Since p is reported to be a critical

Since p53 is reported to be a critical component in autophagy pathways, we studied the role of p53 in fluvastatin-induced autophagy. We found that fluvastatin induced total p53 expression in a dose and time dependent manner (Fig. 4A). Further study showed fluvastatin did not significantly increase the protein level of cytosolic p53, but increase the nuclear p53 level (Fig. 4B). As shown in Fig. 4C, fewer numbers of GFP-LC3 puncta were formed upon fluvastatin treatment in the presence of p53 inhibitor pifithrin-α (PFTα). This was in agreement with the Western blot results (Fig. 4D upper panel) showing the inhibition of LC3II formation by PFTα. Similarly, p53 knockdown in SPC-A-1 bromodomain also blocked fluvastatin-induced LC3 puncta accumulation (Fig. 4C), as well as LC3II expression (Fig. 4D lower panel). These findings demonstrate that p53 is a key mediator that bridges fluvastatin to autophagy. Next, we investigated whether p53 plays an important role in fluvastatin-induced anti-metastatic property in vitro and in vivo. Wound healing experiment showed that the fluvastatin-induced healing inhibition was greatly attenuated by either p53 knockdown or PFTα in SPC-A-1 cells (Fig. 5A). In addition, p53 knockdown or PFTα also markedly blocked fluvastatin-induced anti-invasive effect in Matrigel invasion experiment (Fig. 5B). To further confirm the pivotal role of p53 in fluvastatin-induced anti-bone metastatic activity and survival, we intravenously injected p53 shRNA or negative control shRNA plasmids into mice every week to establish an in vivo p53 knockdown model. As shown in Fig. 5C, the p53 mRNA and protein levels were effectively inhibited in tumor nodes of lung tissue after the in vivo injection of p53 shRNA. As expected, bioluminescence imaging indicated that fluvastatin markedly prevented the dissemination of cancer cells to other tissues especially to the skeleton in the control but not in the p53 knockdown model (Fig. 5D top, and E). Similarly, micro-CT analysis also confirmed that with p53 expression attenuation, fluvastatin did not block the bone damage (BV/TV, Tb.N and Tb.Th decreased and Tb.Sp increased) caused by lung adenocarcinoma cells (Fig. 5D middle and G–J). In accordance with the impaired anti-bone metastatic activity, autophagic level of cancer cells in bone lesion regions was also decreased by p53 knockdown (Fig. Fig. 5D bottom and F). Notably, a 20-week survival follow-up strongly supported that long-term treatment with fluvastatin greatly extended the longevity of normal mice inoculated with SPC-A-1 lung adenocarcinoma cells (Fig. 5K). However, fluvastatin had no protective effect in prolonging the survival when p53 was knockdown in vivo (Fig. 5L). These findings suggest that anti-bone metastatic property and survival-extending ability of fluvastatin are highly dependent on the p53 level in tumors. We next examined the signaling pathway in activation of autophagy employed by fluvastatin. Fluvastatin markedly induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) (Fig. 6A). ACC, a downstream molecule of AMPK, can be phosphorylated and inactivated by AMPK and serves as a marker for AMPK activation. In contrast, fluvastatin neither changed the expression of PTEN nor the phosphorylation of AKT, which is another pathway for autophagy induction (Su et al., 2015b). To further clarify the role of p53 in fluvastatin-induced autophagic pathway, we knockdown p53 by lentivirus-based shRNA or inhibited p53 by PFTα. We found that fluvastatin-induced AMPK phosphorylation and mTOR (Ser-2448) dephosphorylation were mostly blocked showing a p53-dependent pattern (Fig. 6B). It was reported that the phosphorylation of Ser-2448 was dependent on mTOR kinase activity (Chiang and Abraham, 2005), so the reduced phosphorylation of mTOR Ser-2448 is marker for mTOR inhibition and activation of autophagy. These results imply that fluvastatin-triggered autophagy is mediated by the p53-AMPK-mTOR signaling pathway.