Exploring The Role Of Anastasis In Paclitaxel Resistance In Colorectal Cancer

BackgroundColorectal cancer is one of the most prevalent cancers in China.According to the latest statistics in 2020,the incidence of colorectal cancer ranks third in the global tumor incidence,and it is the second leading cause of death among tumors all over the world.The main treatment for patients with early colorectal cancer includes surgery,neoadjuvant radiotherapy and neoadjuvant chemotherapy.For patients with advanced and metastatic colorectal cancer,chemotherapy is the most efficient treatment.In clinic,the most commonly used chemotherapy drugs for colorectal cancer include 5-fluorouracil,oxaliplatin,irinotecan and paclitaxel.Paclitaxel leads to mitotic halt and induces apoptosis by promoting microtubule polymerization.Paclitaxel is effective in the treatment of colorectal cancer,breast cancer,ovarian cancer and other malignant tumors.However,the resistance to paclitaxel has seriously affected the efficacy of chemotherapy,which is also the major reason that leads to recurrence and metastasis in colorectal cancer.Exploring the mechanism of paclitaxel resistance in colorectal cancer is essential for improving patient prognosis and reducing death from colorectal cancer.Inducing apoptosis through activation of Caspase 3 or Caspase 7 is one of the major strategies for tumor chemotherapy.It was believed that once the key checkpoints of apoptosis including the release of cytochrome c from mitochondria,activation of effector caspases,nuclear fragmentation and the formation of apoptotic bodies occur,apoptotic cell death is irreversible.However,some researchers found that some cells can recover from the initiated apoptotic program and executioner caspase activation,by a process called anastasis.It has been reported that compared with their parental tumor cells,anastatic breast cancer cells exhibited enhanced resistance to chemotherapy drugs.It was speculated that anastasis may be an important mechanism for tumor resistance to chemotherapy.Cullin 4B(CUL4B),which assembles the CUL4B-RING E3 ligase complex(CRL4B),was reported to be highly expressed in a variety of solid tumors.It was verified that CUL4B affects the occurrence and progression of tumors through epigenetically repressing the transcription of some tumor suppressor genes.Our previous studies have shown that CUL4B knock-down increases the sensitivity to chemotherapy drugs including cisplatin,mitomycin C,doxorubicin and paclitaxel in bladder cancer,gastric cancer,ovarian cancer and colorectal cancer cells,respectively,which suggested that CUL4B may be involved in the chemotherapy resistance of tumor cells.In this project,we demonstrated that anastasis occurred in colorectal cancer cells after paclitaxel treatment,and studied the role of anastasis in paclitaxel resistance of colorectal cancer cells.We also explored the role and mechanism of CUL4B in regulating paclitaxel resistance in colorectal cancer through anastasis modulation.Objectives1)To determine the role of anastasis in paclitaxel resistance of colorectal cancer cells.2)To determine the role and the related mechanism of CUL4B in regulating paclitaxel resistance via affecting anastasis.MethodsTo mark and trace Caspase-activated cells,we made colorectal cancer cell lines,HCT-116 and HT-29 carrying CasExpress,a lineage tracing system for cells that have experienced executioner caspase activation,HCT-116CasE and HT-29CasE.The expression and cleavage of Caspase-3 and PARP1 were determined by Western blot.FACS(fluorescence-activated cell sorting)was used to detect and sort the anastatic and apoptosis-resistant cells.MTT assay was used to analyze the viability of anastatic and apoptosis-resistant cells.EdU incorporation and clone formation assays were used to evaluate the proliferation rate of anastatic and apoptosis-resistant cells.Transwell assays were applied to test the migration and invasion ability of cells.Nude mice xenograft models were established to observe the tumor-forming ability of anastatic cells and apoptosis-resistant cells.Flow cytometry was employed to detect the proportion of stem cells in xenograft tumors.We established HCT-116CasE and HT-29CasE cell lines with CUL4B knocked down and over-expressed using lentiviral vector transfection.The effect of knocking down or overexpressing CUL4B on the percentage of anastatic cells after paclitaxel treatment was determined by flow cytometry.The expression of anastasis-related genes was examined by qRT-PCR.The protein levels of CUL4B and PIM1 with or without proteasome inhibitor MG 132 was evaluated by Western Blot.To determine the effect of PIM1 inhibition on anastasis,we used PIM1 inhibitor SGI-1776 and small interfering RNA to inhibit the expression and function of PIM1,and measured the percentage of anastatic cells after paclitaxel treatment using flow cytometry.Results(1)Anastatic cell models were established in colorectal cancer cells.Two cell lines,HCT-116CasE and HT-29CasE,which were stably transfected with CasExpress,a lineage tracing system for cells that have experienced executioner caspase activation,were established.Using live-cell imaging technique,we observed the anastatic process of colorectal cancer cells.We found that after treated with 20nM paclitaxel for 24 hours,HCT-116 and HT-29 cells exhibited typical apoptotic changes including disappearance of cell-cell connection,cell shrinkage and membrane blebbing.Green fluorescent protein was expressed in Caspase-3 activated cells and kept in anastatic cells.During the recovering process,cells stretched and began to divide,then grew into clones.The cleaved Caspase-3 and PARP1 were detected in cells under treatment and gradually diminished after drug removal.These results indicated that the anastatic cell model was successfully constructed in colorectal cancer cells,HCT-116 and HT-29,respectively.After induction of anastasis,the survived cells were sorted using FACS.Cells with green fluorescence were anastatic cells and the ones without green fluorescence were apoptosis-resistance cells.(2)Anastatic colorectal cancer cells exhibited increased proliferation ability.The results of MTT assays demonstrated that comparing to control cells,anastatic HCT-116 and HT-29 cells had higher viability.The results of EdU incorporation assay showed that the proliferation rate of anastatic HCT-116 and HT-29 cells was significantly higher than that of control cells.(3)Anastatic colorectal cancer cells are more resistant to chemotherapy drugs.MTT results showed the cell viability of anastatic HCT-116 and HT-29 cells was significantly higher than that of wild-type cells under the treatment of paclitaxel,oxaliplatin or irinotecan,respectively.The results of FACS showed that the proportion of apoptotic cells was significantly lower in anastatic HCT-116 and HT-29 cells treated with paclitaxel compared to that of wild-type cells.All the above results indicated that anastatic HCT-116 and HT-29 cells are more tolerant to chemotherapy drugs.(4)The anastatic colorectal cancer cells are more invasive and migratory than the control cells.The results of Transwell migration assay showed that the ability of anastatic colorectal cancer cells to pass through the chamber was significantly higher than that of wild-type cells.The results of the Transwell invasion assay showed that the invasion ability of anastatic HCT-116 and HT-29 cells was enhanced compared with control cells.(5)The anastatic colorectal cancer cells had higher ability in forming xenograft tumors in nude mice.The results of xenograft experiments showed that the tumor volume and tumor weight formed by anastatic HCT-116 cells were significantly higher than those formed by the control cells and apoptosis-resistant cells.FACS results showed that the proportion of CD44+CD133+cells in the tumors formed by anastatic cells is significantly higher than that in the tumors formed by the control cells.The proportion of CD133+cells in the tumors formed by anastatic HCT-116 cells was significantly higher than that in the tumors formed by the control HCT-116 cells.The proportion of CD44+cells in the tumors formed by anastatic HT-29 cells was significantly higher than that in the control tumors.(6)Knockdown of CUL4B enhances the sensitivity of colorectal cancer cells to paclitaxel.The results showed that,compared with control cells,the viability of the colorectal cancer cells with CUL4B knocked down(HCT116-CUL4BKD and HT29-CUL4BKD cells)was significantly reduced,and the proportion of PI positive cells was significantly increased,which indicated that the cells were more sensitive to the chemotherapy drug paclitaxel.(7)CUL4B promotes anastasis in colorectal cancer cells.The results of FACS showed that the proportion of anastatic cells decreased in HCT116-CUL4BKD cells,while the proportion of anastatic cells increased in CUL4B overexpressing HT-29 compared to control cells.(8)Preliminary research on the mechanism of CUL4B regulation of anastasis.RT-RCR results showed that in HCT116-CUL4BKD and HT29-CUL4BKD cells,the expression of some reported anastasis-related gene including DUSP1,ADM,ID2,NPARP,PIM1 and RHOB was downregulated.PIM1 and its target gene c-MYC were both downregulated in CUL4BKD cells.Western blot confirmed that PIM1 expression was down-regulated in CUL4BKD cells,and upregulated in CUL4B overexpressing cells.Therefore,we speculated that CUL4B might regulate the transcription of PIM1.PIM1 inhibitor SGI-1776 treatment or PIM1-specific siRNA transfection significantly reduced the ratio of anastatic cells after paclitaxel treatment,indicating that PIM1 is involved in the regulation of anastasis in colorectal cells.ConclusionsAnastasis participates in paclitaxel resistance in colorectal cancer.CUL4B may affect anastasis and paclitaxel resistance in colorectal cancer cells through transcriptional regulation of PIM1 expression.