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Abstract Background Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1 (TIMP1), an inflammatory cytokine, under serum-depleted conditions which leads to suppression of lung cancer cell metastasis has been reported. Starvation is also a stimulus of autophagic activity. Herein, we reveal that starvation activates Rab37 and induces autophagy. Methods We used an overexpression/knockdown system to determine the relationship between autophagy and Rab37 in vitro and in vivo. The autophagy activity was detected by immunoblotting, transmission electron microscope, autophagosome purification, and immunofluorescence under the confocal microscope. Lung-to-lung metastasis mouse model was used to clarify the role of autophagy and Rab37 in lung cancer. Clinical lung cancer patient specimens and an online big database were analyzed. Results Initially, we demonstrated that active-form Rab37 increased LC3-II protein level (the marker of autophagosome) and TIMP1 secretion. Accordingly, silencing of Rab37 gene expression alleviated Rab37 and LC3-II levels as well as TIMP1 secretion, and induction of autophagy could not increase TIMP1 exocytosis under such conditions. Moreover, silencing the Atg5 or Atg7 gene of lung cancer cells harboring active-mutant Rab37 (Q89L) led to decreased autophagy activity and TIMP1 secretion. In the lung-to-lung metastasis mouse model, increased TIMP1 expression accompanied by amiodarone-induced autophagy led to decreased tumor nodules and cancer cell metastasis. These phenomena were reversed by silencing the Atg5 or Atg7 gene. Notably, increasing autophagy activity alone showed no effect on TIMP1 secretion under either Rab37 or Sec22b silencing conditions. We further detected colocalization of LC3 with either Rab37 or TIMP1, identified Rab37 and Sec22b proteins in the purified autophagosomes of the lung cancer cells harboring the active-form Rab37 gene, and confirmed that these proteins are involved in the secretion of TIMP1. We reveal that autophagic activity was significantly lower in the tumors compared to the non-tumor parts and was associated with the overall lung cancer patient survival rate. Conclusions We are the first to report that autophagy plays a promoting role in TIMP1 secretion and metastasis in a Rab37-dependent manner in lung cancer cells and the lung-to-lung mouse model.

Abstract Background Esophageal squamous cell carcinoma (ESCC) is widely prevalent in Taiwan, and high metastatic spread of ESCC leads to poor survival rate. Fibronectin (FN) assembly on the cell membrane may induce ESCC mobility. MicroRNAs (MiRNAs) are abundant in and participate in tumorigenesis in many cancers. However, the role of MiRNA in FN assembly-related ESCC mobility remains unexplored. Methods We divided ESCC CE81T cells into high-FN assembly (CE81FN+) and low-FN assembly (CE81FN−) groups by flow cytometry. MiRNA microarray analysis identified miR-146a expression as the most down-regulated miRNA in comparison of CE81FN+ and CE81FN− cells. Results Cell proliferation and migration were decreased when CE81FN+ cells overexpressed transgenic miR-146a compared to the parental cells, indicating an inverse correlation between low miR-146a expression and high proliferation as well as motility of FN assembly ESCC cells. Furthermore, vimentin is the target gene of miR-146a involved in ESCC tumorigenesis. MiR-146a suppressed cell proliferation, migration and invasion of CE81FN+ cells through the inhibition of vimentin expression, as confirmed by real-time PCR, Western blotting and Transwell™ assay. Analysis of one hundred and thirty-six paired ESCC patient specimens revealed that low miR-146a and high vimentin levels were frequently detected in tumor, and that the former was associated with late tumor stages (III and IV). Notably, either low miR-146a expression or high vimentin level was significantly associated with poor overall survival rate among ESCC patients. Conclusions This is the first report to link FN assembly in the cell membrane with miR-146a, vimentin and ESCC tumorigenesis both in vitro and in ESCC patients.