Chemotherapy Exacerbates Cancer Stem Cell-like Characteristics,Repopulation And Metastasis Through GLI1 In Ovarian Carcinoma

Ovarian cancer is the most fatal cancer among all gynecologic malignancies.Although remarkable clinical response has been received through the current taxane/carboplatin chemotherapy treatment protocols at the initial stage,the relapse and metastasis rate of ovarian cancer is distinctively high and leads to the low 5-year survival of the disease.Therefore,exploring the mechanism of the chemotherapyexacerbated recurrence and metastasis in ovarian cancer and blocking the related signal pathway is the key to reduce patient mortality.As a first-line clinical approach for ovarian cancer treatment,chemotherapy has its double-edged effects besides its cytotoxicity.Studies have shown that although classic chemotherapy drugs such as paclitaxel and carboplatin can significantly inhibit the growth of the primary tumor,they can cause increased lung metastasis and secondary tumor proliferation as well.The chemotherapy-exacerbated repopulation and migration of the residual tumor cells may be the leading cause of the high metastasis recurrence rate in ovarian cancer.studies have shown that radiotherapy and chemotherapy can induce tomor cells to acquire CSC-like characteristics and transform into CSC-like cells.As a small and special cell population in tumor tissue,CSC has strong self-renewal and multi-directional differentiation potential,leading to its strong tumorigenicity,multi-drug resistance and high metastasis characteristics.CSC plays an important role in tumor recurrence and metastasis.It is still unclear whether the chemotherapy-exacerbated CSC-like characteristics is involved in the chemotherapy-exacerbated recurrence and metastasis of ovarian cancer.In embryonic or adult stem cells,pluripotency-associated genes such as Nanog,Sox-2,BMI1,and Oct-4 are proved to directly regulate the stemness of cells,and these genes are further regulated by pathways such as Hedgehog(Hh),Wnt,and Notch etc.Under normal conditions in the body,these signaling pathways are under strict regulation.However,when tumors are developed,the signaling pathways and genes will be mutated or abnormally activated,thereby mediating the function of CSC and the maintenance of CSC-like characteristics.In ovarian cancer,which pluripotency-associated gene induces the acquisition of CSC-like characteristics after chemotherapy,and the key regulating transcription factors of it remain to be further explored.Based on the above background,in the first part of this study,(1)the effects of chemotherapy on the repopulation,migration and CSC-like characteristics of ovarian cancer cells;(2)pluripotency-associated genes regulating the chemotherapy-exacerbated CSC-like characteristics of ovarian cancer are investigated:Three different ovarian cancer cell lines SKOV-3,A2780 and KURAMOCHI and two clinical chemotherapy drugs carboplatin and VP-16 were used.First,the chemotherapy-treated dying ovarian cancer cells(Feeder cells)and a small amount of untreated ovarian cancer cells(Receptor cells)are co-cultured in Transwell co-culture chambers,in order to establish the in vitro model to simulate the effect of the chemotherapy-induced massive tumor cell dying process on a small number of residual tumor cells.The results showed that chemotherapy could exacerbate repopulation and migration of the Receptor cells.And the m RNA expression of EMT related markers VIM,TWIST,and Snail were analyzed,showing that VIM and Snail may play an important role in the chemotherapy-exacerbated migration of ovarian cancer cells.It was also shown that the chemotherapy could exacerbate the CSC-like characteristics of ovarian cancer cells by increasing the sphere forming ability and the expression of CSC-like characteristics-related markers CD44 and CD133.The m RNA expression of the reported pluripotency-associated genes in ovarian cancer Nanog,SOX-2,BMI1 and Oct-4 were then investigated.it was shown that SOX-2 and BMI1 may play an important role in the chemotherapy-exacerbated CSC characteristics of ovarian cancer cells.In the second part of this study,we use bioinformatics methods to discover the key transcription factors related to chemotherapy-exacerbated CSC characteristics in ovarian cancer:Bioinformatics methods were used to analyse the clinical data sets before and after chemotherapy in ovarian cancer patients,and 6 candidate transcription factors were obtained.The key transcription factors related to CSC in ovarian cancer were further identified and verified in the clinical patient tissues and the Transwell model,and survival analysis was performed.The GLI1 expression was higher in patients underwent chemotherapy compared with the non-chemotherapy patients.The 5-year overall survival of the GLI1 highly-expressed patients were significantly reduced.Finally,the CSC-regulated transcription factor GLI1 was identified as the key regulator in the chemotherapy-exacerbated CSC-like characteristics in ovarian cancer.Based on this,GLI1 siRNA or its small molecule inhibitor GANT61 were used to investigate the role of GLI1 in the the chemotherapy-exacerbated migration and repopulation of ovarian cancer cells: It was shown that the inhibition of GLI1 could significantly inhibit the chemotherapy-exacerbated migration and reproliferation,as well as the up-regulation of EMT-related genes VIM and Snail in Receptor cells.The inhibition of GLI1 could significantly inhibit the chemotherapy-exacerbated CSC characteristics.The effect of GLI1 on the upregulation of pluripotency-associated genes SOX-2 and BMI1 were further investigated.It was shown that GLI1 siRNA could significantly inhibit the upregulation of BMI1,but didn’t effect the SOX-2 upregulation.Therefore,GLI1 could regulate the CSC-like characteristics,repopulation and migration induced by chemotherapy-altered tumor microenvironment through its downstream stem cell-related gene BMI1.Studies have shown that tumor-derived soluble factors can increase in the stemness of bone marrow mesenchymal cells.Therefore,in the non-contact co-culture system of the Feeder and Receptor cells in our co-culture system,the chemotherapeutics-treated dying Feeder cells may induce the CSC-like characteristics of the Receptor cells by alternating the soluble factors in the tumor microenvironment.Different chemotherapeutics are reported to induce a variety of soluble factors alternation in the tumor microenvironment,our previous study and other studies show that as one of the adverse alterments induced by chemotherapy,the abnormal activation of arachidonic acid(AA)metabolic pathway can leads to the increased prostaglandin E2(PGE2)secretion into the tumor microenvironment.And PGE2 plays an important role in promoting tumor proliferation and migration.Based on this,we further explored the effect of PGE2 on the CSC-like characteristics,migration and repopulation of ovarian cancer cells mediated by GLI1/BMI1 signaling pathway.On the basis of our previous studies,it was confirmed that the AA pathway was activated and the secretion of PGE2 in the tumor microenvironment is significantly increased after chemotherapy.And the exogenous PGE2 can increase the CSC-like characteristics,repopulation and migration of ovarian cancer cells through GLI1/BMI1 signaling pathway.Based on the above results,in the third part of this study,we further propose berberine to target the AA metabolic pathway to rehabilitate the chemotherapyaltered tumor microenvironment,and to inhibit the chemotherapy-exacerbated CSC-like characteristics,repopulation and metastasis,as a potential clinical treatment strategy for ovarian cancer:In our study,berberine was added into the in vitro model indicated above.It is shown that low-dose berberine has no significant killing effect on ovarian cancer cells.While in combination with chemotherapy drugs,it can significantly inhibit the chemotherapy-exacerbated CSC-like characteristics,repopulation,metastasis,as well as the upregulation of EMT-related genes VIM and Snail.And berberine could inhibit the chemotherapy-exacerbated CSC characteristics and the upregulation of BMI1 expression.Berberine could inhibit the increase of PGE2 levels by inhibiting the Caspase 3/iPLA2/AA/COX-2/PGE2 signaling pathway to rehabilitated the chemotherapy-altered tumor microenvironment.Finally,it was shown that berberine can inhibit the abnormal activation of the GLI1/BMI1 signaling pathway after chemotherapy.In summary,this study draws the following conclusions:(1)Chemotherapy altered the tumor microenvironment through activation of the Caspase 3/iPLA2/AA/COX-2/PGE2 signaling pathway and ultimately increasing the PGE2 level in the tumor microenvironment.which played an important role in the chemotherapy-exacerbated CSC-like characteristics,repopulation and metastasis.(2)Berberine can reduce the PGE2 production by inhibiting the key enzymes iPLA2 and COX-2 of the AA metabolic pathway which were abnormally activated after chemotherapy,thereby rehabilitate the chemotherapy-altered tumor microenvironment,inhibit the chemotherapy-exacerbated CSC-like characteristics,repopulation and metastasis.Therefore,this article proposed that the potential interventional strategy for chemotherapy-exacerbated tumor recurrence and metastasis by(1)rehabilitate the chemotherapy-altered soluble factors in the tumor microenvironment and(2)targeting the chemotherapy-induced abnormal GLI1 activation of residual tumor cells,in order to inhibit the chemotherapy-exacerbated CSC-like characteristics.It provides theoretical and experimental basis for the development of new strategies of clinical prevention and treatment for chemotherapy-exacerbated recurrence and metastasis in ovarian cancer.And it may be beneficial to improve the efficiency of chemotherapy,and improve the quality of life of ovarian cancer patients.