Theodora Agalioti

A Tumor-Protective Role for Human Kallikrein-Related Peptidase 6 in Breast Cancer Mediated by Inhibition of Epithelial-to-Mesenchymal Transition

Human kallikrein-related peptidase 6 (KLK6) was cloned as a putative class II tumor suppressor based on its inactivated expression in metastatic breast cancer. Here, we investigated the mechanism(s) underlying the silencing of KLK6 gene in metastatic breast cancer and its putative implications for tumor progression. We present evidence that tumor-specific loss of KLK6 expression is due to hypermethylation of specific CpGs located in the KLK6 proximal promoter. Methylation-dependent binding of methyl CpG-binding protein 2 and the formation of repressive chromatin mediated by localized histone deacetylation are critical components of KLK6 silencing in breast tumors. Re-expression of KLK6 in nonexpressing MDA-MB-231 breast tumor cells by stable cDNA transfection resulted in marked reversal of their malignant phenotype, manifested by lower proliferation rates and saturation density, marked inhibition of anchorage-independent growth, reduced cell motility, and their dramatically reduced ability to form tumors when implanted in severe combined immunodeficiency mice. Interestingly, inhibition of tumor growth was observed at physiologic concentrations of KLK6, but not when KLK6 was highly overexpressed, as observed in a subset of breast tumors. Differential proteomic profiling revealed that KLK6 re-expression results in significant down-regulation of vimentin which represents an established marker of epithelial-to-mesenchymal transition of tumor cells and in concomitant up-regulation of calreticulin and epithelial markers cytokeratin 8 and 19, indicating that KLK6 may play a protective role against tumor progression that is likely mediated by inhibition of epithelial-to-mesenchymal transition. We suggest that KLK6 is an epigenetically regulated tumor suppressor in human breast cancer and provide ways of pharmacologic modulation.

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-1β, 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.

Mutant KRAS promotes malignant pleural effusion formation

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.

NRAS destines tumor cells to the lungs

The lungs are frequently affected by cancer metastasis. Although NRAS mutations have been associated with metastatic potential, their exact role in lung homing is incompletely understood. We cross‐examined the genotype of various tumor cells with their ability for automatic pulmonary dissemination, modulated NRAS expression using RNA interference and NRAS overexpression, identified NRAS signaling partners by microarray, and validated them using Cxcr1‐ and Cxcr2‐deficient mice. Mouse models of spontaneous lung metastasis revealed that mutant or overexpressed NRAS promotes lung colonization by regulating interleukin‐8‐related chemokine expression, thereby initiating interactions between tumor cells, the pulmonary vasculature, and myeloid cells. Our results support a model where NRAS‐mutant, chemokine‐expressing circulating tumor cells target the CXCR1‐expressing lung vasculature and recruit CXCR2‐expressing myeloid cells to initiate metastasis. We further describe a clinically relevant approach to prevent NRAS‐driven pulmonary metastasis by inhibiting chemokine signaling. In conclusion, NRAS promotes the colonization of the lungs by various tumor types in mouse models. IL‐8‐related chemokines, NRAS signaling partners in this process, may constitute an important therapeutic target against pulmonary involvement by cancers of other organs.

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

ABSTRACT 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 osteopontins 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.

ΙκΒ kinase α is required for development and progression of KRAS-mutant lung adenocarcinoma.

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB-activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease.Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939-51. ©2018 AACR.

Myeloid-derived interleukin-1β drives oncogenic KRAS -NF-κΒ addiction in malignant pleural effusion

Malignant pleural effusion (MPE) is a frequent metastatic manifestation of human cancers. While we previously identified KRAS mutations as molecular culprits of MPE formation, the underlying mechanism remained unknown. Here, we determine that non-canonical IKKα-RelB pathway activation of KRAS-mutant tumor cells mediates MPE development and this is fueled by host-provided interleukin IL-1β. Indeed, IKKα is required for the MPE-competence of KRAS-mutant tumor cells by activating non-canonical NF-κB signaling. IL-1β fuels addiction of mutant KRAS to IKKα resulting in increased CXCL1 secretion that fosters MPE-associated inflammation. Importantly, IL-1β-mediated NF-κB induction in KRAS-mutant tumor cells, as well as their resulting MPE-competence, can only be blocked by co-inhibition of both KRAS and IKKα, a strategy that overcomes drug resistance to individual treatments. Hence we show that mutant KRAS facilitates IKKα-mediated responsiveness of tumor cells to host IL-1β, thereby establishing a host-to-tumor signaling circuit that culminates in inflammatory MPE development and drug resistance.Malignant pleural effusion (MPE) is a life-threatening cancer-related disorder. Here, the authors show that KRAS-mutant tumor cells require IKKα, activated via host-provided IL-1β, to promote MPE development and that co-inhibition of both KRAS and IKKα ameliorates the development of MPE in mouse models.

S7 Mouse lung adenocarcinoma cell lines reveal PRL2C2 as a novel lung tumour promoter

Background Carcinogen-inflicted human cancers, including lung tumours harbour thousands of mutations per genome, most of which are unknown (Garraway, LA et al, Cell 2013;153:17–37). Aim To develop a faithful mouse model of human tobacco carcinogen-induced lung adenocarcinoma suitable for the identification of novel oncogenic genes and pathways. Methods We repeatedly managed to obtain several murine lung adenocarcinoma cell lines (MLA) by chronically exposing various mouse strains to different tobacco carcinogens. MLA were characterised for cancer stemness and oncogenes, as well as global gene expression. Results To date, 12 MLA cell lines have been derived from Wt and transgenic mice on the FVB, Balb/c, and C57BL/6 strains by means of urethane or diethylnitrosamine exposure. All MLA were immortal, phenotypically stable, and indefinitely passaged in vitro over a period of over 18 months and/or 60 passages. In addition, all cell lines were oncogenic, transplantable, metastatic, and uniformly lethal in vivo. Interestingly, MLA displayed Kras mutations in codon 61, mono- or bi-allelic Trp53 loss, and expression of lung cancer stemness factors Itgb3 and Lgr6, in amazing similarity to human lung cancers. Microarray revealed that all MLA cell lines heavily overexpressed Prl2c2, encoding proliferin, in comparison to the native lungs. Prl2c2 silencing diminished MLA proliferation and stemness, to a degree comparable with Itgb3 interference. Conclusions MLA are faithful models of human lung adenocarcinoma that led to the discovery of Prl2c2 as a candidate lung tumour promoter. Funding European Research Council Starting Independent Investigator Grant #260524. Respire 2 European Respiratory Society Fellowship, European Respiratory Society Short Term Research Fellowship.

S8 Osteopontin as an airway epithelial tumour promoter

Osteopontin (secreted phosphoprotein 1; SPP1) expression has been identified in human lung cancer and has been linked with enhanced tumour progression. To examine its functional role, we induced lung tumours by repetitive urethane or MCA/BHT lung carcinogens in C57BL/6 mice lacking both (Spp1-/-), one (Spp1+/-), or no (Spp1+/+) copy of the endogenous Spp1 gene. Primary end-points were lung tumour number and size; secondary end-points were SPP1 expression, epithelial cell survival, carcinogen-induced inflammation, and angiogenesis. Data are presented as mean ± SD. Compared with Spp1+/+ mice (n = 22), Spp1-/- mice (n = 25) developed dramatically fewer and significantly smaller lung tumours in response to urethane, while Spp1± mice (n = 12) behaved similar to Spp1-/- mice (number/diameter of lung tumours in Spp1+/+, Spp1+/-, and Spp1-/- mice, respectively: 16.1 ± 12.7/1.2 ± 0.3 mm, 2.4 ± 2.3/0.9 ± 0.2 mm, and 1.3 ± 1.6/0.7 ± 0.2 mm; P < 0.05 for comparison of Spp1+/+ with Spp1-/- and Spp1+/- mice). Spp1-/- mice were also protected from two-hit MCA/BHT-oncogenesis compared with Spp1+/+ controls. Spp1-/- mice displayed decreased epithelial cell survival and reduced numbers of airspace macrophages early after urethane, and enhanced tumour cell apoptosis and limited tumour angiogenesis at late stages of lung tumour progression. SPP1 was expressed in the naïve lung by non-ciliated airway epithelial cells and alveolar macrophages and was significantly up-regulated during multi-stage lung carcinogenesis. Our data indicate that SPP1 is functionally involved in airway epithelial carcinogenesis and may present a target for lung cancer treatment and prevention.