There remains a great need for effective therapies for lung cancer, the majority of which are non–small cell lung cancers (NSCLC). Here, we report the identification of a novel candidate therapeutic target, LUNX, as a molecule overexpressed in primary NSCLC and lymph node metastases that is associated with reduced postoperative survival. Functional studies demonstrated that LUNX overexpression promoted lung cancer cell migration and proliferation by interactions with the chaperone protein 14-3-3. Conversely, LUNX silencing disrupted primary tumor growth, local invasion, and metastatic colonization. The finding that LUNX was expressed on cell membranes prompted us to generate and characterize LUNX antibodies as a candidate therapeutic. Anti-LUNX could downregulate LUNX and reduce lung cancer cell proliferation and migration in vitro. Administered in vivo to mice bearing lung cancer xenografts, anti-LUNX could slow tumor growth and metastasis and improve mouse survival. Together, our work provides a preclinical proof of concept for LUNX as a novel candidate target for immunotherapy in lung cancer. Cancer Res; 75(6); 1–11. ©2015 AACR.

There remains a great need for effective therapies for lung cancer, the majority of which are non-small-cell lung cancers (NSCLC). Here we report the identification of a novel candidate therapeutic target, LunX, as a molecule overexpressed in primary NSCLC and lymph node metastases that is associated with reduced postoperative survival. Functional studies demonstrated that LunX overexpression promoted lung cancer cell migration and proliferation by interactions with the chaperone protein 14-3-3. Conversely, LunX silencing disrupted primary tumor growth, local invasion and metastatic colonization. The finding that LunX was expressed on cell membranes prompted us to generate and characterize LunX antibodies as a candidate therapeutic. Anti-LunX could downregulate LunX and reduce lung cancer cell proliferation and migration in vitro. Administered in vivo to mice bearing lung cancer xenografts, anti-LunX could slow tumor growth and metastasis and improve mouse survival. Together, our work provides a preclinical proof of concept for LunX as a novel candidate target for immunotherapy in lung cancer.

Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. Cancer-associated fibroblasts (CAF) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. CAF also orchestrate tumor-promoting inflammation in multiple tumor types, including breast cancer. However, the mechanisms through which normal tissue fibroblasts are reprogrammed to tumor-promoting CAFs are mainly obscure. Here, we show that mammary fibroblasts can be educated by breast cancer cells to become activated to a proinflammatory state that supports malignant progression. Proteomic analysis of breast cancer cell–secreted factors identified the secreted proinflammatory mediator osteopontin, which has been implicated in inflammation, tumor progression, and metastasis. Osteopontin was highly secreted by mouse and human breast cancer cells, and tumor cell–secreted osteopontin activated a CAF phenotypes in normal mammary fibroblasts in vitro and in vivo. Osteopontin was sufficient to induce fibroblast reprogramming and neutralizing antibodies against osteopontin-blocked fibroblast activation induced by tumor cells. The ability of secreted osteopontin to activate mammary fibroblasts relied upon its known receptors CD44 and αVβ3 integrin. Strikingly, osteopontin silencing in tumor cells in vivo attenuated stromal activation and inhibited tumor growth. Our findings establish a critical functional role for paracrine signaling by tumor-derived osteopontin in reprograming normal fibroblasts into tumor-promoting CAFs. Cancer Res; 75(6); 1–11. ©2015 AACR.

Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. Cancer-associated fibroblasts (CAF) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation and invasion. CAF also orchestrate tumor-promoting inflammation in multiple tumor types including breast cancer. However, the mechanisms through which normal tissue fibroblasts are reprogrammed to tumor-promoting CAFs are mainly obscure. Here we show that mammary fibroblasts can be educated by breast cancer cells to become activated to a pro-inflammatory state that supports malignant progression. Proteomic analysis of breast cancer cell-secreted factors identified the secreted pro-inflammatory mediator Osteopontin (OPN), which has been implicated in inflammation, tumor progression and metastasis. OPN was highly secreted by mouse and human breast cancer cells, and tumor cell secreted OPN activated a CAF phenotypes in normal mammary fibroblasts in vitro and in vivo. OPN was sufficient to induce fibroblast reprogramming and neutralizing antibodies against OPN blocked fibroblast activation induced by tumor cells. The ability of secreted OPN to activate mammary fibroblasts relied upon its known receptors CD44 and αVβ3 integrin. Strikingly, OPN silencing in tumor cells in vivo attenuated stromal activation and inhibited tumor growth. Our findings establish a critical functional role for paracrine signaling by tumor-derived OPN in reprograming normal fibroblasts into tumor-promoting CAFs.

Cigarette smoking is a major risk factor in the development of non–small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. Nicotine, the major addictive component of tobacco smoke, can induce proliferation, invasion, and epithelial-to-mesenchymal transition (EMT) in NSCLC cell lines and promote metastasis of NSCLC in mice. Here, we demonstrate that the scaffolding protein β-arrestin-1 is necessary for nicotine-mediated induction of mesenchymal genes vimentin and fibronectin as well as EMT regulators ZEB1 and ZEB2. Nicotine induced changes in cell morphology and ablate tight junctions consistent with EMT; β-arrestin-1, but not β-arrestin-2, was required for these changes. β-Arrestin-1 promoted the expression of the mesenchymal genes, as well as ZEB1 and ZEB2, through the mediation of the E2F1 transcription factor; this required Src kinase activity. Stimulation of multiple NSCLC cell lines with nicotine led to enhanced recruitment of β-arrestin-1 and E2F1 on vimentin, fibronectin, and ZEB1 and ZEB2 promoters. Furthermore, there was significantly more β-arrestin-1 and E2F1 associated with these promoters in human NSCLC tumors, and β-arrestin-1 levels correlated with vimentin and fibronectin levels in human NSCLC samples. A549-luciferase cells lacking β-arrestin-1 showed a significantly reduced capacity for tumor growth and metastasis when orthotopically implanted into the lungs of SCID-beige mice. Taken together, these studies reveal a novel role for β-arrestin-1 in the growth and metastasis of NSCLC. Cancer Res; 75(6); 1–12. ©2015 AACR.