Osteopontin drives KRAS-mutant lung adenocarcinoma.

Increased expression of osteopontin (secreted phosphoprotein 1, SPP1) is associated with aggressive human lung adenocarcinoma (LADC), but its function remains unknown. Our aim was to determine the role of SPP1 in smoking-induced LADC. We combined mouse models of tobacco carcinogen-induced LADC, of deficiency of endogenous Spp1 alleles, and of adoptive pulmonary macrophage reconstitution to map the expression of SPP1 and its receptors and determine its impact during carcinogenesis. Co-expression of Spp1 and mutant KrasG12C in benign cells was employed to investigate SPP1/KRAS interactions in oncogenesis. Finally, intratracheal adenovirus encoding Cre recombinase was delivered to LSL.KRASG12D mice lacking endogenous or overexpressing transgenic Spp1 alleles. SPP1 was overexpressed in experimental and human LADC and portended poor survival. In response to two different smoke carcinogens, Spp1-deficient mice developed fewer and smaller LADC with decreased cellular survival and angiogenesis. Both lung epithelial- and macrophage-secreted SPP1 drove tumor-associated inflammation, while epithelial SPP1 promoted early tumorigenesis by fostering the survival of KRAS-mutated cells. Finally, loss and overexpression of Spp1 was, respectively, protective and deleterious for mice harboring KRASG12D-driven LADC. Our data support that SPP1 is functionally involved in early stages of airway epithelial carcinogenesis driven by smoking and mutant KRAS and may present an important therapeutic target.

Interleukin-1ß provided by KIT-competent mast cells is required for KRAS-mutant lung adenocarcinoma.

Mast cells (MC) have been identified in human lung adenocarcinoma (LADC) tissues, but their functional role has not been investigated in vivo. For this, we applied three mouse models of KRAS-mutant LADC to two different MC-deficient mouse strains (cKitWsh and Cpa3.Cre). Moreover, we derived MC gene signatures from murine bone marrow-derived MC and used them to interrogate five human cohorts of LADC patients. Tumor-free cKitWsh and Cpa3.Cre mice were deficient in alveolar and skin KIT-dependent (KIT+) MC, but cKitWsh mice retained normal KIT-independent (KIT-) MC in the airways. Both KIT+ and KIT- MC infiltrated murine LADC to varying degrees, but KIT+ MC were more abundant and promoted LADC initiation and progression through interleukin-1ß secretion. KIT+ MC and their transcriptional signature were significantly enriched in human LADC compared to adjacent normal tissue, especially in the subset of patients with KRAS mutations. Importantly, MC density increased with tumor stage and high overall expression of the KIT+ MC signature portended poor survival. Collectively, our results indicate that KIT+ MC foster LADC development and represent marked therapeutic targets.

Tumor-derived osteopontin isoforms cooperate with TRP53 and CCL2 to promote lung metastasis.

The lungs are ubiquitous receptacles of metastases originating from various bodily tumors. Although osteopontin (SPP1) has been associated with tumor dissemination, the role of its isoforms in lung-directed metastasis is incompletely understood. We employed syngeneic mouse models of spontaneous and induced lung-targeted metastasis in C57BL/6 mice competent and deficient in both Spp1 alleles. Tumor-derived osteopontin expression was modulated using either stable anti-Spp1 RNA interference, or forced overexpression of intracellular and secreted Spp1 isoforms. Identified osteopontin's downstream partners were validated using lung adenocarcinoma cells conditionally lacking the Trp53 gene and Ccr2-deficient mice. We determined that host-derived osteopontin was dispensable for pulmonary colonization by different tumor types. Oppositely, tumor-originated intracellular osteopontin promoted tumor cell survival by preventing tumor-related protein 53-mediated apoptosis, while the secretory osteopontin functioned in a paracrine mode to accelerate lung metastasis by enhancing tumor-derived C-C-motif chemokine ligand 2 signaling to cognate host receptors. As new ways to target osteopontin signaling are becoming available, the cytokine may constitute an important therapeutic target against pulmonary involvement by cancers of other organs.

Comprehensive Evaluation of Nuclear Factor-κΒ Expression Patterns in Non-Small Cell Lung Cancer.

Nuclear factor (NF)-κB signalling is required for lung adenocarcinoma development in mice, and both of its subunits RelA and RelB were independently reported to be highly expressed in human non-small cell lung cancer (NSCLC). To comprehensively examine NF-κB expression in NSCLC, we analyzed serial sections of primary tumor samples from 77 well-documented patients (36 adenocarcinomas, 40 squamous cell carcinomas and 3 large cell carcinomas) for immunoreactivity of RelA, RelB, P50, and P52/P100. Tumor and intratumoral stroma areas were discriminated based on proliferating cell nuclear antigen immunoreactivity and inflammatory infiltration was assessed in intratumoral stroma areas. NF-κB immunoreactivity was quantified by intensity, extent, and nuclear localization and was cross-examined with tumor cell proliferation, inflammatory infiltration, and clinical-pathologic data. We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach. Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100. However, RelA and P50 were predominantly expressed in intratumoral stroma, but RelB in tumor cells. Importantly, tumor area RelA expression was correlated with the intensity of inflammatory infiltration, whereas RelB expression was identified in proliferating tumor cells. Using multiple logistic regression, we identified that tumor RelB expression was an independent predictor of lymph node metastasis, and tumor P50 was an independent predictor of TNM6 stage IIB or higher, whereas tumor RelA was an independent predictor of inflammatory infiltration. We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components. Utilizing such an approach, we identified intriguing associations between distinct NF-κB subunits and clinical and pathologic features of NSCLC.

Mast cells mediate malignant pleural effusion formation.

Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1ß, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.

Beneficial impact of CCL2 and CCL12 neutralization on experimental malignant pleural effusion.

Using genetic interventions, we previously determined that C-C motif chemokine ligand 2 (CCL2) promotes malignant pleural effusion (MPE) formation in mice. Here we conducted preclinical studies aimed at assessing the specific therapeutic potential of antibody-mediated CCL2 blockade against MPE. For this, murine MPEs or skin tumors were generated in C57BL/6 mice by intrapleural or subcutaneous delivery of lung (LLC) or colon (MC38) adenocarcinoma cells. Human lung adenocarcinoma cells (A549) were used to induce MPEs in severe combined immunodeficient mice. Intraperitoneal antibodies neutralizing mouse CCL2 and/or CCL12, a murine CCL2 ortholog, were administered at 10 or 50 mg/kg every three days. We found that high doses of CCL2/12 neutralizing antibody treatment (50 mg/kg) were required to limit MPE formation by LLC cells. CCL2 and CCL12 blockade were equally potent inhibitors of MPE development by LLC cells. Combined CCL2 and CCL12 neutralization was also effective against MC38-induced MPE and prolonged the survival of mice in both syngeneic models. Mouse-specific CCL2-blockade limited A549-caused xenogeneic MPE, indicating that host-derived CCL2 also contributes to MPE precipitation in mice. The impact of CCL2/12 antagonism was associated with inhibition of immune and vascular MPE-related phenomena, such as inflammation, new blood vessel assembly and plasma extravasation into the pleural space. We conclude that CCL2 and CCL12 blockade are effective against experimental MPE induced by murine and human adenocarcinoma in mice. These results suggest that CCL2-targeted therapies may hold promise for future use against human MPE.