associated immunosuppressive myeloid populations concomitant with induction of an anti-tumor phenotype. These indicate essential autocrine and paracrine roles for IL-6/STAT3 pathway in promotion of K-ras mutant lung cancer, largely via perpetuating inflammation introducing it as a vulnerability factor for this type of tumors. Here we generated a lung epithelial specific K-ras mutant/STAT3 conditional knockout mouse (LR/STAT3Δ/Δ) to further study the role of epithelial STAT3 activity in K-ras mutant lung cancer. We found that in female mice, lack of epithelial STAT3 inhibited lung cancer, and significantly decreased the lung inflammatory cell population, particularly macrophages, whereas in male mice, STAT3-deficiency surprisingly promoted lung cancer and significantly increased lung neutrophil population. To understand genome-wide mechanisms underlying this intriguing gender disparity, we performed RNA-sequencing of whole lungs from male and female LR/STAT3Δ/Δ and control CC-LR mice. Using a mixed effects model, we found 339 transcripts that were significantly modulated differently between LR/STAT3Δ/Δ and control CC-LR mice in males relative to females. Pathways and gene network analyses of the transcripts demonstrated that lungs of male LR/STAT3Δ/Δ mice exhibited markedly reduced Th1 immune T-cell signatures. We corroborated these findings and found significant phenotypic changes in the lung tumor microenvironment (TME) with males exhibiting increased pro-tumor inflammatory markers. Markedly, we found opposing IL-6 expression patterns with LR/STAT3Δ/Δ females expressing low IL-6 while LR/STAT3Δ/Δ males expressed high levels of IL-6. We then blocked IL-6 in male LR/STAT3Δ/Δ mice, which resulted in a significantly reduced lung tumorigenesis, and reformatted lung TME towards an anti-tumor phenotype with a Th1/CD8+ T cell signature. By contrast, estrogen receptor (ER) signaling blockade by tamoxifen in female LR/STAT3Δ/Δ mice promoted lung cancer, and reprogrammed lung TME toward a pro-tumor phenotype with an increase in Th17/Treg cell signature. Taken together, we conclude that epithelial STAT3 signaling has an important gender-specific role with autocrine and paracrine effects in K-ras induced lung tumorigenesis, which might be mediated by the interplay between ER signaling, and NF-κB mediated cytokine network. Citation Format: Mauricio S. Caetano, Hieu Van, Emmanuel Bugarin, Amber Cumpian, Christina L. McDowell, Huiyuan Zhang, Scott E. Evans, Stephanie Watowich, Humam Kadara, Seyed J. Moghaddam. Gender specific function of epithelial IL-6-STAT3 pathway in K-ras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3974. doi:10.1158/1538-7445.AM2017-3974

Tumor necrosis factor links chronic obstructive pulmonary disease and K-ras mutant lung cancer through induction of an immunosuppressive pro-tumor microenvironment

ABSTRACTTumor necrosis factor (TNF) is known as an important regulator of tumor microenvironment and inflammation. TNF levels are markedly elevated in the bronchoalveolar lavage fluid (BALF) of patients with chronic obstructive pulmonary disease (COPD), which is an independent risk factor for lung cancer. We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model (CC-LR mouse). This was associated with a significant increase of neutrophils in BALF, accompanied by a marked increase in TNF level, suggesting a link between COPD, TNF, and lung cancer promotion. Therefore, we first overexpressed TNF in the airway epithelium of CC-LR mice, which promoted lung cancer by ∼2-fold. This was associated with increased numbers of Ki67 and CD31 positive cells in lung tumors of CC-LR/TNF-Tg mice. We also found a robust increase in NF-κB activation, and numbers of neutrophils and myeloid-derived suppressor cells (MDSCs) in lung. Accordingly, we depleted MDSCs in CC-LR/TNF-Tg mice, which lead to significant tumor suppression emphasizing on the role of TNF-induced MDSCs in K-ras induced lung tumorigenesis. Finally, we targeted TNF expression by crossing CC-LR mice with TNF knock-out mice (CC-LR/TNF-KO), which resulted in a significant decrease in lung tumor burden in the absence or presence of COPD-like airway inflammation. Interestingly, there were less MDSCs and lower Ki67 and CD31 expression in the lung of the CC-LR/TNF-KO mice. We conclude that TNF links COPD to lung cancer promotion by induction of an immunosuppressive MDSC response, and subsequent amplification of proliferation and angiogenesis in tumors.

Abstract 4398: Impact of Interleukin-22 on K-ras mutant lung tumor microenvironment and stemness properties

Abstract Oncogenic K-ras mutations found in ∼ 30% of all non-small cell lung cancers are associated with chemoresistance and poor prognosis. Using a K-ras induced lung cancer mouse model, CC-LR, we previously showed that K-ras mutant lung tumors have intrinsic inflammatory characteristics with activation of NF-kB pathway, release of inflammatory cytokines IL-6, and activation of the IL-6 responsive transcription factor STAT3. We have further shown that IL-6/STAT3 pathway, and IL-17 producing CD4 helper T cells (Th17 cells) through their main cytokine, IL-17A, play critical roles in promotion of lung cancer in this model. IL-22 is another effecter molecule secreted by Th17 cells which is highly expressed in our K-ras mutant mouse model. IL-22 is a unique cytokine, which seems to act exclusively on nonhematopoietic cells, with basal IL-22R expression in the epithelial cells and fibroblast, and mostly signals through STAT3 pathway. Here we found that genetic ablation of IL-22 in CC-LR mice (CC-LR/IL22-KO mice), causes significant reduction in lung surface tumor numbers by ∼54% (2.1-fold). Histopathological analysis of lung sections confirmed a reduction in number and size of tumors in CC-LR/IL22-KO mice, which was associated with significantly lower tumor cell proliferation, angiogenesis and STAT3 activation. IL-22 ablation also reduced the numbers of inflammatory cells in bronchoalveolar lavage fluid, and decreased the expression of pro-tumor inflammatory cytokines such as IL-6, IL-17 and TNFα. This was associated with increased expression of anti-tumor Th1 cells -specific transcription factor (Tbet) and their activation markers, IFNγ, and GZB, and decreased expression of pro-tumor Th17- (RORγ) and T regulatory (FOXP3+) specific transcription factors. Recent studies have shown an association between IL-22 and stem-cell like properties in colon cancer. In lung cancer, cell populations expressing NANOG, SOX2, Oct4 and/or aldehyde dehydrogenase activity are enriched with stemness properties. Interestingly, in CC-LR/IL22-KO mice we found significant reduction in expression of these stemness genes. Thus, we conclude that IL-22 promotes K-ras mutant lung tumorigenesis by inducing a pro-tumor inflammatory microenvironment with proliferative and angiogenic properties as well as protecting stemness characteristic in epithelial/tumor cells. Therefore, we propose pharmacological targeting of IL-22 as a potential therapeutic strategy in combination with conventional cytotoxic therapy, immune check point blockade, or other targeted therapies (e.g. MEK inhibition) for lung cancer patients with K-ras mutation. Citation Format: Nasim Khosravi, Amber M. Cumpian, Soudabeh Daliri, Cynthia De La Garza, Mauricio S. Caetano, Seyed Javad Moghaddam. Impact of Interleukin-22 on K-ras mutant lung tumor microenvironment and stemness properties. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4398.

Abstract 756: Myo-inositol modulates the tumor microenvironment in a mouse model of lung cancer chemoprevention

Abstract Background: Myo-inositol is a nutritional supplement that has been investigated as a chemopreventive agent for lung cancer. We have investigated the effect of myo-inositol in a CC-LR (CCSPCre/LSL-K-rasG12D) mouse lung cancer model. Blood, broncho-alveolar lavage fluid (BALF), and lungs were collected from CC-LR and LR littermate control mice at 14 weeks of age, when mice had a large number of premalignant lesions with only small foci of adenocarcinoma. Methods: Lesion burden and stage was quantified using macroscopically evident lung surface tumor counts as well as histology from paraffin sections. Serial paraffin sections were also used for immunohistochemistry. Plasma was analyzed using high-sensitivity liquid-chromatography coupled tandem mass spectrometry. Cytokine and chemokine concentrations in BALF were quantitated using multiplex immunobead assays. Results: Myo-inositol treated mice (n = 20) had fewer tumors (p < 0.0001) than mice raised on control diet (n = 23). Additionally, lesions were shifted towards lower grade, from adenomas and adenomatous alveolar hyperplasia to bronchial hyperplasia. Several host factors including cytokines and proteins produced by myeloid derived suppressor cells and macrophages were significantly impacted by myo-inositol treatment. In addition to changes in metabolic inflammation derived factors, proteins which are associated with both chronic inflammation and lung tumorigenesis were altered as well. Conclusions: Myo-inositol treatment both reduced tumor burden and lesion grade in a KRAS model of lung cancer. Our findings of a substantial impact of myo-inositol treatment on the host response suggests novel mechanisms for its chemopreventive activitiy. Citation Format: Nese Unver, Kirubel T. Zeleke, Ximing Tang, Samantha Holt, Seyed Javad M. Moghaddam, Hong Wang, Ignacio I. Wistuba, Samir M. Hanash, Edwin J. Ostrin. Myo-inositol modulates the tumor microenvironment in a mouse model of lung cancer chemoprevention. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 756.

Abstract 413: Targeting IL-6 as a preventive and therapeutic strategy for K-ras mutant lung cancer

Abstract Worldwide, lung cancer (LC) leads in cancer mortality and cigarette smoking (CS) is its principal cause. Activating mutations of K-ras are one of the most common alterations in LC and are associated with CS exposure. However, several studies have shown that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have a higher risk of developing LC when compared to smokers without COPD. These facts suggest a strong link between COPD-type inflammatory lung microenvironment and K-ras mutant lung cancer. Unfortunately, attempts to directly target K-ras have thus far failed, clearly stating the need for new approaches to bring clinical benefits to patients with this undrugabble profile. Using a conditional K-ras induced lung cancer mouse model (CC-LR) we showed that K-ras mutant lung tumors have intrinsic inflammatory characteristics, and a bacterial lysate-induced COPD-like airway inflammation promotes lung cancer in this model. This was associated with release of the cytokine IL-6 and activation of the IL-6-responsive transcription factor STAT3. We showed that genetic ablation of IL-6 in this model results in a significant tumor reduction indicating an essential role for IL-6/STAT3 pathway in lung cancer promotion and introducing IL-6 as a druggable target for K-ras mutant lung tumor. Therefore, we blocked IL-6 using a monoclonal anti-IL-6 immunotherapy in our K-ras mutant model. Six week old CC-LR mice were injected intraperitoneally with 20 mg/kg dose of an anti-IL-6 monoclonal IgG1 antibody twice a week for eight weeks, while been exposed to the aerosolized bacterial lysate once weekly for 8 weeks. Anti-IL-6 therapy not only inhibited intrinsic lung cancer development by ∼78%, but also inhibited the tumor promoting effect of the COPD-like airway inflammation. It also decreased the expression of P-STAT3, proliferation marker, Ki67, and pro-angiogenic factors: VEGF, MMP-9 and CD31 in lung tissue. qPCR analysis of lung tissues from anti-IL-6 treated mice showed a decrease in Arginase1, FIZZ1, FOXP3, and IL-17 expression, with increased expression of Th1 markers, IFNγ and TBx21. Flow cytometry analysis of total lung inflammatory cells in CC-LR mouse showed a significant increase in the population of tumor associated macrophages (TAMs) during tumor progression from age 6 to 14 weeks, and COPD-like inflammation induced an increase in myeloid derived suppressor cell (MDSCs) population. Interestingly, we found a significant decrease in TAMs and MDSCs population after anti-IL-6 treatment. Taken together, we conclude that IL-6 blockade not only has cell intrinsic effect in K-ras mutant tumors but also edit the lung microenvironment toward an anti-tumor phenotype. Therefore, we propose pharmacological targeting of IL-6 alone or in combination with conventional cytotoxic therapy, or other targeted therapies for prevention and treatment of lung cancer patients with K-ras mutation. Citation Format: Mauricio S. Caetano, Amber M. Cumpian, Lei Gong, Seon H. Chang, Huiyuan Zhang, Humam N. Kadara, Cinthya Sternberg, Carlos G. Ferreira, Stephanie S. Watowich, Seyed J. Moghaddam. Targeting IL-6 as a preventive and therapeutic strategy for K-ras mutant lung cancer. [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 413. doi:10.1158/1538-7445.AM2015-413

Abstract B43: Targeting tumor microenvironment for treatment of K-ras mutant lung cancer

Abstract Activating mutations of K-ras are one of the most common molecular alterations associated with lung cancer development. Several attempts to develop therapies directly targeting K-ras have been failed thus far, clearly state the need for new strategies to bring clinical benefits to patients displaying such a molecular profile. Using a conditional K-ras mutant lung cancer mouse model (CC-LR) we previously showed that K-ras mutated lung tumors have intrinsic inflammatory characteristics associated with NF-κB pathway activation. Therefore, CC-LR mice were crossed with IKK-β f/f mice to develop a K-ras mutant mouse with lack of NF-κB activity in airway epithelium. This changed the bronchoalveolar lavage fluid (BALF) cellular component of the CC-LR mice (mostly by reduction of macrophages), and resulted in a ∼70% (3.4-fold) reduction in lung surface tumor number. Lung of CC-LR mice also shows high protein level of IL-6 (downstream to NF-κB activation) and increased STAT3 gene expression (downstream signal to IL-6) plus infiltration of myeloid and Th17 cells. Accordingly, we genetically ablated IL-6 in the CC-LR mice. This significantly inhibited lung cancer development (1.7-fold, 41%) suggesting a role for IL-6 mediated signaling in tumor promotion probably through STAT3 pathway activation. IL-6 and STAT3 are required for differentiation of Th17 cells from naïve T cells. Th17 cells mostly produce IL-17A that binds to the IL-17 receptor (IL-17R). IL-17R signaling is required for lung CXC chemokine expression and myeloid cell recruitment. Furthermore, IL-17A induces more production of IL-6 by epithelial cells. So, CC-LR mice were crossed with IL-17A KO mice. Lack of IL-17A in CC-LR mice resulted in a ∼70% (3.4-fold) tumor reduction. It also decreased the expression of pro-inflammatory mediators and reduced recruitment of myeloid cells. These reults suggest that K-ras initiated airway inflammation through NF-κB mediated activation of the IL-6/STAT3/Th17 signaling promotes lung tumorigenesis, and introduce IL-6 as a potential druggable target for lung cancer patients who harbor K-ras mutations. This prompted us to block IL-6 pathway using a monoclonal anti-IL-6 immunotherapy in our K-ras mutant model. Six week old CC-LR mice were injected intraperitoneally with 20 mg/kg dose of an anti-IL-6 monoclonal IgG1 antibody, twice a week, for a period of 8 weeks. Flowcytometry and qPCR analysis of total lung in non-treated CC-LR mice showed 2-fold increase in the population of tumor associated macrophage (TAM) and Arginase-1 mRNA expression (3 fold) during tumor progression from age 6 weeks to 14 weeks suggesting that epithelial signal initiated by K-ras mutation dictates an inflammatory type 2 pro-tumor fate for lung microenvironment. We further found that anti-IL-6 treatment suppressed tumor development by ∼78% (4.6-fold) in CC-LR mice with a significant reduction in the expression levels of Ki-67, VEGF, MMP9, CD31 and phospho-STAT3 in lung tissue. It also reduced TGFβ and KC protein levels in BALF with a significant decrease in Arginase1 mRNA expression (3-fold) and TAM population (5-fold) in total lung homogenate. We conclude that K-ras mutation drives an immunosuppressive pro-tumor response and blocking IL-6 shifts this to an anti-tumor lung microenvironment. Therefore we propose pharmacological targeting of IL-6 as a potential therapeutic strategy alone or in combination with conventional cytotoxic therapy for lung cancer patients with K-ras mutation. Funded by: American Cancer Society, RSG-11-115-01-CNE, and American Lung Association/LUNGevity Foundation, LCD-114696-N Citation Format: Mauricio da Silva Caetano, Seon Hee Chang, Amber M. Cumpian, Soudabeh Daliri, Maria Miguelina De La Garza, Cesar E. Ochoa, Chen Dong, Seyed Javad Moghaddam. Targeting tumor microenvironment for treatment of K-ras mutant lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B43. doi: 10.1158/1557-3125.RASONC14-B43

Abstract A13: Pharmacologic targeting of IL-6 suppresses tumor progression in a non-small cell lung cancer mouse model with K-ras mutation through re-educating the tumor microenvironment.

Abstract Activating mutations of K-ras are one of the most common molecular alterations associated with lung cancer development. Several attempts to develop therapies directly targeting K-ras have failed thus far, clearly state the need for new strategies to bring clinical benefits to patients displaying such molecular profile. Using a conditional K-ras mutated lung cancer mouse model (CC-LR) we previously showed that K-ras mutated lung tumors have intrinsic inflammatory characteristics and we also demonstrated that chronic obstructive pulmonary disease (COPD)-like airway inflammation, an independent risk factor for lung cancer, promotes lung tumor in this model. CC-LR mice shows high protein level of interleukin 6 (IL-6) and increased STAT3 (downstream signal to IL6) gene expression, and we have further shown that genetic ablation of IL-6 in CC-LR mice results in significant tumor reduction. High levels of IL-6 and up-regulation of STAT3 and its target genes have been observed in the lungs of smokers with COPD, and in 50% of human lung adenocarcinomas. These indicate an essential role for IL-6 in lung cancer promotion, and introduce IL-6 as a potential druggable target for lung cancer patients who harbor K-ras mutations. This prompted us to block IL-6 pathway using a monoclonal anti-IL-6 immunotherapy in our K-ras mutated lung cancer mouse model. Six week old CC-LR mice were injected intraperitoneally (I.P.) with 20 mg/kg dose of an anti-IL-6 monoclonal IgG1 antibody, twice a week, for a period of eight weeks. Flowcytometry analysis of total lung inflammatory cell population in non-treated CC-LR mice showed 2-fold increase in the population of tumor associated macrophage (TAM) (CD45+/CD11b+/GR1+/F4/80+/MCSFR+) during tumor progression from age 6 weeks to 14 weeks. qPCR analysis of total lung RNA from non-treated CC-LR mice showed that there is an increase in the level of Arginase-1 (3 fold) with no change in the level of inducible Nitric Oxide Synthetize (NOS2). This suggests that epithelial signal initiated by K-ras mutation may dictate the fate of lung microenvironment by creating an inflammatory pro-tumor type 2 response. Then we found that anti-IL-6 treatment suppresses tumor progression by ~78% (4.6-fold) in CC-LR mice which is associated with a significant reduction in the quantity of inflammatory cells in bronchoalveolar lavage fluid (BALF). It also decreased the expression of proliferation marker: Ki-67, and pro angiogenic factors: VEGF, MMP9 and CD31 and phospho-STAT3 in lung tissue. ELISA analysis of BALF from CC-LR mice also showed that anti-IL-6 treatment reduces TGFβ and KC protein levels with no change in level of IL-17. qPCR and flowcytometry analysis of total lung homogenate of CC-LR mice further showed that IL-6 blockade induces a significant decrease in Arginase1 expression (3-fold) and TAM population (5-fold). Taking together here we showed that K-ras mutated lung tumor drives an immunosuppressive pro-tumor microenvironment, with an M2 TAM polarization and blocking the suppressive actions of IL-6 shifts this to an anti-tumor lung microenvironment. Therefore we propose pharmacological targeting of IL-6 as a potential therapeutic strategy alone or in combination with conventional cytotoxic therapy for lung cancer patients with K-ras mutation. Funded by: American Cancer Society, RSG-11-115-01-CNE, and American Lung Association/LUNGevity Foundation, LCD-114696-N Citation Format: Mauricio S. Caetano, Amber M. Cumpian, Lei Gong, Cesar E. Ochoa, Seon H. Chang, Huiyuan Zhang, Stephanie Watowich, Cinthya Sternberg, Carlos G. Ferreira, Seyed J. Moghaddam. Pharmacologic targeting of IL-6 suppresses tumor progression in a non-small cell lung cancer mouse model with K-ras mutation through re-educating the tumor microenvironment. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A13.

Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase.

BackgroundTumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. Neutrophils are among the first cells to arrive at sites of inflammation, and the increased number of tumor-associated neutrophils is linked to poorer outcome in patients with lung cancer.ResultsWe have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with severe lung neutrophilic influx due to the increased level of neutrophil chemoattractant, KC. To further study the role of neutrophils in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody. This resulted in a significant reduction in lung tumor number. We further selectively inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2. Similarly, this resulted in suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant tumor reduction. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. We crossed the CC-LR mice with NE knock-out mice, and found that lack of NE significantly inhibits lung cancer development. These were associated with significant reduction in tumor cell proliferation and angiogenesis.ConclusionWe conclude that lung cancer promotion by inflammation is partly mediated by activation of the IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trials using the IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer.