Heat Shock Proteinβ And Enterolactone Impact Focal Adhesion Kinase And Regulate Cell Migration And Invasion In Breast Cancer Cells

Part Ⅰ HSP90β stabilizes FAK and regulates cell migration and invasion in breast cancer cellsObjectives:HSP90 is a highly conserved molecular chaperone which participates in variety of molecular processes through regulating folding, maturation and degradation of client proteins. However, as an important tyrosine kinase, FAK has not yet been generally considered as a client of HSP90. Focusing on the interaction between HSP90β and FAK and the mechanism within FAK regulating cell growth, migration and invasion in MDA-MB-231 breast cancer cell, this study is to find novel tumorigenesis associated client protein for HSP90, which will provide a potential drug target in combination with HSP90 inhibitors.Methods:1.Interaction between FAK and HSP90β were determined by plasmids construction, coimmunoprecipitation and immunolfluorescence assays; HSP90β binding domain for FAK was confirmed by GST-pull down;2.Impacts of HSP90β on FAK stability were observed in MDA-MB-231 cells treated with 17-AAG, a HSP90 inhibitor and cycloheximide(CHX), a protein synthesis inhibitor. To analyze whether FAK is degraded through the ubiquitin/proteasome pathway, ubiquitin protein was immunoprecipitated with FAK antibody and then detected in MDA-MB-231 cells treated with 17-AAG, MG-132 alone or together.3.The growth and metastasis of MDA-MB-231 cells treated with HSP90β and FAK inhibitors were detected with establishment of stable cell lines, soft-agar, cell scratch assay, transwell assay and cytoskeleton staining.Results:1.Expression plasmids of full length HSP90β and three domain mutant (N terminal 1-232 fragment, middle 233-620 fragment, C terminal 621-724 fragment) were successfully constructed.2.Coimmunoprecipitation assay confirmed the interaction between endogenous and exogenous HSP90β and FAK in MDA-MB-231 cells. Immunofluorescence assay showed that HSP90β and FAK were colocalized in MDA-MB-231 and H4 cells.3.Region of the 233-620 amino acid residues of HSP90β is the main binding site for FAK.4.FAK is degraded by the ubiquitin/proteasome pathway. HSP90β stabilizes FAK and protects it from degradation.5.PF-573228 and 17-AAG inhibited the growth, migration and invasion of MDA-MB-231 cells; specific inhibition of FAK or HSP90β expression by RNA interference decreased growth and migration of MDA-MB-231 cells.6.PF-573228 and 17-AAG decreased MDA-MB-231 and H4 cell migration through interfering with cell cytoskeleton.Conclusion:1.HSP90β interacts with FAK. The middle region of HSP90β is the main binding domain for FAK.2.HSP90β plays a critical role in FAK stability. As an oncogenic client protein of HSP90, FAK is degraded through proteasome-dependent pathway.3.HSP90β regulated FAK signaling pathway, which plays an important role in promoting the process of growth and metastasis of breast cancer cells.4.Breast cancer cells treated with inhibitors of FAK or HSP90β exhibited decreased cell growth and metastasis, which provides a novel way contributing for cancer therapy.Part Ⅱ Inhibitory effects of Enterolactone on growth and metastasis inbreast cancer cellsObjective:Lignan rich diet has been confirmed to reduce the risk of human breast cancer. Enterolactone, one of the active polyphenol metabolites of lignan, was reported to have antitumor effect for estrogen receptor (ER) negative breast cancer. However, the mechanism underlined of ENL effects on breast cancer cell growth and metastasis is not clear. The purpose of this experiment study is to explore the molecular mechanism of Enterolactone against tumor.Methods:1.The anti-proliferative effects of Enterolactone were assessed by a methyl thiazolyltetrazolium (MTT) assay. The effects of Enterolactone on the cell cycle in MDA-MB-231 cells were determined by flowcytometry. Q-PCR was used to confirm the mRNA level of genes associated with cell proliferation and cell cycle in Enterolactone treated MDA-MB-231 cells.2.Phosphorylation of FAK was detected by western blot. Metastasis and cytoskeleton of MDA-MB-231 cells treated with Enterolactone were detected by cell scratch assay, transwell assay and cytoskeleton staining.Results:1.Enterolactone inhibited growth of MD A-MB-231 cells in dose and time-dependent manner, the IC50 of Enterolactone on MDA-MB-231 cell at 48 hours treatment was 261.9±10.5 μM.2.Enterolactone induced cell accumulation in the S phase. The percentage of cells at the S phase significantly increased from 7.03% to 19.35%,21.67%, 28.32% after treatment with 50,100 and 200μM Enterolactone, respectively. The percentage of cells in G1 and G2/M phase were decreased compared with the control group.3.Ki67, PCNA, and FoxMl which are necessary for cellular proliferation were decreased with the increase of the concentration of Enterolactone. Meanwhile, Cyclin El, Cyclin A2, Cyclin B1 and Cyclin B2, which are associated with S and G2/M phase of cell cycle were decrease, but CDK4, CDK6 and Cyclin D1 that participate in G0/G1 phase regulation had no obvious change.4.Enterolactone significantly reduced phosphorylation of FAK and Paxillin, which is an important downstream effector of FAK.5.Enterolactone inhibited MDA-MB-231 cell motility by interfering with the cytoskeleton.Conclusion:1. Enterolactone affects cell proliferation via inhibiting expression of PCNA, Ki-67 and FoxMl, and cell cycle phase via regulating the expression of genes associate with the S and G2/M phases of cell cycle. These findings provide new insights into the molecular mechanisms that Enterolactone exerts antitumor effect.2. Enterolactone interfered with the cytoskeleton, suppressed migration and invasion of breast cancer cells, at least partially, through inhibiting phosphorylation of FAK/paxillin signaling pathway. These findings suggest that Enterolactone may serve as a potentially anticancer agent for breast cancer progression.