Mauricio S. Caetano

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.

STAT3 restrains RANK- and TLR4-mediated signalling by suppressing expression of the E2 ubiquitin-conjugating enzyme Ubc13

The transcriptional regulator STAT3 curbs pro-inflammatory cytokine production mediated by NF-κB signaling in innate immune cells, yet the mechanism by which this occurs has been unclear. Here we identify STAT3 as a pivotal negative regulator of Ubc13, an E2 ubiquitin-conjugating enzyme that facilitates TRAF6 K63-linked ubiquitination and NF-κB activation. Ubc13 accumulates intracellularly in the absence of STAT3. Depletion of Ubc13 in Stat3-deficient macrophages subdues excessive RANKL- or LPS-dependent gene expression, indicating Ubc13 overexpression mediates enhanced transcriptional responses in the absence of STAT3. In RANKL-activated macrophages, STAT3 is stimulated by autocrine IL-6 and inhibits accrual of Ets-1, Set1 methyltransferase and trimethylation of histone H3 lysine 4 (H3K4me3) at the Ube2n (Ubc13) promoter. These results delineate a mechanism by which STAT3 operates as a transcriptional repressor on Ube2n, thus modulating NF-κB activity by regulation of Ubc13 abundance. Our data suggest this pathway plays important roles in bone homeostasis and restraint of inflammation.

Redox Imbalance and Pulmonary Function in Bleomycin-Induced Fibrosis in C57BL/6, DBA/2, and BALB/c Mice

The development of bleomycin-induced pulmonary fibrosis (BLEO-PF) has been associated with differences in genetic background and oxidative stress status. The authors’ aim was to investigate the crosstalk between the redox profile, lung histology, and respiratory function in BLEO-PF in C57BL/6, DBA/2, and BALB/c mice. BLEO-PF was induced with a single intratracheal dose of bleomycin (0.1 U/mouse). Twenty-one days after bleomycin administration, the mortality rate was over 50% in C57BL/6 and 20% in DBA/2 mice, and BLEO-PF was not observed in BALB/c. There was an increase in lung static elastance (p < .001), viscoelastic/inhomogeneous pressure (p < .05), total pressure drop after flow interruption (p < .01), and ΔE (p < .05) in C57BL/6 mice. The septa volume increased in C57BL/6 (p < .05) and DBA/2 (p < .001). The levels of IFN-γ were reduced in C57BL/6 mice (p < .01). OH-proline levels were increased in C57BL/6 and DBA/2 mice (p < .05). SOD activity and expression were reduced in C57BL/6 and DBA/2 mice (p < .001 and p < .001, respectively), whereas catalase was reduced in all strains 21 days following bleomycin administration compared with the saline groups (C57BL/6: p < .05; DBA/2: p < .01; BALB/c: p < .01). GPx activity and GPx1/2 expression decreased in C57BL/6 (p < .001). The authors conclude that BLEO-PF resistance may also be related to the activity and expression of SOD in BALB/c mice.

Endotoxin-induced acute lung injury is dependent upon oxidative response

The aim of the present study was to investigate the involvement of oxidative stress in acute lung injury (ALI) induced by lipopolysaccharide (LPS) and its effects upon cell structure, function and inflammation. In total, 108 male C57BL/6 mice were divided into seven groups: CTR Group (50 µL of saline) administered intratracheally (i.t.), LPS 6 h (10 µg of LPS − i.t.), LPS 12 h (10 µg of LPS − i.t.), LPS 24 h (10 µg of LPS − i.t.), LPS 48 h (10 µg of LPS − i.t.), LPS 24 h (10 µg − i.t.) + NAC 40 mg/kg (gavage) and 24 h LPS (10 µg − i.t.) + NAC 100 mg/kg (gavage). The antioxidant treatment protected the lungs from stress in the first 12 h, but significant oxidative stress induction was observed at the 24-hour time point, and, after 48 h, there was no protection exerted by the antioxidant treatment. NAC (N-acetylcysteine) reversed the elastance parameters, and ΔP1 and ΔP2 compared with 24 h LPS alone. NAC reduced the number of inflammatory cells in histology analysis when compared with the 24 h LPS alone-treated group. NAC also inhibited the transcription of NFκB, IL-6, TNF-α and COX2 usually induced by LPS. Our results suggest that oxidative stress plays an important role in structural, functional and inflammatory responses in the ALI model.

Efficient blockade of Akt signaling is a determinant factor to overcome resistance to matuzumab.

BackgroundClinical studies have shown antineoplastic effectiveness of monoclonal antibodies (MAbs) against EGFR for different indications. Several MAbs directed to EGFR were developed recently, such as matuzumab, but there is still lack of information on preclinical data on its combination with chemo-radiation. Thus, the present study intended to examine the molecular pathways triggered by matuzumab alone or associated to chemo-radiotherapy in gynecological cell lines and its impact on cell growth and signaling.ResultsCombination of matuzumab with radiation and cisplatin did not enhance its cytostatic effects on A431, Caski and C33A cells (high, intermediate and low EGFR expression, respectively) in clonogenic assays, when compared to controls. The lack of effect was mediated by persistent signaling through EGFR due to its impaired degradation. In spite of the fact that matuzumab inhibited phosphorylation of EGFR, it had no effect upon cell viability. To analyze which downstream molecules would be involved in the EGFR signaling in the presence of matuzumab, we have tested it in combination with either PD98059 (MAPK inhibitor), or LY294002 (PI3K inhibitor). Matuzumab exhibited a synergic effect with LY294002, leading to a reduction of Akt phosphorylation that was followed by a decrease in A431 and Caski cells survival. The combination of PD98059 and matuzumab did not show the same effect suggesting that PI3K is an important effector of EGFR signaling in matuzumab-treated cells. Nonetheless, matuzumab induced ADCC in Caski cells, but not in the C33A cell line, suggesting that its potential therapeutic effects in vitro are indeed dependent on EGFR expression.ConclusionsMatuzumab combined with chemoradiation did not induce cytotoxic effects on gynecological cancer cell lines in vitro, most likely due to impaired EGFR degradation. However, a combination of matuzumab and PI3K inhibitor synergistically inhibited pAkt and cell survival, suggesting that the use of PI3K/Akt inhibitors could overcome intrinsic resistance to matuzumab in vitro. Altogether, data presented here can pave the way to a rational design of clinical strategies in patients with resistant profile to anti-EGFR inhibitors based on combination therapy.

Indoleamine 2,3-dioxygenase 1 inhibition targets anti-PD1-resistant lung tumors by blocking myeloid-derived suppressor cells

Indoleamine 2,3-dioxygenase 1 (IDO1), involved in the catabolism of tryptophan (Trp) to kynurenine (Kyn) is an important regulator of tumor-mediated immunosuppression implicated in resistance to anti-PD1 immunotherapy. We investigated the role of IDO1 in an anti-PD1-resistant lung cancer model (344SQ_R) compared to the parental 344SQ tumors (344SQ_P). IDO1 was overexpressed in tumor-infiltrating leukocytes, and plasma Kyn levels were increased, in 344SQ_R vs. 344SQ_P. The IDO1 inhibitor INCB023843 retarded tumor growth and reduced lung metastases in 344SQ_R. IDO1 was expressed at higher levels in F4/80+Gr1intCD11b+ myeloid-derived suppressor cells (MDSCs) that were prominent in 344SQ_R. The INCB023843 reduced IDO1 expression and percentages of these MDSCs while increasing CD8+ T cells infiltration, hence reactivating antitumor T-cell responses in 344SQ_R. Therefore, IDO1 inhibition holds promise for treating lung cancer that does not respond to anti-PD1 therapy.

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.

Radiation Followed by OX40 Stimulation Drives Local and Abscopal Antitumor Effects in an Anti–PD1-Resistant Lung Tumor Model

Purpose: Radiation is used extensively to treat localized cancer, but improved understanding of its effects on the immune system has increased interest in its potential systemic (abscopal) effects, particularly in combination with checkpoint inhibitors such as anti-PD1. The majority of patients either do not respond or develop resistance to monotherapy over time. Here, we investigated the efficacy of OX40 (CD134) stimulation as an alternative immunotherapeutic approach in combination with radiotherapy (XRT) in a murine model of anti–PD1-resistant lung tumors. Experimental Design: We established a bilateral tumor model in 129Sv/Ev mice using an anti–PD1-resistant lung tumor cell line. Primary tumors were treated with intratumoral injection of an OX40 agonist antibody, given as adjuvant therapy after XRT (36 Gy in three 12-Gy fractions), whereas secondary tumors were left untreated to investigate abscopal outcomes. Results: The combination of XRT followed by OX40 stimulation effectively inhibited local and systemic antitumor growth, limited lung metastases, and improved survival rates. This treatment regimen augmented CD4+ and CD8+ T-cell expansion. XRT induced the expression of OX40 on T cells in tumors and spleens and increased the percentages of splenic CD103+ dendritic cells. Conclusions: Our data extend the benefits of radiation to systemic disease control, especially when combined with anti-OX40 agonist to promote immunologically mediated abscopal effects. Moreover, this study provides a rational treatment approach and sequence to overcome anti–PD1-resistant poorly immunogenic tumors. Clin Cancer Res; 24(22); 5735–43. ©2018 AACR.