Agr2 is a disulfide isomerase residing in the endoplasmic reticulum (ER), which physiologically regulates protein folding and mediates resistance to ER stress. Agr2 is overexpressed in adenocarcinomas of various organs, where it participates in neoplastic transformation and metastasis, therefore acts as a pro-oncogenic protein. Besides its normal localization in the ER, Agr2 is also found in the serum and urine of cancer patients, although the physiological significance of extracellular Agr2 is poorly understood. In this study, we demonstrated that extracellular Agr2 can activate stromal fibroblasts and promote fibroblast-associated cancer invasion in gastric signet-ring cell carcinoma (SRCC), where Agr2 is highly expressed. Agr2 secreted from SRCC cells was incorporated by the surrounding gastric fibroblasts and promoted invasion by these cells. In turn, activated fibroblasts coordinated the invasive behavior of fibroblasts and cancer cells. Our findings suggested that Agr2 drives progression of gastric SRCC by exerting paracrine effects on fibroblasts in the tumor microenvironment, acting also to increase the growth and resistance of SRCC cells to oxidative and hypoxic stress as cell autonomous effects. Cancer Res; 75(2); 1–11. ©2014 AACR.

Agr2 is a member of the endoplasmic reticulum (ER) protein disulfide isomerase, which physiologically regulates protein folding and plays a pivotal role in resistance to ER stress. Agr2 is expressed primarily in adenocarcinomas of various organs, and Agr2 protein overexpression participates in neoplastic transformation and metastasis, therefore acts as a pro-oncogenic protein. Besides the normal ER-localization, extracellular Agr2 is present in serum and urine of cancer patients. However, the physiological significance of extracellular Agr2 is poorly understood. In this study, we demonstrated a novel function of extracellular Agr2 that activated stromal fibroblasts and promoted fibroblast-associated cancer invasion. Agr2 is highly expressed in gastric signet-ring cell carcinoma (SRCC). Agr2 secreted from SRCC cells was incorporated by the surrounding gastric fibroblasts and promoted invasion by these cells. In turn, activated fibroblasts promote the coordinated invasion by fibroblasts and cancer cells. Thus Agr2 plays pivotal roles in the progression of gastric SRCC by exerting paracrine effects on the surrounding fibroblasts. Furthermore, Agr2 increased the growth and resistance of SRCC cells to oxidative and hypoxic stress as cell autonomous effects. Our results indicate that Agr2 may be a suitable therapeutic target for gastric SRCC.

The bradykinin receptor B1R is overexpressed in many human cancers where it might be used as a general target for cancer imaging. In this study, we evaluated the feasibility of using radiolabeled kallidin derivatives to visualize B1R expression in a preclinical model of B1R-positive tumors. Three synthetic derivatives were evaluated in vitro and in vivo for receptor binding and their ability to visualize tumors by PET. Enalaprilat and phosphoramidon were used to evaluate the impact of peptidases on tumor visualization. While we found that radiolabeled peptides based on the native kallidin sequence were ineffective at visualizing B1R-positive tumors, peptidase inhibition with phosphoramidon greatly enhanced B1R visualization in vivo. Two stabilized derivatives incorporating unnatural amino acids (68Ga-SH01078 and 68Ga-P03034) maintained receptor-binding affinities that were effective, allowing excellent tumor visualization, minimal accumulation in normal tissues, and rapid renal clearance. Tumor uptake was blocked in the presence of excess competitor, confirming that the specificity of tumor accumulation was receptor mediated. Our results offer a preclinical proof of concept for noninvasive B1R detection by PET imaging as a general tool to visualize many human cancers. Cancer Res; 75(2); 1–7. ©2014 AACR.

The bradykinin receptor B1R is overexpressed in many human cancers where it might be used as a general target for cancer imaging. In this study, we evaluated the feasibility of using radiolabeled kallidin derivatives to visualize B1R expression in a preclinical model of B1R-positive tumors. Three synthetic derivatives were evaluated in vitro and in vivo for receptor binding and their ability to visualize tumors by positron emission tomography (PET). Enalaprilat and phosphoramidon were used to evaluate the impact of peptidases on tumor visualization. While we found that radiolabeled peptides based on the native kallidin sequence were ineffective at visualizing B1R-positive tumors, peptidase inhibition with phosphoramidon greatly enhanced B1R visualization in vivo. Two stabilized derivatives incorporating unnatural amino acids (68Ga-SH01078 and 68Ga-P03034) maintained receptor-binding affinities that were effective, allowing excellent tumor visualization, minimal accumulation in normal tissues and rapid renal clearance. Tumor uptake was blocked in the presence of excess competitor, confirming that the specificity of tumor accumulation was receptor-mediated. Our results offer a preclinical proof of concept for non-invasive B1R detection by PET imaging as a general tool to visualize many human cancers.

Crosstalk between tumor and stromal cells in the tumor microenvironment alter its properties in ways that facilitate the invasive behavior of tumor cells. Here, we demonstrate that cancer-associated fibroblasts (CAF) increase the stiffness of the extracellular matrix (ECM) and promote anisotropic fiber orientation, two mechanical signals generated through a Snail1/RhoA/αSMA–dependent mechanism that sustains oriented tumor cell migration and invasiveness. Snail1-depleted CAF failed to acquire myofibroblastic traits in response to TGFβ, including RhoA activation, αSMA-positive stress fibers, increased fibronectin fibrillogenesis, and production of a stiff ECM with oriented fibers. Snail1 expression in human tumor–derived CAF was associated with an ability to organize the ECM. In coculture, a relatively smaller number of Snail1-expressing CAF were capable of imposing an anisotropic ECM architecture, compared with nonactivated fibroblasts. Pathologically, human breast cancers with Snail1+ CAF tended to exhibit desmoplastic areas with anisotropic fibers, lymph node involvement, and poorer outcomes. Snail1 involvement in driving an ordered ECM was further confirmed in wound-healing experiments in mice, with Snail1 depletion preventing the anisotropic organization of granulation tissue and delaying wound healing. Overall, our results showed that inhibiting Snail1 function in CAF could prevent tumor-driven ECM reorganization and cancer invasion. Cancer Res; 1–12. ©2014 AACR.