P62/SQSTM1 Interacts With Vimentin To Enhance Breast Cancer Metastasis

1.BackgroundCancer metastasis is a complex multi-step cell biological process termed the invasion-metastasis cascade,by which primary tumour cells acquire the invasive ability and then spread to distant organs.Metastases are responsible for more than 90%of cancer-related deaths in various solid malignancies,including breast cancer.Despite the early diagnostic methods,surgical resection and adjuvant therapy have improved,metastatic disease is largely incurable.A large amount of evidence demonstrates that cancer stem cells?CSCs?,a tumor subpopulation which is critical for tumour metastasis,relapse and drug resistance,possesses indefinite potential for self-renewal that drive tumorigenesis.Therefore,the characteristics of cancer stem cells are closely related to tumour metastasis phenotype.Recent studies have shown that the primary cause for cancer cells to drive the invasion-metastasis process is the acquisition of the genetic or epigenetic alterations.Thus,the exploration of key molecules and mechanisms underlying breast cancer metastasis and breast cancer stemness regulation is urgently needed.The adaptor protein p62?also known as SQSTM1?is initially identified as a cytosolic 62kDa protein which can bind to the isolated src homology 2?SH2?domain of p56^lck.This multidomain protein interacts selectively with different signalling intermediaries,such as Raptor,Nrf2-binding site on Keap1,ubiquitin and LC3,to regulate metabolic reprogramming,antioxidant response and selective autophagy,respectively.Abnormal p62 overexpression has been documented in various neoplasms,especially in breast cancer.For example,high p62 expression is associated with breast tumours exhibiting the clinicopathological features of aggressive disease,as well as overexpression of EGF receptor?EGFR?,HER2,HER3 and HER4.In triple-negative breast cancers,patients with p62 accumulation exhibit a higher risk of positive lymph node and lymphovascular invasion.The preliminary results of our research group revealed that p62 signaling adaptor protein plays an important regulatory role in the phenotypic characteristics of breast cancer stem cells,and targeting this protein can effectively inhibit the characteristics of breast cancer stem cells.These findings highlight the potential of p62 as a therapeutic target during cancer progression.However,the detailed mechanism by which p62-mediates cancer cell invasion and metastasis remains largely unknown.Studies of epithelial malignancies indicate that the acquisition of invasion and metastasis potential by the incipient cancer cell may depend on the transition of a mesenchymal phenotype.Vimentin,a Type?intermediate filament,serves as a classical mesenchymal phenotype biomarker.Furthermore,vimentin protein overexpression positively correlates with cell motility,induction of EMT,metastatic disease and poor prognosis.Vimentin-deficient?-/-?mice reveal weakened wound healing ability in all stages of growth as a result of the seriously impaired fibroblasts in their capacity to migrate.In this study,we found that p62 binds to vimentin and regulates vimentin protein level,which in turn contributes to cancer cell invasion and metastasis.Thus,this p62-vimentin interaction may be a promising target and provide new opportunities for therapeutic intervention.2.Methods?1??1?We collected 5 pairs of clinical metastatic breast cancer and adjacent normal tissues,and conducted Western blot analysis of p62 expression levels.?2?We analyzed the gene expression value of normal/primary tumour/metastatic tumour tissues from TCGA database.?3?Using Kaplan-Meier survival analysis to evaluate the clinical relevance of p62.?2??1?We used shRNA to mediate p62 depletion in metastatic MDA-MB-231 cells and lentivirus to facilitate ectopic overexpression of p62 in non-metastatic MCF-10A cells.The efficiencies of p62 knockdown and overexpression were assessed by Western blot.?2?We built up a microfluidic model to observe local invasion of cancer cells in real time to assess the contribution of p62 in promoting breast cancer invasive ability.?3?In vivo experiment:?1?In zebrafish embryo xenograft assay,we engrafted MCF-7 siNC and MCF-7 sip62 cells,stably expressing red fluorescent proteins,in a zebrafish embryo host.?2?In mice xenograft models,control shRNA MDA-MB-231and p62 shRNA MDA-MB-231 cells were injected into the lateral tail vein of 4⁶weeks old BALB/C?nu/nu?female nude mice to investigate the effect of p62 on breast cancer metastasis.?3?Equal number of control and p62 knockdown MDA-MB-231cells?1×10⁶ cells?were subcutaneously injected into 4⁶ weeks old BALB/C?nu/nu?female nude mice?5 mice in each group?to determine the effect of p62 knockdown on tumour growth.Tumour xenografts were then measured every week and mice sacrificed after 8 weeks.?4??1?Taking advantage of the proteomics-based approach,we analyzed endogenous p62 coimmunoprecipitated?Co-IP?proteins by mass spectrometry to identify putative p62 protein binding partners.?2?Co-IP assay was conducted to examine whether endogenous p62 bound to the target protein.?3?The p62-target protein interaction was further confirmed by glutathione S-transferase pull-down assay.?4?Immunofluorescence studies using p62 and the target protein specific antibodies.?5?Western blot assay was performed to detect the effect of p62 on the expression of target protein.RT-qPCR assay was performed to detect the effect of p62 on the expression of target gene.?5??1?We overexpressed target gene in both MDA-MB-231-shNC and MDA-MB-231-shp62 cells and conducted shRNA-mediated target gene suppression in both MDA-MB-231-Ctrl and MDA-MB-231-p62-OE cells.Western blot assays were conducted to evaluate the protein levels of p62 and target gene.?2?We performed the microfluidic assay to verify the effect of target gene on p62-mediated cancer cells invasion.?3?We performed the transwell assay to verify the effect of target gene on p62-mediated cancer cells invasion.?6??1?Western blot assay was conducted to evaluate the protein levels of p62 and target gene in clinical breast cancer specimens.?2?Linear regression analysis of p62 and target protein relative expression values.3.Results?1?p62 expression was elevated in metastatic breast cancer and its overexpression correlated with poor prognosis:?1?Compared with normal tissues,p62 protein levels were up-regulated at varying degrees in all metastatic breast cancer tissues.?2?Analysis of gene expression value from TCGA showed that p62 mRNA expression levels in normal tissues?n=110?were significantly lower than that in both primary tumour?n=1065;-0.4355 versus-0.1485?and metastatic tumour tissues?n=7;-0.4355 versus-0.1423?.?3?Kaplan-Meier survival analysis of 87 breast carcinoma specimens revealed a correlation between the higher p62 expression and reduced metastasis-free survival times?P=0.011,GSE6532 from the GEO database?.104 breast cancer samples from GEO database?GSE42568?by univariate analysis showed that high p62expression was associated with decreased relapse-free survival times?P<0.0001?.?2?p62 promoted invasive phenotypes of breast cancer cells in vitro:?1?p62 was effectively suppressed by two different p62 shRNA species in MDA-MB-231 cells,and significantly increased by lentivirus-infection in MCF-10A cells.?2?Silencing p62expression dramatically reduced the invasive capacity of MDA-MB-231 cells as indicated by a decrease in both the area and distance of invasion.Overexpressing p62 in non-metastatic MCF-10A cells resulted in the acquisition of invasive ability as indicated by the microfluidic assay.?3?Inhibition of p62 attenuated breast cancer metastasis and led to decreased tumorigenicity in vivo:?1?At 3 days post-implantation?dpi?,silencing p62 expression in MCF-7 cells exhibited an obviously reduced invasive behavior when compared to control cells.In addition,zebrafish embryo engrafted with p62 siRNA MCF-7 cells had a lower metastases incidence?20/90,22.2%?compared to control cells?49/111,44.1%?.Quantification of the fluorescence intensity of invasive cells showed a significant decrease elicited by p62 knockdown.?2?Eight weeks after tail vein injection,mice injected with MDA-MB-231-shp62 cells had a markedly decreased metastatic burden as measured by macrography and H&E staining.Consistently,the area and number of metastatic nodules in the lung of mice injected with MDA-MB-231-shp62 cells were significantly reduced as compared with that injected with MDA-MB-231-shNC cells.?3?Comparing with mice inoculated with MDA-MB-231-shNC cells,which formed large tumours within 56 days,the mice inoculated with MDA-MB-231-shp62-1 and shp62-2 cells showed a significant reduction in the tumour growth as indicated by the decreased tumour volumes and tumour weights.?4?Identification of vimentin as a novel p62 binding partner and p62 suppression downregulated vimentin protein expression:?1?Combined Co-IP with mass spectrometry to identify putative p62 protein binding partners.?2?We detected vimentin after immunoprecipitation with a p62 antibody,but not with the control IgG,indicating that endogenous p62 interacted with vimentin.?3?The p62-vimentin interaction was further confirmed by glutathione S-transferase pull-down assay,by which GST-tagged p62 co-precipitated with His-tagged vimentin,as well as GST-tagged vimentin cooperated with Flag-tagged p62.?4?Immunofluorescence studies using p62and vimentin specific antibodies showed that both proteins co-localized in the cytoplasm in MDA-MB-231 cells.?5?shRNA or RNAi mediated depletion of p62downregulated the protein expression levels of vimentin in both MDA-MB-231 and SK-BR-3 cells.In contrast,lentivirus-mediated overexpression of p62 upregulated vimentin protein levels in both MCF-10A and MCF-7 cells.Interestingly,vimentin mRNA levels exhibited no significant differences whether in p62 knockdown MDA-MB-231 and SK-BR-3 cells or in p62 overexpression MCF-10A and MCF-7 cells compared with their relative controls.?5?Vimentin played an important role in p62-mediated breast cancer cells invasion:?1?Depletion of p62 attenuated vimentin expression,but overexpression of vimentin increased vimentin protein levels in both shNC and shp62 cells.Overexpression of p62promoted vimentin expression,but suppression of vimentin decreased vimentin protein levels in both Ctrl and p62-OE cells.?2?Re-constitution of vimentin expression in MDA-MB-231-shp62 cells abolished the reduction in invasive capacity.Knockdown of vimentin in MDA-MB-231-p62-OE cells attenuated the increased invasive ability and exhibited no significant difference in the capacity of invasion compared with vimentin knockdown MDA-MB-231-Ctrl cells.?3?Transwell invasion assay showed that both the vimentin overexpressed shNC cells and the vimentin overexpressed shp62 cells exhibited an increased invasive ability when compared to their relative shNC and shp62cells,and the difference in the invasive ability of vimentin overexpressed shNC and shp62 cells was not so dramatically as the difference in shNC and shp62 cells.Knockdown of vimentin in MDA-MB-231-p62-OE cells attenuated the increased invasive ability.?6?p62 expression was positively correlated with vimentin protein levels in clinical breast cancer specimens:?1?The expression of vimentin was relatively higher in samples which showed much more abundant p62 expression.?2?Linear regression analysis showed a positive correlation between p62 and vimentin protein expression?R²=0.7539,P=0.0011?.4.Conclusions?1?p62 expression is elevated in metastatic breast cancer and its overexpression correlates with poor prognosis.?2?p62 enhances breast cancer invasion and metastasis.?3?Identification of vimentin as a novel p62 binding partner and p62 suppression downregulates vimentin protein expression.?4?Vimentin plays an important role in p62-mediated breast cancer cells invasion.?5?p62 expression is positively correlated with vimentin protein levels in clinical breast cancer specimens.