CRC is the most common malignancy with the third largest incidence and mortality among all diagnosed cancers in the worldwide [
15,
16]. In this study, we find that Rab11-FIP2 expression is overexpressed in colorectal cancer and is associated with nodal metastasis. The log rank test demonstrated that tumors with the high Rab11-FIP2 expression were associated with short overall patient survival, whereas patients with tumors displaying low level of Rab11-FIP2 expression showed a better clinical outcome. Metastasis that makes colorectal cancer difficult to treat is the leading cause of cancer mortality. We investigated whether Rab11-FIP2 positively regulates colorectal cancer cells invasion. Our results demonstrated that overexpression of Rab11-FIP2 accelerated cell migration in vitro and tumor metastasis in vivo.
It was reported that Rab11-FIP2 regulated CXCR2 recycling and receptor-mediated chemotaxis, suggesting that Rab11-FIP2 may play a role in chemotaxis [
6]. We used an anti-phosphotyrosine receptor antibody array to assess whether RTKs were induced in response to Rab11-FIP2 overexpression. PAI-1, an endogenous inhibitor of urokinase-type plasminogen activator (uPA), is known to play a major role in benign disorders such as deep vein thrombosis, myocardial infarction, atherosclerosis, and stroke, and more recently has been linked to some cancers [
17], including colorectal cancer. Perturbation of PAI-1 and the uPA system has been shown to be involved in a number of cancer models, primarily by regulating migration, invasion, apoptosis, and angiogenesis [
18,
19]. For example, it was reported that plasma PAI-1 level was increased in CRC patients with liver metastasis, and PAI-1 silencing may suppress colorectal cancer progression and liver metastasis in vitro and in vivo [
20]. We found that overexpression of Rab11-FIP2 results in an increased secretion of PAI-1 in colorectal cells. Formation of new blood vessels is crucial for solid tumor growth and metastasis [
21]. We found that the microvessel density (MVD) was positively correlated with the expression of Rab11-FIP2. Tumor cells actively release pro-angiogenic factors such as vascular endothelial growth factor to promote endothelial cell proliferation, survival and migration for the formation of new blood vessels [
22,
23]. Inhibition of PAI-1 limits tumor angiogenesis regardless of angiogenic stimuli in malignant pleural mesothelioma [
9]. Similarly, we found that overexpression of Rab11-FIP2 promotes endothelial cell tube formation in a PAI-1-dependent manner. Collectively, we showed that overexpressed Rab11-FIP2 can induce tumor angiogenesis in CRC, and PAI-1 might mediate this process.
We also found that inhibition of PAI-1 by tiplaxtinin, a specific inhibitor of PAI-1, resulted in the reduction of cell migration and colony formation, and the induction of apoptosis in Rab11-FIP2 overexpression colorectal cancer cells but not the negative control cells. In parallel, we found that there was no significant difference between the PAI-1-treated HCT116 cells with those untreated cells in phosphorylation of signaling molecules (data not shown). It was reported that overexpression of Rab11-FIP2 suppresses the internalization of epidermal growth factor receptors [
24]. We postulated that Rab11-FIP2 played an important role in phosphorylation of signaling molecules. Rab11-FIP2 interaction with MYO5B regulates movement of Rab11a-containing recycling vesicles. Rab11-FIP2 has been implicated as a regulator of the recycling of several receptors such as transferrin receptor, the AMPA-type glutamate receptor subunit GluR1, the M4 muscarinic acetylcholine receptor and CXCR2 [
5,
25]. PAI-1 binds to the low density lipoprotein receptor-related protein 1 (LRP1) to regulate LRP1-dependent cell motility. We also found that PAI-1 may increase cell migration in an LRP1-dependent manner. We postulated that Rab11-FIP2 activated the phosphorylation of signaling molecules through PAI-1/LRP1 pathway in a vicious cycle manner. Because there were lots works to be done to confirm this postulation, some data was not presented in this article. Importantly, we found that treatment with rhPAI-1 increase the phosphorylation of a group of signaling molecules, including ERK1/2 and AKT/mTOR in 116/FIP2 cells but not the negative control cells, suggesting that overexpression of Rab11-FIP2 increases sensitivity of CRC to PAI-1.