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Cell migration is a highly coordinated process that involves not only integrin-mediated adhesion but also de-adhesion. We previously found that a cryptic de-adhesive site within fibronectin molecule, termed FNIII14, weakens cell adhesion to the extracellular matrix by inactivating β1-integrins. Surprisingly, eukaryotic translation elongation factor-1A (eEF1A), an essential factor during protein biosynthesis, was identified as a membrane receptor that mediates the de-adhesive effect of FNIII14. Here, we demonstrate that FNIII14-mediated de-adhesion causes enhanced migration and invasion in two types of highly invasive/metastatic cancer cells, resulting in the initiation of metastasis. Both in vitro migration and invasion of highly invasive human melanoma cell line, Mum2B, were inhibited by a matrix metalloproteinase (MMP)-2/9 inhibitor or a function-blocking antibody against FNIII14 (anti-FNIII14 Ab), suggesting that MMP-mediated exposure of the cryptic de-adhesive site FNIII14 was responsible for Mum2B cell migration and invasion. The MMP-induced FNIII14 exposure was also shown to be functional in the migration and invasion of highly metastatic mouse breast cancer cell line 4T1. Overexpression and knockdown experiments of eEF1A in Mum2B cells revealed that the migration and invasion were dependent on the membrane levels of eEF1A. In vivo experiments using tumor xenograft mouse models derived from Mum2B and 4T1 cell lines showed that the anti-FNIII14 Ab has a significant anti-metastatic effect. Thus, these results provide novel insights into the regulation of cancer cell migration and invasion and suggest promising targets for anti-metastasis strategies.

To investigate the adrenal toxicity of a novel inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, compound X (CX), histopathological examinations, fat staining, adrenal cholesterol measurement, blood biochemistry, plasma corticosterone and ACTH measurement, ACTH-stimulation assay, and adrenal gene-expression analyses were done in rats in repeated-dose studies (experiment 1: 0, 3, 10, 30 and 150mg/kg for 4, 8, 15 and 28 days; experiment 2: 0, 3, 10,30 and 150mg/kg for 28 days; experiment 3: 0, 10, 30, 100 and 300mg/kg for 28 days). CX induced morphologic changes such as vacuolation and hypertrophy in the zona fasciculata (ZF) at ≥10mg/kg, and eosinophilic changes in the ZF at 150mg/kg. Vacuolation decreased in a dose-dependent manner and was replaced by eosinophilic changes. Inflammatory and fibrous changes were observed at ≥30mg/kg. These changes were expressed at early stages of dosing and were not exacerbated by extension of the administration period. Oil-red-O/Filipin staining showed depletion of cholesterol ester in dose-dependent manner and enabled adrenal cholesterol measurement. Filipin staining also revealed vacuoles to be composed of cholesterol esters. No significant changes were observed during the dosing period of CX for plasma corticosterone and ACTH levels. Gene-expression analyses showed up-regulation of Star and Abca1 mRNA levels at 300mg/kg. In conclusion, CX induced adrenal toxicity, but CX did not influence adrenocortical functions, and exacerbation of adrenal toxicities by extension of the administration period was not observed. Up-regulation of genes related to the transport of FC, such as Star and Abca1, were observed in CX groups, and these genes may be involved in the maintenance of adrenal structure and function in rats given CX.

The acquisition of drug resistance mediated by the interaction of tumor cells with the extracellular matrix (ECM), commonly referred to as cell adhesion-mediated drug resistance (CAM-DR), has been observed not only in hematopoietic tumor cells but also in solid tumor cells. We have previously demonstrated that a 22-mer peptide derived from fibronectin, FNIII14, can inhibit cell adhesion through the inactivation of β1 integrin; when coadministered with cytarabine, FNIII14 completely eradicates acute myelogenous leukemia by suppressing CAM-DR. In this study, we show that our FNIII14 peptide also enhances chemotherapy efficacy in solid tumors. Coadministration of FNIII14 synergistically enhances the cytotoxicity of doxorubicin and aclarubicin in mammary tumor and melanoma cells, respectively. The solid tumor cell chemosensitization induced by FNIII14 is dependent upon the upregulation and activation of the pro-apoptotic protein, Bim. Furthermore, the metastasis of tumor cells derived from ventrally transplanted mammary tumor grafts is suppressed by the coadministration of FNIII14 and doxorubicin. These results suggest that the coadministration of our FNIII14 peptide with chemotherapy could achieve efficient solid tumor eradication by increasing chemosensitivity and decreasing metastasis. The major causes of tumor recurrence are the existence of chemotherapy-resistant primary tumor cells and the establishment of secondary metastatic lesions. As such, coadministering FNIII14 with anti-cancer drugs could provide a promising new approach to improve the prognosis of patients with solid tumors.

Anoikis, apoptosis because of loss of cell anchorage, is crucial for tissue homeostasis. Fibronectin not only provides a scaffold for cell anchorage but also harbors a cryptic antiadhesive site capable of inducing β1-integrin inactivation. In this study, this cryptic antiadhesive site is implicated in spontaneous induction of anoikis. Nontransformed fibroblasts (NIH3T3) adhering to a fibronectin substratum underwent anoikis during serum starvation culture. This anoikis was caused by proteolytic exposure of the cryptic antiadhesive site in fibronectin by matrix metalloproteinase. Eukaryotic elongation factor 1A (eEF1A) was identified as a membrane receptor for the exposed antiadhesive site. Serum starvation raised the membrane residence of eEF1A, and siRNA-based disruption of this increase rendered cells anoikis-resistant. By contrast, cells became more susceptible to anoikis in parallel with increased membrane residence of eEF1A by enforced expression. These results demonstrate that eEF1A acts as a membrane receptor for the cryptic antiadhesive site of fibronectin, which contributes to cell regulation, including anoikis, through negative regulation of cell anchorage.