Protein-protein Interactions Between Ezrin And P65/Smad2 And Their Effects On Invasion And Proliferation Of Breast Cancer Cells

BackgroundBreast cancer is one of the most common malignant tumors in the world, which is with the increasing tendency of 0.8% each year. Most of the patients with breast cancer are the females with age between 50 and 54, but there is still 1% of the breast cancer happened in males. The metastasis of breast cancer tumor is the main cause leading to death. The mechanism of tumor metastasis is complicated, with multiple signal pathways acting and being regulated by different factors. Although lots of previous studies reported about the clinical situation of breast cancer, the mechanisms of the tumor metastasis still lacked of systematic and comprehensive investigations, especially on the critical proteins and protein-protein interactions (PPIs), which was lacking for systematic statistics and reports.The interactions between different proteins compose the large and complicated signal network in vivo and in vitro, which precisely regulates and controls the physiological process in human body. As the basis of tumor pathogenesis and development, the changes of tumor genome are usually related to the changes of PPIs network, especially the proteins related to signal pathways. Therefore, PPIs provide the interventions and therapies on different tumors with the feasible molecular targets. Wei Wang et al. summarized that there were more than 30 kinds of proteins related to and interacted with nuclear factor kappa-light-chain-enhance of activated B cells (NF-kB). EZH2 interacting with p65/RelA could induce and activate NF-κB in breast cancer (Estrogen Receptor-, ER-), but with opposite effects in breast cancer (ER+). ER and p65 could inhibit the activity of the other. Notch-I interacted with IKK-a, which inhibited the activation of NF-κB; β-TRCP interacted with IκB-α, which induced the recombination of IκB-α by protease system, then promoted the degradation of proteasome and activated NF-κB signal pathway. HSP-27 interacted with IKKα/β, which could inhibit NF-κB activity.Therefore, that starting from the PPIs and further investigations on the mechanisms in tumor metastasis are important for the therapy and prevention in breast cancer.Ezrin, one of the family members of Ezrin-Radxin-Moesin (ERM), is the protein encoded by vil2 gene, containing 586 amino acids (AAs). The N-terminal of Ezrin is the FERM domain with 296 AAs, and the C-terminal is the C-ERMAD domain, which could change the inactive Ezrin into active Ezrin. By changing the structure of microvilli, Ezrin could change the morphology of the cells, regulate the adhesion among cells, and play in the different physiological activities, which would influence the tumor metastatic and invasive abilities. For example, the activated Ezrin is the critical factor in tumor metastasis, not only in the cell morphological changes, but also in the responding to epidermal growth factor (EGF).Ezrin was also reported to be related to breast cancer metastasis and identified as one of the influencing factors in the prognosis of breast cancer. In MCF-10A, as the carcinoma cells in situ, and MDA-MB-231, as the invasive carcinoma cells, the knockdown of Ezrin could both decrease the invasive abilities of the two. Mak et al. found that Src/Ezrin interaction could regulate the metastasis and invasion of breast cancer in situ, while Wan et al. found that β4 integrin/Ezrin interaction could regulate the lung metastasis of osteosarcoma. Therefore, it is important to investigate on Ezrin and its interactions with other proteins in the mechanisms of breast cancer metastasis and invasion, which involves the activation and interaction of different signal pathways.In different kinds of malignant tumors, Ezrin and its interactions with other proteins make many effects on the tumor progression. Harrison et al. found that in the prostate cancer cells, Ezrin interacted with CD44 and regulated the interaction between prostate cancer cells and epithelial cells by co-translocation, which influenced the metastasis and invasion of prostate cancer. Brownd et al. studied on oral cancer cells and showed that Ezrin interacted with Dsg3, which could promote the potential metastasis of oral cancer.However, there are few researches on Ezrin interacting with NF-κB. NF-κB is the nuclear transcription factor protein family, whose the activated signal pathway is considered as the critical link in the stress response, regulating the expressions of many genes relating to the cell apoptosis, virus replication, tumor pathogenesis and development, inflammatory response, autoimmune diseases and so on. According to previous researches, NF-κB was with highly constitutive expression in inflammatory breast cancer (IBC). Another breast cancer subtype with high constitutive NF-κB is triple negative breast cancer (TNBC). Nowadays, the clinical investigations on the relationship between NF-κB and breast cancer show that the activation of NF-κB is related to the tumor size, lung metastasis, brain metastasis and HER2 expression.NF-κB is the protein complex containing 5 subunits, including Rel (cRel), p65 (RelA, NF-κB3), RelB, p50 (NF-κB 1) and p52 (NF-κB2). The most common dimer of NF-κB consists of p50 and p65, widely distributing in the variety kinds of cells. NF-κB is correlated to the transcription and expression of some genes, which could regulate immune responses and inflammatory reactions. In the non-malignant breast cancer, MCF-10A cell lines, the overexpression of p65 subunit could promote epithelial-mesenchymal transition (EMT). On the other hand, NF-KB-p65 is related to the tumor pahogenesis, proliferation, metastasis and invasion, apoptosis and immune activity. Therefore, whether the interaction between Ezrin and NF-κB could influence the metastasis and invasion in breast cancer is needed further attentions and investigations.On the other hand, the recent researches on the relationship between Ezrin and Smad protein family is very few. Smad protein family is the important cytokine of transforming growth factor-β (TGF-β) superfamily, mediating different members of TGF-β family transporting from cytoplasm into nucleus and regulating the physiological processes including tumor growth, development and apoptosis. Smad2 belongs to the receptor regulated Smads (R-Smad2), which participates in the regulation on signal transduction in mammal cells. Petersen et al. studied on the bone metastasis of breast cancer and found that knocking down Smad2 could promote the breast cancer metastasis. Chen et al. investigated and found that phosphorylated-Smad2 (p-Smad2) could be the index of poor prognosis and survival in the patients with non-small cell lung cancer in differently clinical stages. Liao et al. discovered that SIAH2 interacted with Smad2, which regulated the degradation and ubiquitin. However, there is no research on the relationship between Ezrin and Smad2, as well as whether the interaction between the two could influence tumor metastasis. Therefore, it is worth to study on the relationship and interaction between Ezrin and Smad2 in the metastatic cancer.Otherwise, there are many techniques using in study PPIs, including Co-Immunoprecipitation (Co-IP), tandem affinity purification (TAP), yeast 2 hybrid (Y2H), pull down, two-dimensional electrophoresis (2DE), Immunofluorescence (IF) and so on. The immunoprecipitation (IP) combined with Liquid Chromatography Mass Spectrometry (LCMS) could be used to screen the proteins in the PPIs network. In our study, we used IP combined with LCMS to find the interacting proteins with Ezrin in MDA-MB-231 cell. Ezrin/p65 and Ezrin/Smad2 were found and identified by Co-IP combined with IF. The effects of Ezrin/p65 and Ezrin/Smad2 on the invasion and proliferation of breast cancer cells were studied.Objectives1. To investigate the PPIs network between Ezrin and its binding proteins in breast cancer cells, and then select p65 and Smad2 as the subjectives to identify their interactions with Ezrin; 2. To investigate the effects of Ezrin/p65 and Ezrin/Smad2 on the invasion and proliferation of breast cancer cells.Methods1. Breast cancer cells cultureMDA-MB-231 cell was cultured with Leibovitz-15 (L-15) medium containing 10% fetal bovine serum (FBS), 100U/mL penicilin,100μg/mL streptomycin and 2mmol/L glutamine (Gln) in the conditions of 37℃ and saturated humidity without CO2, while MCF-7 was cultured with dulbecco minimum essential medium (DMEM) containing 10% FBS, 100U/mL penicilin, 100μg/mL streptomycin and 2mmol/L Gln in the conditions of 37 ℃,5% CO2 and saturated humidity. Passaged and cultured the cells with 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid (EDTA) when with 90% of confluence.2. IP combined with LCMSFirst IP combined with LCMS detection:MDA-MB-231 cell lysis was obtained by IP and analyzed by sodium dodecylsulphate-polyacrylamide gel electrophresis (SDS-PAGE) combined with (coomassie brilliant blue) CBB staining, in order to confirm the enrichments of Ezrin and its binding proteins. The IP sample was sent to BGI Company (Shenzhen, China) for LCMS. The target proteins were selected.Second IP combined with LCMS detection:MDA-MB-231 cell lysis was obtained by IP and analyzed by SDS-PAGE combined with silver staining, in order to confirm the enrichments of Ezrin and its binding proteins. The IP sample and IgG control were sent to BGI Company for LCMS, in order to confirm the PPIs.3. Co-IP, Western Blot (WB) and IF3.1 Cytoplasmic and nuclear proteins extraction and Co-IPExtracted the cytoplasmic and nuclear proteins in MDA-MB-231 and MCF-7 cells, respectively. Co-IP was carried on to detect the PPIs of Ezrin/p65, Ezrin/Smad2.3.2 WBWB was used to detect the expressions and PPIs of Ezrin/p65, Ezrin/Smad2 in MDA-MB-231 and MCF-7 cells.3.3 IF detecting co-localizationsEzrin/p65, Ezrin/Smad2 in MDA-MB-231 and MCF-7 cells were with IF staining. Confocal microscopy was used to detect and confirm the expressions and co-localizations of Ezrin/p65, Ezrin/Smad2.4.3xFlag-Ezrin-6xHis plasmid construction and transfection4.13×FIag-Ezrin-6xHis plasmid constructionpcDNA3.1 vector was used to construct the plasmid containing 3 X Flag and 6X His tags, as well as the complete Ezrin encoding sequence. The 3 X Flag-Ezrin-6 X His plasmad was amplified by DH5a and extracted.4.2 Transfection, Co-IP and IFTransfected MDA-MB-231 and MCF-7 cells with the plasmids according to the following groups: ①co-transfected with 3 X Flag-Ezrin-6 X His and GFP-RelA (GFP-p65); ②co-transfected with 3 X Flag-Ezrin-6 X His and Smad2-HA.Co-IP:Extracted and separated the cytoplasmic and nuclear proteins, and then carried on Co-IP to identify the PPIs of Ezrin/p65, Ezrin/Smad2.IF:Ezrin/p65, Ezrin/Smad2 were with IF staining. Confocal microscopy was used to detect and confirm the expressions and co-localizations of Ezrin/p65, Ezrin/Smad2.5. Transwell detecting breast cancer cells invasion5.1 Silencing and/or overexpressing target proteinsIn order to identify the effect of Ezrin/p65 PPI on cell invasion, the groups were followed: ①vector group; ②silencing Ezrin; ③silencing p65; ④overexpressing p65; ⑤silencing Ezrin and overexpressing p65.In order to identify the effect of Ezrin/Smad2 PPI on cell invasion, the groups were followed: ① vector group; ② silencing Ezrin; ③ silencing Smad2; ④ overexpressing Smad2; ⑤ilencing Ezrin and overexpressing Smad2.5.2 Transwell detecting breast cancer cells invasionMDA-MB-231 and MCF-7 cells, which were with treatments for silencing and/or overexpressing according to the groups mentioned above, were transferred into the Transwell chambers with Matrigel. After culturing for 18h, cleared the Matrigel and cells in the upper chambers. Stained the cells in the lower chambers with crystal violet and observed by microscopy. Counted the cells and analyzed the differences among groups.6. Cell counting kit-8 (CCK-8) method detecting breast cancer cells proliferation6.1 Silencing and/or overexpressing target proteinsIn order to identify the effect of Ezrin/p65 PPI on cell proliferation, the groups were followed: ①vector group; ②silencing Ezrin; ③silencing p65; ④silencing Ezrin and p65; ⑤silencing Ezrin and overexpressing p65; ⑥silencing p65 and overexpressing Ezrin.In order to identify the effect of Ezrin/Smad2 PPI on cell proliferation, the groups were followed: ①vector group; ②silencing Ezrin; ③silencing Smad2; ④ silencing Ezrin and p65; ⑤silencing Ezrin and overexpressing Smad2; ⑥silencing Smad2 and overexpressing Ezrin.MDA-MB-231 and MCF-7 cells were cultured in 96-well plates,5 plates in each cell line, and transfected for 24h,48h,72h,96h and 120h.6.2 CCK-8 detecting breast cancer cells proliferationAccording to the points of time after transfection, added CCK-8 liquid into the breast cancer cells and incubated for 20min. Enzyme labeling instrument was used to detect the OD value of MDA-MB-231 and MCF-7 cells at 450nm.Results1. PPIs network of Ezrin in MDA-MB-231 cellPrimary IP combined with CBB identified Ezrin enrichment. The LCMS results showed that there were totally 1889 proteins binding to Ezrin, including p65 and Smad2.Secondary IP combined with silver staining identified Ezrin and its binding proteins involving p65 and Smad2 by LCMS, comparing to IgG control.2. Ezrin/p65 PPI identification in breast cancer cells2.1 Endogenous Ezrin/p65 PPI identificationThe results of Co-IP and WB showed that, in both MDA-MB-231 and MCF-7 cells, Ezrin/p65 PPI was mainly in cytoplasm but not in nucleus. IF results showed that Ezrin/p65 was co-localized in cytoplasm but not in nucleus. The mutual corroboration was found in the two results mentioned above.2.2 Exogenous Ezrin/p65 PPI identificationCo-IP and WB results showed that, in both MDA-MB-231 and MCF-7 cells, Ezrin/p65 PPI was mainly in cytoplasm but not in nucleus. IF results showed that Ezrin/p65 was co-localized in cytoplasm but not in nucleus. The mutual corroboration was found in the two results mentioned above.3. Ezrin/Smad2 PPI identification in breast cancer cells3.1 Endogenous Ezrin/Smad2 PPI identificationThe results of Co-IP and WB showed that, in MDA-MB-231 cell, Ezrin/Smad2 PPI was in both cytoplasm and nucleus, while in MCF-7 cell, Ezrin/Smad2 PPI was mainly in cytoplasm but not in nucleus. IF results showed that, in MDA-MB-231 cell, Ezrin/Smad2 was co-localized in both cytoplasm and nucleus, while in MCF-7 cell, Ezrin/Smad2 was mainly co-localized in cytoplasm but not in nucleus. The mutual corroboration was found in the two results mentioned above.3.2 Exogenous Ezrin/Smad2 PPI identificationCo-IP and WB results showed that, in MDA-MB-231 cell, Ezrin/Smad2 PPI was in both cytoplasm and nucleus, while in MCF-7 cell, Ezrin/Smad2 PPI was mainly in cytoplasm but not in nucleus. IF results showed that, in MDA-MB-231 cell, Ezrin/Smad2 was co-localized in both cytoplasm and nucleus, while in MCF-7 cell, Ezrin/Smad2 was mainly co-localized in cytoplasm but not in nucleus. The mutual corroboration was found in the two results mentioned above.4. Ezrin/p65, Ezrin/Smad2 promoting breast cancer cells invasionIn both MDA-MB-231 and MCF-7 cells, ①Not matter in silencing Ezrin or in silencing p65 or Smad2 groups, the cell counting was more than in vector group, significantly (P＜0.05); ②Overexpressing p65 or Smad2, the cell counting was more than in vector group, significantly (P<0.05); ③In silencing Ezrin and overexpressing p65 or Smad2 groups, the cell counting was more than in silencing Ezrin group, significantly (P<0.05); ④In silencing Ezrin and overexpressing p65 or Smad2 groups, the cell counting was less than in overexpressing p65 or Smad2 groups, significantly (P<0.05). The results showed that silencing Ezrin could inhibit the promotion on breast cancer cells invasion by overexpressing p65 or Smad2, which indicated that the promotions of p65 or Smad2 on breast cancer cells invasion depended on Ezrin expression, and the PPIs of Ezrin/p65, Ezrin/Smad2 could promote breast cancer cells invasion.5. No influences of PPIs of Ezrin/p65, Ezrin/Smad2 on breast cancer cells proliferation5.1 No influences of PPIs of Ezrin/p65, Ezrin/Smad2 on MDA-MB-231 cell proliferationFor detecting the effect of Ezrin/p65 on MDA-MB-231 cell proliferation, factorial analysis showed that there was interaction effect between days and groups (F=8.29, P=0.00). By adjusting R2=0.93 and paired comparison, there was no significant differences between groups in day 1 (transfecting for 24h)(F=0.03, P=0.99). From day 2 to day 5 (transfecting for 48h-120h), there were significant differences between groups (P<0.05). By multiple comparisons, there were significant differences between the experiment groups and vector group (P<0.05), while there was no significant difference among the experiment groups.For detecting the effect of Ezrin/Smad2 on MDA-MB-231 cell proliferation, factorial analysis showed that there was interaction effect between days and groups (F=7.24, P=0.00). By R2=0.92 and paired comparision, there was no significant differences between groups in day 1 (transfection for 24h)(F=0.03, P=0.92); From day 2 to day 5 (transfection for 48h-120h), there were significant differences between groups (P<0.05); By multiple comparisons, there were significant differences between the experiment groups and vector group (P<0.05), while there was no significant difference among the experiment groups.5.2 No influences of PPIs of Ezrin/p65, Ezrin/Smad2 on MCF-7 cell proliferationFor detecting the effect of Ezrin/p65 on MCF-7 cell proliferation, factorial analysis showed that there was interaction effect between days and groups (F=8.29, P=0.00). By adjusting R2=0.96 and paired comparision, there was no significant differences between groups in day 1 (transfecting for 24h)(F=0.03, P=0.99) and day 2 (transfecting for 48h)(F=1.09, P=0.38). From day 3 to day 5 (transfecting for 72h～120h), there were significant differences between groups (P＜0.05). By multiple comparisons, there were significant differences between the experiment groups and vector group (P＜0.05), while there was no significant difference among the experiment groups.For detecting the effect of Ezrin/Smad2 on MCF-7 cell proliferation, factorial analysis showed that there was interaction effect between days and groups (F=11.84, P=0.00). By adjusting R2=0.98 and paired comparision, there was no significant differences between groups in day 1 (transfecting for 24h)(F=0.94, P=0.46) and day 2 (transfecting for 48h)(F=1.97, P=0.10). From day 3 to day 5 (transfecting for 72h～120h), there were significant differences between groups (P＜0.05). By multiple comparisons, there were significant differences between the experiment groups and vector group (P＜0.05), while there was no significant difference among the experiment groups.Conclusions1. There were 1889 proteins binding to Ezrin in MDA-MB-231 cell lysis totally, including p65 and Smad2.2. In MDA-MB-231 and MCF-7 cells, the PPI and co-localization between Ezrin and p65 were mainly in cytoplasm while not obvious in nucleus.3. In MDA-MB-231 cell, the PPI and co-localization between Ezrin and Smad2 were in both cytoplasm and nucleus, while in MCF-7 cell, the PPI and co-localization were mainly in cytoplasm but not obvious in nucleus.4. The PPIs between Ezrin and p65, Ezrin and Smad2 could both promote MDA-MB-231 and MCF-7 cells invasion.5. There was no obvious effect of the PPIs between Ezrin and p65, Ezrin and Smad2 on MDA-MB-231 or MCF-7 cells proliferation.