Since fluvastatin has a role in

Since fluvastatin has a role in the suppression of lung adenocarcinoma bone metastasis, we subsequently explored the underlying mechanisms. We found that fluvastatin can induce autophagy in both SPC-A-1 and A549 cells. Electron microscope images showed an increased autophagosomes upon fluvastatin stimulation (Fig. 2A). Furthermore, after the fluvastatin induction, a larger number of GFP-LC3 puncta formed indicating the presence of autophagosomes (Fig. 2B). These data were in accordance with the dose-dependent increase of LC3IIexpression upon fluvastatin stimulation as shown in Fig. 2C. Another tool to track autophagy is using the tandem monomeric mCherry-GFP-LC3 tagged protein. The GFP fluorescent signal is very sensitive to acidic conditions of the lysosome lumen and easily quenched, while the mCherry fluorescence is more stable in the lysosome. Therefore, colocalization of GFP and mCherry fluorescence (yellow puncta) indicates that the tandem protein is on the phagophore or within the autophagosome; strong mCherry signal but weak/no GFP signal (red puncta) indicates that the protein is within the autolysosome. As shown in Fig. 2D, stimulation with fluvastatin showed a strong increase in yellow LC3 puncta at 24h and decrease from 36h, suggesting the formation of autophagosome and subsequent fusion with lysosome. The lysosomal inhibitor chloroquine (CQ) raised the lysosomal pH and increased the autophagic flux (more GFP and mCherry puncta) (Fig. 2D). The quantification of yellow and red LC3 puncta is shown in Fig. 2E. In accordance with LC3 fluorescence results, fluvastatin induced strong LC3-IIexpression between 12 and 24h, but the expression decreased from 24h; pretreatment with CQ delayed the degradation of LC3II (Fig. S1A). Next, we established autophagy-defective SPC-A-1 kras-pdeδ inhibitor by deleting Atg5 or Atg7 gene using CRISPR/Cas9 system and there was no LC3II induction in response to fluvastatin or rapamycin in these cells. The knockout effect of Atg5 or Atg7 was validated by Western blot (Fig. S1B, C). Taken together, these findings demonstrate that fluvastatin induces autophagy in lung adenocarcinoma cells in a time and dose dependent manner. Since fluvastatin can both trigger autophagy and inhibit lung adenocarcinoma bone metastasis, we next investigated the causal relationship between induction of autophagy and prevention of lung adenocarcinoma bone metastasis. We first determined the effect of fluvastatin on lung adenocarcinoma cell migration and invasion in vitro. Wound healing experiment showed that the fluvastatin-induced healing inhibition was greatly attenuated by autophagy inhibitor 3-methyadenine (3-MA) or Bafilomycin A1 (Baf A1) in either A549 or SPC-A-1 cells (Fig. 3A). In addition, 3-MA or Baf A1 also markedly blocked fluvastatin-induced anti-invasive effect in Matrigel invasion experiment (Fig. 3B). Results were similar in Atg5 or Atg7 SPC-A-1 cells, in which fluvastatin lost the ability to inhibit the migration and invasion of SPC-A-1 cells (Fig. 3C, D). These findings suggest the critical role of autophagy in fluvastatin-induced inhibition of migration and invasion in lung adenocarcinoma cells. We next investigated the function of autophagy in fluvastatin-induced suppression of bone metastasis in vivo. In accordance with the in vitro data, 3-MA or Baf A1 treatment also reversed the protective effect of fluvastatin on bone lesions. As shown in Fig. 3E, bone sections from untreated mice showed a large number of tumor cells in tibia. Bone sections from fluvastatin-treated mice revealed tumor cell reduction. However, pretreatment with 3-MA and Baf A1 inhibited the protective effect of fluvastatin on tumor invasion and bone destruction. Micro-CT images showed alleviated bone damage from fluvastatin treatment with significantly increased the bone volume/total volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th), but decreased the trabecular separation (Tb.Sp); inhibition of autophagy by 3-MA or Baf A1 blocked the fluvastatin-induced anti-bone metastatic function (Fig. 3F–I). To further validate the essential role of autophagy in this process, we used autophagy defective cells, Atg5 or Atg7 SPC-A-1 to establish lung adenocarcinoma bone metastasis to show that fluvastatin lost the protective effect on bone metastasis; X-ray imaging and H&E staining confirmed this finding (Fig. 3J, K). Bone sections from mice inoculated with the Atg5 or Atg7 SPC-A-1 cells displayed remarkable tumor-induced osteolysis, and fluvastatin lost the ability to protect the integrity of bones in autophagy-defective tumors (Fig. 3J). The quantification of lesion area is shown in Fig. 3K. These data support that the anti-metastatic function of fluvastatin in vitro and in vivo is greatly mediated by autophagy in lung adenocarcinoma cells.