The Role Of Insulin Like Growth Factor 1 Receptor(IGF1R)Inhibitor Linsitinib In Esophageal Squamous Cell Carcinoma And The Mechanism Of Drug Resistance

Esophageal cancer is a highly aggressive malignancy with a global mortality rate ranking sixth.China is one of the countries with high incidence of esophageal cancer and it has the highest morbidity and mortality of esophageal squamous cell carcinoma(ESCC)all over the world that nearly 70%of ESCC cases occur in China.Nowadays,the treatment of ESCC is based on radical resection combined with perioperative chemotherapy with or without radiotherapy.However,there are still no effective molecular therapeutic targets.Thus,the 5 year survival rate of ESCC was only 10%-25%.So that molecular targeted therapy guided by driver gene is an important development direction in the treatment of ESCC.At present,the biggest barrier to the accurate study of ESCC is the lack of an effective research model.There are few commercially available cell lines in ESCC,and the common esophageal cancer cell lines are mainly from Japan,including KYSE series and TE series.These cell lines have been used for long time in vitro.Some of them have lost the characteristics of the original tumor tissue in order to adapt to the environment in vitro.Due to racial differences,habits,environmental factors and so on,it is a great deal of doubt whether these cells can represent the type and characteristics of esophageal cancer in China.In contrast,the patient derived cells(PDC)from fresh tumor tissue preserve the main features of primary tumors such as histology,genome characteristics,cell heterogeneity and drug response characteristics.Compared with the commercial cell lines,PDC can simulate the real environment in vivo,which is expected to break the bottleneck of esophageal cancer treatment.Our team collected fresh tumor tissue after operation and tumor cells in pleural fluid from ESCC patients since May 2014 to December 2015 in Zhejiang Cancer Hospital for primary cell culture.We have successfully established 10 ESCC cell lines via tissue adherent method and trypsin digestion method.And we have analyzed 10 cell lines' characteristics including chromosome karyotype,short tandem repeats(STR),cell proliferation,tumor marker detection,clone formation and nude xenograft.Further,we picked up the 3-5 generation primary cell lines for the screening of 12 common used chemotherapeutic agents and 9 targeted drugs in clinical trials.We found that among the 16 primary cells,25%(4/16)cells were sensitive to insulin-like growth factor 1 receptor(IGF1R)tyrosine kinase inhibitor Linsitinib,while the relative resiatance rate was 25%(4/16).Then we chosen 6 commercial ESCC cell lines(Eca-109,EC-9706,KYSE-510,KYSE-410,TE-1 and TE-13)treated with Linsitinib.We found that TE-13 was sensitive to Linsitinib,while the other 5 cell lines were resistant.Furthermore,we used Western Blot to detect the downstream signal pathway of IGF1R,and found that Linsitinib could play a biological role by inhibiting the activation of PI3K/AKT/mTOR and Ras/MEK/ERK signaling pathways.But in sensitive cell lines,the activation of NF-kappa B p65 was inhibited,while p65 was enhanced in resistant cell lines so as the apoptosis was inhibited.And the combination of NF-kappa B inhibitor JSH-23 and Linsitinib can effectively increase the sensitivity of drug-resistant strains to Linsitinib.In summary,the primary ESCC cell lines we established provide a good model for understanding the Chinese primary esophageal cancer mechanism.And we used these primary cell lines for drug screening and found that Linsitinib may be a potential target drug for ESCC.The activation of NF-kappa B signaling pathway may be a potential drug resistance mechanism of Linsitinib in ESCC,and p65 may be used as a molecular marker for the prediction of ESCC.PART I stablishment of primary cell lines of esophageal squamous cell carcinoma(ESCC)Objective:To establish the method of primary isolation,purification and culture of human esophageal squamous cell carcinoma(ESCC),and to provide the necessary research models for the study of the biological characteristics,carcinogenesis and drug screening of esophageal carcinoma.Methods:Our study collected surgical resection of the primary tumor tissues and pleural fluid from patients with ESCC since May 2014 to December 2015 in Zhejiang Cancer Hospital to establish primary ESCC cell lines by tissue adherent method and trypsin digestion method.And we described 10 ESCC cell lines through morphological observation,chromosome karyotype and STR type.Real-Time Cell Analyzer(RTCA)was used to describe the cell proliferation.We detected the expression of tumor markers like cytokeratin5/6(CK5/6),p63,cytokeratin 14(CK14),CgA,Sy and CD56 in these cell lines by immunohistochemical.Clone formation and nude xenograft were used to analyse the proliferation ability of the new established cell lines.Results:We successfully established 10 ESCC cell lines which could passage stably in vitro(ZEC-014,ZEC-056,ZEC-134,ZEC-157,ZEC-166,ZEC-043,ZEC-061,ZEC-118,ZEC-127,ZEC-145).The 10 ESCC cell lines had been observed under microscope showing that they were irregular polygon cells,closely linked to each other and matched to the characteristics of epithelioid cells.The results of STR were compared with the data of ATCC,DSMZ and other cell banks showing that 10 esophageal squamous cell carcinoma cell lines were all newly constructed cell lines.Karyotype examination showed that the number of chromosomes was mostly distributed between 34?85.In 10 cell lines,CK5/6,p63 and CK14 were positive,otherwise,CgA,Sy and CD56 were negative.The clone formation assay showed that the cell clone formation ability was stronger when the density of the cells was 4500.In 10 cells subcutaneously implanted on nude back,ZEC-014,ZEC-056,ZEC-134,ZEC-157 and ZEC-166 could formulate xenograft,but ZEC-043,ZEC-061,ZEC-118,ZEC-127 and ZEC-145 had poor abilities of tumor formation.Conclusions:We successfully established the method of isolation,purification and culture of primary ESCC.The established cell lines have their own biological characteristics,which provide a research tool for the study of esophageal cancer in the future.PART ? The role of Insulin like growth factor 1 receptor(IGF1R)inhibitor Linsitinib in esophageal squamous cell carcinomaObjective:To explore the molecular targeted drugs for esophageal squamous cell carcinoma(ESCC),and to explore the effect of insulin-like growth factor 1 receptor(IGF1R)inhibitor Linsitinib in ESCC.Methods:Using MTT assay to detect how 12 clinical chemotherapeutic drugs including Topotecan,Irinotecan,Oxaliplatin,Fluorouracil,Epirubicin,Doxorunicin,Gemcitabine,Methotrexate,Paclitaxel,Capecitabine,Carboplatin and Cisplatin,as well 9 targeted drugs tested in clinical trials including Afatinib,Bortezomib,Navitoclax,Sunitinib,Trametinib,Eriotinib,GDC-09419 MK-2206 and Linsitinib affect the proliferation of 16 new established ESCC cell lines and 6 commercial cells lines(Eca-109/EC-9706/KYSE-510/KYSE-410/TE-1/TE-13).Immunoblotting was used to explore the mechanism of how Linsitinib inhibited the proliferation of ESCC.Results:The MTT assay showed that in primary ESCC cell lines 25%(4/16)cells were found to be sensitive to Linsitinib,while 25%(4/16)cells were resistant.And in commercial cells lines,TE-13 was found sensitive to Linsitini,while the other 5 strains were resistant.The results of immunoblotting showed that Linsitinib could significantly decrease the phosphorylation levels of pAKT308,pAKT473,p-mTOR and p-ERK1/2 in TE-1,TE-13 and KYSE-510 cells.Conclusions:Linsitinib may play a biological role by inhibiting the activation of PI3K/AKT/mTOR and Ras/MEK/ERK signaling pathways which are downstreams of IGF1R,which may be a potential target for ESCC.PART III The resistance mechanism of Insulin like growth factor 1 receptor(IGF1R)inhibitor Linsitinib in esophageal squamous cell carcinomaObjective:To characterize special biomarker to screen Linsitinib-sensitive patients as well as explore the molecular resistant mechanism to Linsitinib in esophageal squamous cell carcinoma.Methods:After Linsitinib treatment,the expressions of downstream signaling molecules and apoptosis pathways were measured by western blot.And the anti-tumor effect of Linsitinib and(Nuclear Factor-KB)NF-?B inhibitor JSH-23,was analyzed both as single agent and in combination in ESCC.Apoptosis,cell viability and clonogenic survival analysis were also investigated.Results:Phosphorylation level of NF-?B p65 was obviously activated to reduce apoptosis effect in Linsitinib-resistant cell lines.Most importantly,blockage of NF-?B activityby JSH-23 could sensitize resistant cells to Linstinib treatment.Conclusions:Results from this study demonstrated the intrinsic resistance to Linsitinib was predominantly mediated by NF-?B activation in ESCC.Moreover,combination of Linsitinib and JSH-23 as therapy provides a novel strategy to overcome resistance to Linsitinib in ESCC.