Mechanisms Underlying TBC1D3 Oncogene-induced Migration Of Human Breast Cancer Cells

Objective:We recently found that the TBC1D3 oncogene promotes the migration of breast cancer cells,however,litter is known regarding the mechanism by which TBC1D3 induces the migration of cancer cells.Here,we demonstrated that TBC1D3 stimulated the oxidized low density lipoprotein receptor 1(OLR1),a stimulator of cell migration,in breast cancers at the transcritional level.TBC1D3 is a Hominoid-specific oncogene highly expressed in tumor cells and tissue,which interacts with a variety of protein.It is necessary to find new protein interact with TBC1D3 and explore the biological significance between them.Methods:In order to exprole the mechanism by which TBC1D3 induces the migration of cancer cells,MCF-7 cells were transfected with Flag-TBC1D3 or control Flag vector,then total RNA was extracted,and subjected to high-throughput sequencing.Q-RT-PCR was used to validate the RNA level and Western blot was used to validate the protein level.In order to define the role of OLR1 in where TBC1D3 affect the migration of MCF-7 cells,we build OLR1 shRNA lentivirus and control lentivirus NC shRNA.MCF-7 and BT549 cells were infected with NC and OLR1 shRNA lentiviruses,OLR1 gene expression was detected by Western blot to determine the effectiveness of specific shRNA.To perform transwell migration experiment,stable OLR1 knockdown MCF-7 were transfected with Flag-TBC1D3.In order to explore the role of TNF signaling pathway in the TBC1D3-induced up-regulation of OLR1,TNF inhibitor pomalidomide and NF-κB inhibition using its specific inhibitor caffeic acid phenethyl ester were chosen.Since activation of TNF signaling pathway is intricately regulated by TNFα itself and TNFα receptors as well as a variety of adaptor proteins such as TRAFs,we examined the release of TNFα from MCF-7 ad BT549 cells using the enzyme-linked immunosorbent assay.We also examined the expression of TNFR and multiple TRAFs by Western blotting.Since protein levels in cells are regulated y the rate of both their synthesis and degradation,we next sought to determine whether TBC1D3 protects TNFR1 and TRAFs from intracellular protein degradation.Since ubiquitination has been shown to play a vital role in TNF-induced degradation of TNFR1 via the proteasome pathway,we examined the ubiquitination fo TNFR1 in MCF-7 cells transfected with Flag-TBC1D3.In order to find new proteins interacting with TBC1D3,MCF-7 cells were transfected with Flag-TBC1D3 or control Flag vector,then seperation of cytoplasm and nuclear protein were subjected to immunoprecipitation using anti-Flag and beads,Precipitated was separated by SDS-PAGE and colored by coomassie brilliant blue dyestuff.Then,proteins that probably associated with TBC1D3 was analyzed by MS/MS.Results:we demonstrated that TBC1D3 stimulated the expression of OLR1,a stimulator of cell migration,in breast cancer cells at the transcriptional level.Depletion of OLR1 by siRNA or down-regulation of OLR1 expression using pomalidomide,significantly decreased TBC1D3-induced migration of these cells.Notably,TBC1D3 overexpression activated NF-κB,a major effector of TNFα signalling suppressed the effects of TBC1D3.Consistent with this,NF-κB inhibition using its specific inhibitor caffeic acid phenethyl ester decreased both TBC1D3-induced OLR1 expression and cell migration.TBC1D3-overexpressing MCF-7 and BT549 cells exhibited approximately 1.4-and 2.5-fold increases in TNF secretion,respectively.We also examined the expression of TNFR and TRAFs by Western blotting.Transfection with Flag-TBC1D3 considerably increased the expression of TNFR1,TRAF1,TRAF5 and TRAF6 proteins in MCF-7 and BT549 cells,while there was no significant change in the expression of TNFR2 and TRAF3.Consistent with these results,overexpression of TBC1D3 stimulated the Mrna expression of TNFR1,TRAF1,TRAF5 and TRAF6,but no TRAF3 in MCF-7 cells.we next sought to determine whether TBC1D3 protects TNFR1 and TRAFs from intracellular protein degradation.we foud MCF-7 cells transfected with control Flag vector showed a rapid degradation of TNFR.In contrast,TNFR1 degradation was significantly delayed in MCF-7 cells expressing TBC1D3.However,TBC1D3 failed to delay the degradation of TRAF1,TRAF3,TRAF5 and TRAF6 in MCF-7 cells.Afer cytoplasm and nuclear protein were subjected to immunoprecipitation using anti-Flag and beads,some possible proteins binding to TBC1D3 were found.Among them,42 and 66 KDa proteins were obvious and repeatable.On the contrary,there were no obvious protein bands with the same molecular weight for negative control.Then,the 42 and 55 KDa proteins were cut and sent for MS/MS assay.According to the results,two bands were Lamin B and Beta-actin respectively.TBC1D3 and β-actin interact in the cytoplasm and nuclear of MCF-7 cells and TBC1D3 interacts with Lamin B in the nuclear of MCF-7 cells by co-immunonrecitation.Conclusion:1.TNF signaling contributes to TBC1D3-induced OLR1 expression and cell migration2.TBC1D3 induces activation of TNF signaling on multiple levels,including by increasing the release of TNF,elevating the transcription of TNFR1,TRAF1,TRAF5 and TRAF6,and decreasing the degradation of TNFR13.TBC1D3 interacts with Beta-actin in the cytoplasm and nuclear of MCF-7 cells4.TBC1D3 interacts with Lamin B in the nuclear of MCF-7 cells...