IL-33 increases ST2+ Tregs and promotes metastatic tumour growth in the lungs in an amphiregulin-dependent manner.

Regulatory T cells (Tregs) facilitate primary and metastatic tumour growth through the suppression of anti-tumour immunity. Emerging evidence suggests a distinct role for Tregs in mediating tissue repair and barrier integrity in the lungs by IL-33 mediated production of the growth factor amphiregulin (AREG). Dependent on the type of cancer and local microenvironment, AREG may induce tumour cell proliferation, invasion, migration or resistance to apoptosis by signaling through the epidermal growth factor receptor (EGFR). We have found that IL-33 is dramatically increased in and around metastatic tumour foci in the lungs of mice bearing orthotopic murine mammary tumours. We observed that Tregs express significantly more of the IL-33 receptor, ST2, relative to conventional T cells, that ST2+ Tregs accumulate in the lungs of metastatic tumour-bearing mice, and that ST2+ Tregs produce significantly more AREG than ST2- Tregs. The intranasal administration of recombinant IL-33 increased the proportion of AREG producing ST2+ Tregs and enhanced the level of phosphorylated EGFR in the metastatic lungs. While recombinant AREG did not impact mammary tumour cell proliferation in vitro despite inducing a dose-dependent increase in phosphorylated EGFR, intranasal administration of AREG resulted in a ten-fold increase in pulmonary metastatic tumour burden in vivo. Further, the intranasal administration of recombinant IL-33 significantly increased metastatic tumour burden in the lungs in an amphiregulin-dependent manner. These data identify ST2+ Tregs as a microenvironmental source of AREG in the lungs of mice with orthotopic metastatic mammary tumours and highlight an important role for AREG in promoting metastatic tumour growth in the lungs.

The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion.

Carbonic anhydrase IX (CAIX) is a hypoxia inducible factor 1-induced, cell surface pH regulating enzyme with an established role in tumor progression and clinical outcome. However, the molecular basis of CAIX-mediated tumor progression remains unclear. Here, we have utilized proximity dependent biotinylation (BioID) to map the CAIX 'interactome' in breast cancer cells in order to identify physiologically relevant CAIX-associating proteins with potential roles in tumor progression. High confidence proteins identified include metabolic transporters, ß1 integrins, integrin-associated protein CD98hc and matrix metalloprotease 14 (MMP14). Biochemical studies validate the association of CAIX with α2ß1 integrin, CD98hc and MMP14, and immunofluorescence microscopy demonstrates colocalization of CAIX with α2ß1 integrin and MMP14 in F-actin/cofilin-positive lamellipodia/pseudopodia, and with MMP14 to cortactin/Tks5-positive invadopodia. Modulation of CAIX expression and activity results in significant changes in cell migration, collagen degradation and invasion. Mechanistically, we demonstrate that CAIX associates with MMP14 through potential phosphorylation residues within its intracellular domain, and that CAIX enhances MMP14-mediated collagen degradation by directly contributing hydrogen ions required for MMP14 catalytic activity. These findings establish hypoxia-induced CAIX as a novel metabolic component of cellular migration and invasion structures, and provide new mechanistic insights into its role in tumor cell biology.

Maraviroc decreases CCL8-mediated migration of CCR5(+) regulatory T cells and reduces metastatic tumor growth in the lungs.

Regulatory T cells (Tregs) play a crucial physiological role in the regulation of immune homeostasis, although recent data suggest Tregs can contribute to primary tumor growth by suppressing antitumor immune responses. Tregs may also influence the development of tumor metastases, although there is a paucity of information regarding the phenotype and function of Tregs in metastatic target organs. Herein, we demonstrate that orthotopically implanted metastatic mammary tumors induce significant Treg accumulation in the lungs, which is a site of mammary tumor metastasis. Tregs in the primary tumor and metastatic lungs express high levels of C-C chemokine receptor type 5 (CCR5) relative to Tregs in the mammary fat pad and lungs of tumor-free mice, and Tregs in the metastatic lungs are enriched for CCR5 expression in comparison to other immune cell populations. We also identify that C-C chemokine ligand 8 (CCL8), an endogenous ligand of CCR5, is produced by F4/80(+) macrophages in the lungs of mice with metastatic primary tumors. Migration of Tregs toward CCL8 ex vivo is reduced in the presence of the CCR5 inhibitor Maraviroc. Importantly, treatment of mice with Maraviroc (MVC) reduces the level of CCR5(+) Tregs and metastatic tumor burden in the lungs. This work provides evidence of a CCL8/CCR5 signaling axis driving Treg recruitment to the lungs of mice bearing metastatic primary tumors, representing a potential therapeutic target to decrease Treg accumulation and metastatic tumor growth.

EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk.

In an effort to identify novel biallelically inactivated tumor suppressor genes (TSGs) in sporadic invasive and preinvasive non-small-cell lung cancer (NSCLC) genomes, we applied a comprehensive integrated multiple 'omics' approach to investigate patient-matched, paired NSCLC tumor and non-malignant parenchymal tissues. By surveying lung tumor genomes for genes concomitantly inactivated within individual tumors by multiple mechanisms, and by the frequency of disruption in tumors across multiple cohorts, we have identified a putative lung cancer TSG, Eyes Absent 4 (EYA4). EYA4 is frequently and concomitantly deleted, hypermethylated and underexpressed in multiple independent lung tumor data sets, in both major NSCLC subtypes and in the earliest stages of lung cancer. We found that decreased EYA4 expression is not only associated with poor survival in sporadic lung cancers but also that EYA4 single-nucleotide polymorphisms are associated with increased familial cancer risk, consistent with EYA4s proximity to the previously reported lung cancer susceptibility locus on 6q. Functionally, we found that EYA4 displays TSG-like properties with a role in modulating apoptosis and DNA repair. Cross-examination of EYA4 expression across multiple tumor types suggests a cell-type-specific tumorigenic role for EYA4, consistent with a tumor suppressor function in cancers of epithelial origin. This work shows a clear role for EYA4 as a putative TSG in NSCLC.

Drug-induced alterations in tumour perfusion yield increases in tumour cell radiosensitivity.

The perfusion of human tumour xenografts was manipulated by administration of diltiazem and pentoxifylline, and the extent that observed changes in tumour perfusion altered tumour radiosensitivity was determined. 2 tumour systems having intrinsically different types of hypoxia were studied. The responses of SiHa tumours, which have essentially no transient hypoxia, were compared to the responses of WiDr tumours, which contain chronically and transiently hypoxic cells. We found that relatively modest increases in net tumour perfusion increased tumour cell radiosensitivity in WiDr tumours to a greater extent than in SiHa tumours. Moreover, redistribution of blood flow within WiDr tumours was observed on a micro-regional level that was largely independent of changes in net tumour perfusion. Through fluorescence-activated cell sorting coupled with an in vivo-in vitro cloning assay, increases in the radiosensitivity of WiDr tumour cells at intermediate levels of oxygenation were observed, consistent with the expectation that a redistribution of tumour blood flow had increased oxygen delivery to transiently hypoxic tumour cells. Our data therefore suggest that drug-induced changes in tumour micro-perfusion can alter the radiosensitivity of transiently hypoxic tumour cells, and that increasing the radiosensitivity of tumour cells at intermediate levels of oxygenation is therapeutically relevant.