Rab3D is necessary for tumor metastasis and progression in vivo

To further test the significance of Rab3D in tumor progression, we next investigated the function of Rab3D in tumor metastasis and progression in vivo. At first, we established the cancer cell lines stably overexpressing recombinant human Rab3D (Fig. 6A and Fig. S4A) and stably transfecting with GFP-Rab3D shRNA, respectively (Fig. 6B and Fig. S4B). We injected these cells separately into mammary fat pads of nude mice to establish spontaneous metastasis model and monitored tumor growth and metastasis. Clear boarders without obvious invasion to surrounding tissues were observed in the control group, while tumors formed by Rab3D-MCF-7 cells invaded surrounding muscles, mammary fat pads as well as lung tissues (Fig. 6C and Fig. S4C), demonstrating that Rab3D-MCF-7 cells became more invasive. In the metastatic lung, we observed small metastatic clones with GFP density near the blood vessels in the Rab3D-MCF-7 group. Conversely, stable knockdown of Rab3D in xenograft tumor model is sufficient to reduce metastatic colonies in the lung (Fig. 6D and Fig. S4D). In addition, there were no obvious differences of cell proliferation in vitro (Fig. S5A and S5B) and tumor growth in vivo (Fig. S5C-F) among different groups, although the overexpression of Rab3D decreased apoptosis to some extent (Fig. S5G), demonstrating that Rab3D is highly specific for promoting tumor invasion and metastasis but not due to changes in proliferation.

Consistent with our observations in vitro, the levels of Rab3D expression were positively correlated with the levels of N-cadherin, phosphorylated GSK3β, snail, but negatively correlated with E-cadherin in vivo by IHC analysis (Fig. 6E and Fig. S6A-E). These data have further emphasized the positive correlation between Rab3D and EMT. Fig. 6F also showed that the level of plasma Hsp90α in Rab3D-MCF-7 xenografts was twice as much as that in the control group (n=5). Furthermore, stable Rab3D knockdown in the xenografts showed much lower level of Hsp90α in blood plasma compared with controls.

Taken together, our results support that Rab3D is necessary and sufficient to promote tumor metastasis. A working model for the effect of Rab3D on metastasis is shown in Fig. 6G.

Figure 6: In vivo effects of Rab3D on tumor metastasis. (A-B). Rab3D expression in stably transfected tumor cell lines. (C). Representative images of primary tumors and lung section stained with hematoxilin and eosin in control and Rab3D-MCF-7 xenografts bearing mice (n = 5 mice per group). (D). H & E staining of lung in control and shRab3D-MDA-MB-231 xenografts bearing mice. (E). Immunohistochemical staining images and quantification of EMT-related signaling activation in control and Rab3D-MCF-7 xenografts. Scale bar, 50μm. (F). The level of plasma Hsp90α in nude mice detected by ELISA assay (n = 5 mice per group). (G). The working model for Rab3D-induced tumor cell invasion. In response to hypoxia, intracellular Rab3D is increased and its expression is correlated with tumor malignancy. Rab3D regulates exosomes release and Hsp90α secretion, which promotes EMT and tumor metastasis. eHsp90α indicates extracellular Hsp90α.