Amber M. Cumpian

T helper 17 cells play a critical pathogenic role in lung cancer

Significance One of the challenging problems in lung cancer is to understand how inflammation pathways influence on the development of lung cancer and to identify early immune mediators. In this study, we functionally dissect the critical events occurring at the interface between endogenously arising lung tumors and the host immune system that determine tumor progression. Our findings greatly advance our knowledge on the function of T helper 17 cells in lung cancer and help understand the mechanisms of inflammatory mediators that promote lung cancer. Lung cancer development is associated with extensive pulmonary inflammation. In addition, the linkage between chronic obstructive pulmonary disease (COPD) and lung cancer has been demonstrated in population-based studies. IL-17–producing CD4 helper T cells (Th17 cells) play a critical role in promoting chronic tissue inflammation. Although Th17 cells are found in human COPD and lung cancer, their role is not understood. We have thus used a mouse model of lung cancer, in which an oncogenic form of K-ras (K-rasG12D), frequently found in human lung cancer, is restrictedly expressed in lung epithelial cells [via Clara cell secretory protein (CCSPcre)]. In this model, Th17 and Treg but not Th1 cells were found enriched at the tumor tissues. When CCSPcre/K-rasG12D mice were weekly challenged with a lysate of nontypeable Haemophilus influenza (NTHi), which induces COPD-type inflammation and accelerates the tumor growth, they showed greatly enhanced Th17 cell infiltration in the lung tissues. Lack of IL-17, but not IL-17F, resulted in a significant reduction in lung tumor numbers in CCSPcre/K-rasG12D mice and also those treated with NTHi. Absence of IL-17 not only resulted in reduction of tumor cell proliferation and angiogenesis, but also decreased the expression of proinflammatory mediators and reduced recruitment of myeloid cells. Depletion of Gr-1+CD11b+ myeloid cells in CCSPcre/K-rasG12D mice suppressed tumor growth in lung, indicating Gr-1+CD11b+ myeloid cells recruited by IL-17 play a protumor role. Taken together, our data demonstrate a critical role for Th17 cell-mediated inflammation in lung tumorigenesis and suggest a novel way for prevention and treatment of this disease.

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.

IL6 Blockade Reprograms the Lung Tumor Microenvironment to Limit the Development and Progression of K-ras–Mutant Lung Cancer

Activating mutations of K-ras are the most common oncogenic alterations found in lung cancer. Unfortunately, attempts to target K-ras-mutant lung tumors have thus far failed, clearly indicating the need for new approaches in patients with this molecular profile. We have previously shown NF-κB activation, release of IL6, and activation of its responsive transcription factor STAT3 in K-ras-mutant lung tumors, which was further amplified by the tumor-enhancing effect of chronic obstructive pulmonary disease (COPD)-type airway inflammation. These findings suggest an essential role for this inflammatory pathway in K-ras-mutant lung tumorigenesis and its enhancement by COPD. Therefore, here we blocked IL6 using a monoclonal anti-IL6 antibody in a K-ras-mutant mouse model of lung cancer in the absence or presence of COPD-type airway inflammation. IL6 blockade significantly inhibited lung cancer promotion, tumor cell-intrinsic STAT3 activation, tumor cell proliferation, and angiogenesis markers. Moreover, IL6 inhibition reduced expression of protumor type 2 molecules (arginase 1, Fizz 1, Mgl, and IDO), number of M2-type macrophages and granulocytic myeloid-derived suppressor cells, and protumor T-regulatory/Th17 cell responses. This was accompanied by increased expression of antitumor type 1 molecule (Nos2), and antitumor Th1/CD8 T-cell responses. Our study demonstrates that IL6 blockade not only has direct intrinsic inhibitory effect on tumor cells, but also reeducates the lung microenvironment toward an antitumor phenotype by altering the relative proportion between protumor and antitumor immune cells. This information introduces IL6 as a potential druggable target for prevention and treatment of K-ras-mutant lung tumors. Cancer Res; 76(11); 3189-99. ©2016 AACR.

Reduced IL-6 levels and tumor-associated phospho-STAT3 are associated with reduced tumor development in a mouse model of lung cancer chemoprevention with myo-inositol

Several promising chemopreventive agents have for lung cancer emerged in preclinical models and in retrospective trials. These agents have been shown to modulate pathways altered in carcinogenesis and reduce markers of carcinogenesis in animal and cell culture models. Cancer‐prone transgenic mice with oncogenic Kras expressed in the airway epithelium (CcspCre/+; KrasLSL‐G12D/+) were raised on diets compounded with myo‐inositol. These animals form lung premalignant lesions in a stereotypical fashion over the ten weeks following weaning. Mice raised on myo‐inositol containing diets showed potent reduction in the number, size, and stage of lesions as compared to those raised on control diets. myo‐inositol has previously been reported to inhibit phosphoinositide 3‐kinase (PI3K) signaling. However, in mice raised on myo‐inositol, total PI3K signaling was largely unaffected. Proteomic and cytokine analyses revealed large reduction in IL‐6 related pathways, including STAT3 phosphorylation. This effect was not due to direct inhibition of IL‐6 production and autocrine signaling within the tumor cell, but rather through alteration in macrophage recruitment and in phenotype switching, with an increase in antitumoral M1 macrophages.

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

ABSTRACT Tumor 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.

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.

Requirement for MUC5AC in KRAS-dependent lung carcinogenesis

With more than 150,000 deaths per year in the US alone, lung cancer has the highest number of deaths for any cancer. These poor outcomes reflect a lack of treatment for the most common form of lung cancer, non-small cell lung carcinoma (NSCLC). Lung adenocarcinoma (ADC) is the most prevalent subtype of NSCLC, with the main oncogenic drivers being KRAS and epidermal growth factor receptor (EGFR). Whereas EGFR blockade has led to some success in lung ADC, effective KRAS inhibition is lacking. KRAS-mutant ADCs are characterized by high levels of gel-forming mucin expression, with the highest mucin levels corresponding to worse prognoses. Despite these well-recognized associations, little is known about roles for individual gel-forming mucins in ADC development causatively. We hypothesized that MUC5AC/Muc5ac, a mucin gene known to be commonly expressed in NSCLC, is crucial in KRAS/Kras-driven lung ADC. We found that MUC5AC was a significant determinant of poor prognosis, especially in patients with KRAS-mutant tumors. In addition, by using mice with lung ADC induced chemically with urethane or transgenically by mutant-Kras expression, we observed significantly reduced tumor development in animals lacking Muc5ac compared with controls. Collectively, these results provide strong support for MUC5AC as a potential therapeutic target for lung ADC, a disease with few effective treatments.

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

IL22 promoted Kras mutant lung cancer by providing a tumor cell–supporting, inflammatory microenvironment. Its knockout in a murine lung cancer model restored antitumor responses, highlighting the potential to target this cytokine early in the treatment of this cancer. 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

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

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