CD24 Regulated The Properties Of Self-renewal, Drug-resistant, And Tumorigencity Of Colorectal Cancer Stem Cell

ObjectiveTraditional models of carcinogenesis posit that cancers might originate in any cell through a series of stochastic genetic events resulting in clonal selection. By contrast, the stem cell model proposes that cancers originate in select cell population from normal tissue stem cells or progenitor cells and that initiating events disrupt the process that regulates stem cell self-renewal,This is so called cancer stem cell or cancer stem-like cell. Self-renewal, one of characteristics of cancer stem cell, is a cell division in which one or both of the resulting daughter cells remains undifferentiated, and retains the ability to give rise to another stem cell with the same capacity to proliferate as the parental cells; In additional, cancer stem cell have the capacity to differentiate, a property that generates the bulk of cells within a tumor. In addition to the property of self-renewal, cancer stem cells might also be resistant to the radiotherapy and chemotherapy, and capacity of tumorigenesis. Because cancer stem cells is not sensitive to chemotherapy and radiotherapy, the current treatments only kill the different lineages of terminally differentiated tumor cells, while no obvious effect on the cancer stem cells. After treatment, residual tumor stem cell with self-renewal has resulted in tumor recurrence.Then, a new neoadjuvant treatment will be require targeting the cancer stem cell against the self-renewal, tumorigenicity and drug resistance.Many studies have verified that there were several special genes in the cancer stem cell.The presence or absence of the genes in the cancer stem cell can regulate the self-renewal capacity and enhance the sensitivity to radiotherapy and chemotherapy, and block the tumorigenesis in the immunodeficiency mice.For example,inceased let-7 paralleled reduced H-RAS and HMGA2,can reduced proliferation,mammosphere formating,and the proportion of undifferentiated cells in vitro and tumor formation and metastasis in NOD/SCID mice in breast cancer stem cell.ABCG2,one of MDRs (Multiple Resistance Drug family), was expressed in kinds of cancer stem cells. Silencing ABCG2 in cancer stem cell can increased the sensitivity to chemotherapy and radiotherapy. In additional,Blocking interleukin-4 signaling can senditize colorectal cancer stem cells to apoptotic stimuli and increase the in vivo efficacy of cytotoxic therapy.In total, these studies improved outcomes of therapies might require targeting cancer stem cell subgroup.CD24 was regarded as a prominent role in many genes associated with tumorigensis.The CD24 gene encoded a heavily glycosylated cell surface protein anchored to the membrane by phosphatidylinositol, which consisted of three 31 amino acids with 16 potential O-glycosylation and N-lycosylation sites and was capable of mucin-like characteristics. Under normal physiologic conditions, CD24 transiently facilitated primary hematopoietic cell selection and promoted their progeny maturation during hematopoiesis.It also plays a role during the embryonic development of pancreatic cells. Silencing CD24 expression could inhibit the phenotype transformation in lymphocytes precursors.CD24 played a vital role not only in normal tissue but also in various cancers. Some published literatures have verified that CD24 was related to kinds of human cancer carcinogenesis. Furethermore,cancer stem cells showed the properties of self-renewal and the ability to produce differentiated progeny in dependence of the CD24 expression in breast cancer and panceatic cancer. CD24 also suggested in some studies to contribute to malignant transformation in colorectal cancer and was limited to CD133+ subgroup, but little or no expression in CD 133- cell while cancer cells were isolated. Other document also vertified the results. These data suggested that CD24 promotes tumorigenesis, and acted as a marker of cancer stem cell in some cancers.But,no study searching for markers and function for colorectal cancer stem cells has been preformed to data.In present study, the role of CD24 in the properties of self-renewal, drug-resistant, and tumorigenicity of colorectal cancer stem cell was investigated. Our study suggested that CD24 was a potential colorectal cancer stem cell marker and a candidate for therapy alone or in combination with chemotherapy drugs in colorectal cancer.Methods and Results1. A stable cell line expressing CD24 was establishedCD24 recombinant plasmid pcDNA3.1 (+)-CD24, which was constructed using expression vector pcDNA3.1 (+) in previous study, was transfected into HCT116 cells and cultured in medium including G418. After selection, resistant colonies to G418 were chosen and named as HCT116CD24 and HCT116vector. MRNA level and protein expression were assessed by real-time PCR and FCS. Our results showed that the mRNA level and protein expression of CD24 increased significantly compared with control groups (p<0.05).2. Up-regulating CD24 expression increased the expression of CD133 and EPcamTo investigate the expression of CD133 after CD24 up-regulated, CD133 expression was detected by FCS in HCT116CD24 and HCT116vector cells. Our data showed that CD133 expression increased significantly in HCT116CD24 cells compared with in HCT116vector cells (p<0.001). Epcam was used as a marker of epithelial cell phenotype, there was no change in Epcam expression level and suggested that the cell phenotype still kept (p=0.531).3. Up-regulating CD24 expression enhanced the resistance to 5-FU and OxaliplatinTo investigate the effect of CD24 expression on chemotherapy drug, the cell viability was detected by CCK-8 assay after 5-FU and/or Oxaliplatin treated in HCT116CD24 and HCT116vector cells. We results showed that cell viability increased in HCT116CD24cells in a dose-dependent manner after 5-FU treated. The same trend was found in oxaliplatin treated groups. Our data suggested that up-regulating CD24 expression enhanced the resistance to 5-FU and oxaliplatin.4. Up-regulating CD24 expression induced tumor mammosphere formationAs we all know, tumor mammospheres generation is an in vitro assay of self renewal potency in cancer stem cell. We detected the mammospheres formating in serum-free medium after CD24 recombinant plasmid was transfected.After 15 days, tumor mammospheres in HCT116CD24 group were significantly increased 10 fold than control one (.207±11vs.26±5/1000 cells) Collectively, our data suggested that up-regulating CD24 increased the proportion of cancer stem-like cell in HCT1165.Up-regualting CD24 regramming CD133- cells into cancer stem-like cell and increased the resistance to 5-FU and Oxaliplatin in CD133- cells subgroupTo further clarify the mechanism which CD24 increased CRC stem-like cell, we isolated CD133+ cell group and CD133" cell group with MACS. CD24 recombinant plasmid was stably transfected respectively into the two groups.The endogenous expression of CD24 is 35% in CD133+ cell group,and no change after overexpression of CD24;In the contrary, there was almost no expression of CD24 in CD133- cell group, but obviously increased after transfection.Moreover,the expression of CD133 also increased in parallel with the expression of CD24. Consistence with the theory that tumor stem cell was not sensitive to chemodrugs, and CD133- cells was senstive to 5-FU and Oxaliplatin, the results showed that CD133+cells resistance to chemotherapeutic was similar to CD 133+ cells with the treatment of CD24 recombinant plasmid (p>0.05), but the resistance to chemotherapeutic of CD133- cells was obviously enhanced compared to CD133-cells with the treatment of CD24 recombinant plasmid (p<0.05).6. CD24 increased DNA synthesis in CD133- cell just as cancer stem cellIn addition, the self-renewal of cancer stem-like cells was evaluated using BrdU, which marked cancer stem-like cells. We found that the BrdU positive cells in CD133- cells was increased from 3±0.25% to 27+3.4% after up-regulating CD24 expression. the same results was not see in CD133+ cells and CD133+ cells with the treatment of CD24 recombinant plasmid. There was no change of the the positive cells of BrdU in CD133+cells and CD133+ cells with the treatment of CD24 recombinant plasmid (85±5.4% vs.86±4.3%;p>0.05).7. CD133- cell can formatting mammosphere after gain-function of CD24Furthermore, tumor mammospheres was obtained with spheroid culture in serum-free DMEM/F12 medium,containing B27,20ng/ml FGF-2 and 60ng/ml EGF.The mammospheres derived from CD133+ cells was approximatively 830±40.5 in 1000 CD133+ cells, the same as derived from CD 133+ with the treatment of CD24 recombinant plasmid. In the contrary, the mammospheres was increased after overexpression of CD24 in CD133-cells (20±4 vs.140+15, p<0001).8.CD24 enhanced CD133- cells tumorigenesis in vivoIn accordance with in vitro results, in vivo exnograft tumor further verified that up-regulating CD24 enhanced the tumorigenesis capability of CD133- cells compared with the control (p<0.001). Collectively,these data suggested that Up-regulating CD24 regrammed CD133- cells into cancer stem-like and helped CD133-cells capacible of the charactistic of self-renewal,chemotherapeutic resistance, tumor mammospheres formating,and tumorigenesis.9. Silencing CD24 resulted in tumor mammosphere differentiationGiven the increasing the proportion of CRC stem-like cell after up-regulation of CD24,we next test the possibility that down-regulating CD24 affects the features of CRC stem cell. CD133+ cell was isolated combination with spheroic culture in serum-free medium. Using the shRNA method with lentiviral vector for knockdown of CD24 expression in CD133+cells,the data showed the treatment of CD24 shRNA could diminish the CD133 expression, and facilitate tumor mammosphere differetiation.Furthermore,tumor mammosphere formating was blocking after CD24 shRNA transfectiong compared with scramble shRNA.Without the treatment of CD24 shRNA,67±10.3% CD133+ cells could format tumor mammospheres;in the contrary, only 13±3.5% CD133+ cells could format mammosphere (p<0.05). There was significantly diffferent between two groups.CK20 indicated cell differetiation,our data shows that CK20 expression increased after the treatment of CD24 shRNA,but OCT-4 decreased.10. Silencing CD24 expression induced in the cancer stem cell apoptosisIn addtion, we also investigated the role of CD24 in apoptosis. Freshly isolated CD133+ cells, which derived from HCT116, was trasfected respectively with CD24 shRNA and scramble shRNA.Hoechst 33342 staining was used to evaluate the apoptosis morphology. Nuclear enrichment indicated cell apoptosis.There was also significantly increased with CD24 shRNA treatment compared with scramble shRNA treatment.In a word, these data indicated that CD24 expression played a crucial role in maintaining self-renewal and cancer stem cell properities in CD133+ cells. Since down-regulation of CD24 expression induced in tumor mammospheres differetiation, CD24 shRNA may be a attractive candidate as a potential adjuvant to chemotherapy for colon cancer.11. Silencing CD24 enhanced the tumor mammmosphere sensitivity to chemotherapy in vitroTo vertify whether CD24 shRNA overcome resistance to chemotherapy in CD133+ cells, using the treatment CD24 shRNA, the results exhibited that the knockdown of CD24 in CD133+ cells can significantly improve the anti-cancer effect in single or combination with 5-FU or oxaliplatin in vitro (F=804.769.p<0.001).Moreover, we detected the expression of ABCG2, one of MDRs, which mainly responsible for cancer stem cell resistance to chemotherapy and irradiation treatment. The ABCG2 expression was reduced-5 fold in the CD24 shRNA than the scramble shRNA.These data suggested that the up-regulating CD24 could effectively strengthen the chemotherapeutic sensitivity of cancer stem cells by regulating ABCG2 expression.12. Silencing CD24 blocked the tumorigencity of mammosphere in vivo1000 tumor mammospheres was injected in the nude mice to generate human colorectal cancer.At day 15 and 22, thery were injected with lenti-CD24 shRNA or lenti-scramble shRNA or PBS. In vivo xenograft tumor results confirmed that injection lenti-CD24 shRNA could block the tumorigencity of CD133+ cell or tumor mammmosphere, and CD133 expression was decreased than lenti-scramble shRNA or blank control (F=804.769,P<0.001).. Further, we found that CD24 and CD 133 expression obviously decreased in the xenograft with the treatment of lenti-CD24 shRNA by western blotting.these data collectively suggested that CD24 could act as an effective therapy target.13. Co-expression of CD24 and CD133 associated with the low grade and metastasis, and predicted poor overall survival in CRC patients To investigate the correlation between the co-expression of CD24 and CD133 and clinicopathologic feature, immunohistochemical staining was performed in serial sections of CRC paraffin-embedded colorectal cancer specimens. As a result, the CD24 expression was found to be significantly associated with the expression of CD133 (r=0.658,p=0.000),and the co-expression of CD24 and CD133 was dramatically different between positive distal metastasis and negative distal metastasis (p<0.05) compared with the negative expression of CD24 and CD133,the same as positive nodal metastasis (p<0.05),and low tumor grade (p=0.038).These results suggested that co-expression of CD24 and CD 133 was closely correlated with low tumor grade, positive nodal and distal metastasis in colon cancer tissues.To determine whether the co-expression of CD24 and CD133 was a significant prognostic factor for the survival of patients with surgically resected colorectal cancer, we performed a log-rank test with Kaplan-Meier estimates. Among the 213 patients analyzed,89 patients were followed up. The 100 months overall survival rate was significantly lower with co-expression of CD24 and CD 133 than negative expression in primary colorectal cancer patients (p=0.01). These results clearly indicated that co-expression of CD24 and CD 133 was associated with the 100 months survival of colorectal cancer cases and suggested that co-expression of CD24 and CD 133 might be a biomarker of poor prognosis.ConclusionIn the present study, using gain of function and loss of functiong,we demonstrated that CD24 expression induced tumor spheres formating,and increased drμg-resistant;Strikingly, CD133-cell subgroup gained cancer stem cell Properities after gain of function of CD24 with BrdU intake. we also showed that Knocking down CD24 resulted in colorectal cancer sphere differentiated,and obviously increased apoptosis. Our data suggested that CD24 mainly maintained the properities of colorectal cancer stem cell by regulating the self-renew, drug-resistant, and tumorigenesis. Collectively, our data suggested that CD24 was a key role in maintaining the properities of CRC stem cell; Strikely, CD24 could regramming the CD133 negative cells into cancer stem cell.Combination with CD133 positive expression, CD24 was correlated with poor overall survival in CRC patients. These data taken together highlight a function of CD24 in CRC stem cell, and indentify CD24 as a potential novel candidate for therapeutic intervention alone or in combination with chemotherapy drugs.