The Dual Roles And Mechanisms Of KIF22in Breast Cancer Cell Proliferation And Progression

Background:The kinesin family member22(KIF22), also known as kinesin-like DNA binding protein, is a plus-end-directed microtubule-based motor and binds to both microtubules and chromosomes. Although the function of KIF22as a motor protein during mitosis has been well defined, the expression status, function and underlying mechanism of KIF22during carcinogenesis and progression remain to be explored. As a DNA-binding protein, KIF22can bind to the promoter region, however, its transcriptional regulatory function still has not been defined. In our previous study, we found that KIF22is upregulated in primary breast cancer tissues compared with the paired adjacent normal breast, but downregulated in lymph node metastasis tissues compared with the paired primary breast cancer tissues. Thus, we speculated that KIF22plays different roles during carcinogenesis and progression, and regulates the breast cancer proliferation and metastasis by different mechanisms. In addition, our previous study indicated that CDC25C is the target gene of KIF22by ChIP-on-chip analysis, suggesting that KIF22may participated in tumorignesisn and progression in CDC25C-dependent manner. This study aims to clarify the opposite role of KIF22in cancer proliferation and metastasis. To further clarify the mechanism of KIF22in cancer proliferation medicated by CDC25C and the mechanism of KIF22in cancer metastasis medicated by TGF-β.Methods:1. RNA interference was used to knock down the KIF22expression. To determine the effect of KIF22depletion on the proliferation and metastasis of breast cancer cells, MTT, Fluorescence activated cell sorter (FACS), Immunofluorescence (IFC) and Transwell assays were performed to evaluate the biological behavior of KIF22-depleted and control cancer cells.2. To assess the role of KIF22in cell cycle progression, cell synchronization was performed to synchronize cells in G0/G1, G1/S and G2/M phases, respectively. FACS, Immunoblot and IFC assays were used to analyze the effect of KIF22depletion on the cell cycle.3. To determine the effect of KIF22depletion on the EMT of breast cancer cells, RT-QPCR, Immunoblot and IFC assays were used to detect the expression of EMT biomarkers and EMT-related transcription factors.4. Chromatin immunoprecitation (ChIP) and dual-luciferase reporter assays were used to validate the transcriptional regulation of KIF22on CDC25C. To further clarify the mechanism of KIF22-regulated cell cycle progression, RT-QPCR, Immunoblot and site-directed mutation assays were performed.5. To clarify the mechanism of induction of EMT by KIF22depletion, SMAD luciferase reporter, RT-QPCR, Immunoblot and IFC assays were performed.Results:1. Depletion of KIF22inhibits cancer cell proliferation and leads to G2/M arrest, accompanied by an increased number of binucleated and multinucleated cells.2. After serum starvation and release, FACS analysis showed that depletion of KIF22did not affect G1/S transition, and the expression of Cyclin A, P-Rb and CDK1is not changed. Furthermore, cells were synchronized using a double-thymidine block to arrest cells at G1/S phases, we noted an increased accumaulation of G2/M cells in KIF22-depleted cells and the expression of Cyclin B1and Cyclin E is significantly changed accompanied with an increased number of mitotic metaphase cells and a decreased number of mitotic telophase cells. To further clarify the role of KIF22in M phase progression, we synchronized cells at G2/M phases using a thymidine-nocodazole block. W obsercved a decreased accumulation of G1phaes cells in KIF22-depleted cells by FACS analysis. The CDK1activity is also changed and the number of mitotic metaphase cells increased in KIF22-depleted cells. Together, these results indicated that depletion of KIF22leads to a faster S to G2/M progression but a slower mitotic exit.3. ChIP assay showed that KIF22can bind to the promoter region of CDC25C. The dual-luciferase reporter assay indicated that KIF22suppress the transcriptional activity of CDC25C depedent on Thr463phosphorylation and the phosphorylated KIF22suppresses CDK1activivy. These results demonstrated that KIF22suppressed CDK1activation through the down-regulation of CDC25C expression.4. KIF22inhibits CDK1activity by transcriptionally downregulation of CDC25C expression in a KIF22Thr463site phosphorylation dependent manner.5. Depletion of KIF22promotes breast cancer cell migration and invasion. Depletion of KIF22up-regulates the expression level of the genes involved in mesenchymal phenotype (Vimentin and N-cadherin) and EMT-related transcription factor (Twistl,Snail and Slug), and down-regulates the expression level of the genes involved in epithelial phenotype (E-cadherin and (3-catenin). These results indicated that depeltion of KIF22induces an EMT phenotype.6. Depletion of KIF22up-regulates TGF-β1expression. The SMAD luciferase reporter analysis showed that depletion of KIF22induces TGF-β signaling. Depletion of KIF22up-regulates pSmad3expression accompanied with an increased proportion of Smad3nuclear localization. These results indicated that depletion of KIF22induces TGF-β signaling by upregulating TGF-β1expression.Conclusions:1. Depletion of KIF22expression suppresses cancer cell proliferation by delaying mitotic exit through upregulating CDC25C expression.2. Depletion of KIF22expression promotes breast cancer migration and invasion by induction of EMT through activating TGF-β signaling.