Seyedeh Golsar Mirabolfathinejad

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.

Curcumin inhibits COPD-like airway inflammation and lung cancer progression in mice

Recent studies have demonstrated that K-ras mutations in lung epithelial cells elicit inflammation that promotes carcinogenesis in mice (intrinsic inflammation). The finding that patients with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer after controlling for smoking suggests a further link between lung cancer and extrinsic inflammation. Besides exposure to cigarette smoke, it is thought that airway inflammation in COPD is caused by bacterial colonization, particularly with non-typeable Hemophilus influenzae (NTHi). Previously, we have shown that NTHi-induced COPD-like airway inflammation promotes lung cancer in an airway conditional K-ras-induced mouse model. To further test the role of inflammation in cancer promotion, we administered the natural anti-inflammatory agent, curcumin, 1% in diet before and during weekly NTHi exposure. This significantly reduced the number of visible lung tumors in the absence of NTHi exposure by 85% and in the presence of NTHi exposures by 53%. Mechanistically, curcumin markedly suppressed NTHi-induced increased levels of the neutrophil chemoattractant keratinocyte-derived chemokine by 80% and neutrophils by 87% in bronchoalveolar lavage fluid. In vitro studies of murine K-ras-induced lung adenocarcinoma cell lines (LKR-10 and LKR-13) indicated direct anti-tumoral effects of curcumin by reducing cell viability, colony formation and inducing apoptosis. We conclude that curcumin suppresses the progression of K-ras-induced lung cancer in mice by inhibiting intrinsic and extrinsic inflammation and by direct anti-tumoral effects. These findings suggest that curcumin could be used to protract the premalignant phase and inhibit lung cancer progression in high-risk COPD patients.

Interleukin 6, but Not T Helper 2 Cytokines, Promotes Lung Carcinogenesis

Several epidemiologic studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer compared with smokers without COPD. We have shown a causal role for COPD-like airway inflammation in lung cancer promotion in the CCSPCre/LSL-K-rasG12D mouse model (CC-LR). In contrast, existing epidemiologic data do not suggest any definite association between allergic airway inflammation and lung cancer. To test this, CC-LR mice were sensitized to ovalbumin (OVA) and then challenged with an OVA aerosol weekly for 8 weeks. This resulted in eosinophilic lung inflammation associated with increased levels of T helper 2 cytokines and mucous metaplasia of airway epithelium, similar to what is seen in asthmatic patients. However, this type of inflammation did not result in a significant difference in lung surface tumor number (49 ± 9 in OVA vs. 52 ± 5 in control) in contrast to a 3.2-fold increase with COPD-like inflammation. Gene expression analysis of nontypeable Haemophilus influenzae (NTHi)-treated lungs showed upregulation of a different profile of inflammatory genes, including interleukin 6 (IL-6), compared with OVA-treated lungs. Therefore, to determine the causal role of cytokines that mediate COPD-like inflammation in lung carcinogenesis, we genetically ablated IL-6 in CC-LR mice. This not only inhibited intrinsic lung cancer development (1.7-fold) but also inhibited the promoting effect of extrinsic COPD-like airway inflammation (2.6-fold). We conclude that there is a clear specificity for the nature of inflammation in lung cancer promotion, and IL-6 has an essential role in lung cancer promotion. Cancer Prev Res; 4(1); 51–64. ©2010 AACR.

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.

Abstract 4977: An essential role for neutrophils in lung cancer promotion .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Lung cancer is the leading cause of cancer death worldwide, and cigarette smoking is the principal cause of it. Cigarette smoke-induced lung cancer is characterized by a deregulated inflammatory tumor microenvironment. Tumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes (innate and adaptive immune cells) can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. In addition, smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer compared to smokers without COPD. These facts suggest a strong link between COPD-type inflammation and lung cancer promotion. In histopathologic specimens of lung and in bronchoalveolar lavage fluid (BALF) from COPD patients, neutrophils and macrophages are prominent. 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. We 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 neutrophilic influx due to increased level of neutrophil chemoattractant, KC, which was partially inhibited by using a natural non-specific anti-inflammatory agent, curcumin 1% in diet, and resulted in significant tumor suppression. To further study the role of neutrophil in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody, mLy6G, 5mg/kg twice a week by intraperitoneal injection. This resulted in complete lung neutrophil depletion and 68% (3.2-fold) reduction in lung surface tumor number. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. It participates actively in COPD development and might contribute to tumor progression by activating proteolytic cascades. The concentration of NE in tumor extracts was also found to be linked to the invasiveness of lung cancer. Therefore, we crossed the CC-LR mice with NE knock out (KO) mice. We have found that lack of NE significantly inhibited lung cancer development by 43% (1.7-fold) without changing the BALF inflammatory cell component of the CC-LR mice. We further inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2, using a selective inhibitor, SB332235Z, 50 mg/kg orally twice daily by gavage. Similarly, this also resulted in significant suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant (∼64%, 2.8-fold) tumor reduction. We conclude that lung cancer promotion by inflammation partly mediated by activation of IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trails using IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer. Citation Format: Lei Gong, Amber C. Cumpian, Cesar E. Ochoa, Daniel J. Lapid, Seyedeh Golsar Mirabolfathinejad, Maria Miguelina De la Garza, Seyed Javad Moghaddam. An essential role for neutrophils in lung cancer promotion . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4977. doi:10.1158/1538-7445.AM2013-4977

Abstract A32: Mechanistic Dissection of Lung Cancer Promotion by Airway Inflammation

Cigarette smoking is the principal cause of lung carcinogenesis, however, smokers with chronic obstructive pulmonary disease (COPD) have an increased risk of lung cancer (3 to 10 fold) compared to smokers with comparable cigarette exposure but without COPD. Histopathologic studies have clearly demonstrated lung inflammation in COPD, and it persists even after cessation of cigarette smoking. These facts suggest a strong link between COPD-related airway inflammation and lung cancer promotion independent of smoking but the precise mechanistic link is not known. We developed a COPD-like mouse model of airway inflammation through repeated aerosol challenge to a lysate of non-typeable Haemophilus influenzae (NTHi) which commonly colonizes the airways of COPD patients. Then, we showed that this type of airway inflammation, but not asthma-like airway inflammation, promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with NF-kB pathway activation, and up-regulation of its downstream target genes, including IL-6 and T helper 17 (Th17) response. Therefore, we studied the role of NF-kB in lung cancer promotion by targeting its expression in airway epithelium. CC-LR mice were crossed with IKK-βf/f mice to develop a mouse with lack of NF-kB activity in airway secretory cells (CC-LR-IKKβ mice). NF-kB deficiency in the airway epithelium changed the bronchoalveolar lavage fluid (BALF) cellular component (mostly reduction in macrophage number) of the CC-LR mice, and resulted in a ∼70% (3.4-fold) reduction in lung surface tumor number compared to age and sex matched control CC-LR mice. It also significantly reduced the number of visible tumors on the lung surface of the CC-LR mice by >60% (2.6-fold) after inducing COPD-like airway inflammation using weekly NTHi exposure for 8 weeks. We then genetically ablated IL-6 in the CC-LR mice. Lack of IL-6 did not change the BALF inflammatory cell component of the CC-LR mice. However, it not only inhibited intrinsic lung cancer development (1.7-fold, 41%), but also inhibited the promoting effect of extrinsic NTHi-induced COPD-like airway inflammation (2.6-fold, 62%) on lung carcinogenesis, suggesting a role for IL-6 mediated epithelial cell signaling in tumor promotion by COPDlike airway inflammation. This is under further investigation by our group. IL-6 is required for differentiation of Th17 cells from naive T cells. Th17 cells mostly produce IL-17A, and IL-17F. These both bind to the IL-17 receptor (IL-17R), and IL-17R signaling is required for lung CXC chemokine expression and neutrophil recruitment, however, we found that only the IL-17R KO and IL-17A KO but not IL-17F KO mice showed lower levels of neutrophilic influx into the BALF and less inflammatory cell infiltration in lung tissue in response to NTHi. 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 resulted in a ∼70% (3.4-fold) reduction in lung surface tumor number compared to age and sex matched control CC-LR mice. It also significantly reduced the number of visible tumors on the lung surface of the CCLR mice by ∼54% (2.2-fold) after inducing COPD-like airway inflammation using weekly NTHi exposure for 4 weeks from the age of 10 weeks. We conclude that airway inflammation through NF-kB mediated activation of the IL-6/Th17 signaling pathway promotes lung carcinogenesis.