The Role Of Inhibitor Of Differentiation 1(ID1) In The Regulation Of Stemness Induced By Chemotherapy In Hepatocellular Carcinoma Cell

BackgroundHepatocellular carcinoma (HCC) ranks fifth among the most prevalent cancers in the world and is the third most common cause of cancer related mortality. Surgical re-section, liver transplantation and radiofrequency ablation(RFA) are regarded as poten-tially curative treatment modalities with 5 year overall survival rate of about 50%. Un-fortunately, only less than 30% of patients are candidates for curative therapy, most of HCC patients have to receive transcatheter hepatic arterial chemoembolization (TACE), sorafenib(an inhibitor of tyrosine protein kinases)or systemic chemotherapy due to their advanced-stage at diagnosis or disease replase/refractory. Recent studies have uncovered the presence of cancer stem cells(CSCs) with the ability to regenerate tu-mors. Similarto normal tissue stem cells, CSCs have the ability to self-renewand the ca-pacity todifferentiate into the multitude of cells that comprise the bulktumormass. Moreover, CSCs are resistant to conventional chemotherapies and current targetedthe-rapeutics.These properties of CSCs are believed tobe fundamentally responsible for continued malignant growth,cancer metastasis and recurrence and cancer drug resis-tance.Despite the increasingly recognized importance of CSCs in thepathogenesis of most tumors, the origin of these cells remains controversial. In particular, whether they originatefrom normal tissue stem cells that have undergone oncogenictransformation or from more differentiated cancer cells that haveacquired the ability to self-renew is currently unknown.It has been reported that the residual tumor cells which survive from chemotherapy will cause Epithelial-to-mesenchymal transition (EMT) andstem-ness enhancement.ID1 is regarded as one of the oncogenes due to its critical role in promoting stem-ness transformation. In our previous study, expression profiling Chip was used to screen for differential genes and it was verified by IHC and Western blot that there was an in-cresed expression of ID1 in the tissues after oxaliplatin treatment. Based upon this pre-liminary result, the goal of our study was to investigate the role of ID1 in regulation of acquired sternness for HCC cells after chemotherapy and its possible related molecular mechanism. This will provide the theoretical basis for the clinical application of stem-cell targeted therapy to overcome chemo-resistance of HCC.MethodsThe human HCC cell lines MHCC-97H with high metastatic potential was treated with oxaliplatin from lowconcentration to high concentration sequentially. Stable resistant cell lineMHCC97H-OXA wasevaluated for its sensitivity to oxaliplatin by CCK8. The ex-pression of stemness related molecular markers, functions of metastasis and stemness were also analyzed to verify the features of stemness for resistance cell line. Further-more, knock-down and over-expressing of ID1 into MHCC-97H cell lineby lentiviral transfection was used to investigate the role of ID1 in the regulation of stemness trans-formation. Following a highly selective Aurora-A inhibitor VX-689 treatment on ID1 over-expressing MHCC-97H cell line and MHCC97H-OXA, the stemness related functions and oxaliplatin sensitivity were evaluated to investigate the role of Aurora-A in the modulation of stemness induced by ID1. Finally, Aurora-A and ID1 expression were measured in 152 HCC surgical resection tissue array by immunohistochemistry assay. Overall survival and relapse-free survival was analyzed by Kaplan-Meier model, and Log-rank method was used to compare the difference between high and low expression of ID1 and Aurora-A kinase.Results1. Oxaliplatin-treated hepatocellular cell line MHCC97H-OXAshowed decreased prolife-ration rate (P<0.0001), but enhanced migration (P<0.0001),invasion (P<0.0001), colony forming ability (P=0.0002) and decreased sensitivity to oxaliplatin (P<0.0001). Expres-sion of Aurora-A was upregulated in parallel with ID1.2. By knockingdown and over-expression of ID1 gene, we generate of stably-transfected cell lines as ID1-KD and ID1-OE from MHCC-97H. Cell migration, invasion and clonal forming function changed correspondingly, accompanied by Aurora-Aand stem cell markers as ID1, OCT4 and SOX2. For ID1-KD cell,the migration rate was 23.5000± 3.9076%, invasion rate was 27±5.8651 cells/field,number of clonies was 21.4±3.9076 compared with vector control 41.1000±1.7030%(P=0.002),50±3.7427(P=0.01), 52.5±1.7030(P<0.0001),respectively. On the contrary, the clony forming ability was re-markably increased for ID1-OE cell line (P<0.0001), IC50 for oxaliplatin was also in-creased from 32.42±1.619 to 91±1.824μM (P<0.0001).3. After treatment with highly selective Aurora-A inhibitor VX-689 500nM on ID1-OE cell line/the number of clonies was decreased from 83.7±4.4509/well to 16.3421± 6.1609/well in the control arm (P<0.0001). Similarly, the IC50 to oxaliplatin in the VX-689 arm was also decreased (P<0.0001). Furthermore, treatment with VX-689 on MHCC97H-OXA, it showed inhibition of Aurora-A activity, while migration rate in the VX-689 treated arm was 59.5±1.31% vs 100±1.98% in the control arm (P<0.0001); invasion rate was 46±4.58 cells/field in the VX-689 treatment group vs 89±6.22 in the control arm (P<0.0001), colony-forming number was decreased from 83.7±4.45/well to 16.34±6.16/well (P<0.0001).4. The Aurora-A and ID1 expression were measured by IHC in the tissues of 152 HCC pa-tients who underwent resection with a median follow-up of51.17±21.46 months. Pa-tients with high Aurora-A expression were 58 cases, while patients with high ID1 ex-pression were 68 cases. The mean OS for high or low Aurora-A expression was 41.545± 4.948 vs 57.658±2.723months, P<0.001; mean RFS was 22.771±3.198 vs 44.773± 2.585months, P<0.001. The mean OS for ID1 high or low expression was 34.467±3.137 vs 66.244±3.423months, P<0.001; mean RFS was 30.218±3.285 vs 41.652± 2.841months, P=0.0013. By Pearson Correlation analysis, Aurora-A and ID1 were closely related, the Correlation coefficient was 0.285, P<0.001. Univariate analysis showed patients with high expression of either Aurora-A or ID1 has a shorter OS and RFS. Both markers are independent prognostic factor for OS (P=0.045 and 0.003 respectively), while only Aurora-A was an independant prognostic factor associated with RFS (p=0.001). Patients with both high expression of Aurora-A and ID1 have the worst prognosis with the mean OS and RFS was 25.17±4.21m,20.60±2.21m,respectively, P<0.001.Conclusion:1. ID1 is a key regulator of sternness in HCC cells and down-regulation of its expression could inhibit sternness and chemo-resistance.2. It was confirmed the increased sternness of MHCC97H-OXA cell line, accompanying by upregulation of Aurora-A expression3. Aurora-A plays an important role in mediation the "stemness"induced by ID1 upre-gulation. The undetermined mechanism between them is worthy further study4. Either Aurora-A or ID1 expression is a significant predictor for overall survival in HCC. High expression of Aurora-A predicts a rapid disease progression with shorter RFS. There might be interplay between Aurora-A and ID1 in the progression of HCC, with concurrently high expression of Aurora-A and ID1 patients have the worst prognosis.Potential application of this work1. It was confirmed that the "stemness"of MHCC97H cell line after treatment by oxa-liplatin was enhanced, which may help to develop novel therapeutic agents targeted on cancer stem cell to overcome the secondary resistance induced by conventional chemotherapy2. It was confirmed that ID1 is a key regulator of sternness transformation. This war-rants further investigation of ID1 as a promising therapeutic target.3. Aurora-A palys an important role in mediation "sternness" induced by ID1 upregula-tion. Use VX-689 could block the activity of Aurora-A kinase and abrogate ID1-induced sternness. This will support further clinical trials with Aurora-A inhibi-tors in the treatment of HCC.4. Because of the prognostic significance of Aurora-A and ID1 expression in HCC, we can develop a new model to predict both the potential riskof recurrence and death from HCC after surgical resection.Originalities of this work1. For the first time, it was demonstrates Aurora-A is one of the molecular mecha-nismsinvolved in the regulation of ID1-induced sternness in HCC2. For the first time, it was reported that high expression of Aurora-A and ID1 were independent prognostic factors for OS in HCC.