The Phenotypic Characteristics And TKIs-therapeutic Responses Of CK19-positive Hepatocellular Carcinoma

Background: Hepatocellular carcinoma(HCC)is one of the most common cancers worldwide,and around half of HCC population occur in China.The molecular heterogeneity of HCC results in a high degree of difference in response to treatment in patients with the same clinical stage.Molecular subtype researches elaborate the classification of HCC,and further to guide clinical individualized precise treatment.Cytokeratin 19(CK19)is a marker of "progenitor/cholangiocytes".HCC with positive expression of CK19 is named as “biliary phenotype”,indicating poor prognosis.However,the molecular characteristics and therapeutic regimens of CK19-positive HCC have not been fully revealed.Aim: The aim of this study was to study the phenotypic characteristics and tyrosine kinase inhibitors(TKIs)-therapeutic responses of CK19-positive HCC,which were based on fluorescence activated cell sorting(FACS)and patient-derived xenograft(PDX)models.Finally,we aimed to provide theoretical basis for elaborating the therapy-guided molecular subtypes in HCC.Methods: 1.A total of 206 HCC tumors were collected from patients undergoing liver transplantation.The expression of CK19 was detected by immunohistochemistry,and the correlation between CK19 expression and clinicopathological parameters and prognosis was analyzed.The fluorescent reporter of CK19 promoter was constructed and transfected into Huh7 and PLC/PRF/5 cell lines.CK19-negative(CK19-)and CK19-positive(CK19+)cells were isolated by FACS,and their proliferation,clone formation and apoptosis phenotypes were detected.2.Sorafenib,regorafenib,apatinib and 5-fluorouracil(as a negative control)were used to test the cytotoxicity and apoptosis of CK19-and CK19+ cells in vitro.Tumor-bearing mice from a CK19+ PDX were used to test the efficacy of the four drugs in vivo.3.We explored the mechanism of regorafenib preferentially inhibiting CK19+ cells using RNA sequencing and GO analysis.The inhibitory effects of regorafenib on the mitochondrial function of CK19+ cells were detected by q PCR,mitochondrial morphology and Seahorse Mito Stress Test.Small interfering RNA(si RNA)of signal transduction and activation of transcription 3(STAT3)and STAT3 inhibitor were used to detect the role of STAT3 signaling in the preferential inhibition of regorafenib in CK19+ cells.4.We constructed the HCC PDX model platform using 76 HCC tumors,and selected 10 PDX models with different CK19 expression(five with CK19-tumors and five with CK19+ tumors)to evaluate the efficacy of regorafenib.We also constructed nanoparticles containing regorafenib and explore its potential efficacy against CK19+ HCC.Results:1.Immunohistochemistry results showed that 23.8%(49/206)HCC presented CK19 positivity.Chi-square analysis showed that the positive expression of CK19 was significantly correlated with increased AFP level(> 400 ng/ml,P = 0.019),poor differentiation(P = 0.007)and microvascular invasion(P = 0.034).The recurrence free survival rate of patients with CK19+ HCC decreased significantly(P = 0.029).The ability of proliferation,clone formation and anti-apoptosis of CK19+ cells were significantly stronger than those of CK19-cells.These indicate that CK19+ HCC has more aggressive phenotypes and poor outcome than CK19-HCC.2.Regorafenib preferentially increased the growth inhibition and apoptosis of CK19+ cells.Whereas,sorafenib,apatinib and 5-fluorouracil had stronger effect on inhibiting the proliferation and promoting the apoptosis of CK19-cells.The IC50 of regorafenib on CK19+ cells were significantly lower than that of CK19-cells.The anti-tumor effect of regorafenib on CK19+ HCC PDX was better than that of sorafenib,apatinib and 5-fluorouracil.The data suggest that CK19+ HCC are sensitive to regorafenib treatment.3.RNA sequencing and GO analysis showed that CK19+ cells had more mitochondrial ribosomal proteins(MRPs)than CK19-cells.And the q PCR test validated that the m RNA expression of MRPSs were specifically downregulated by regorafenib treatment.Regorafenib treatment changed the mitochondrial morphology and reduced the oxygen consumption rate,which were related to mitochondrial respiration in CK19+ cells.Western bolt results showed that CK19+ cells had strong expressions of phosphorylation of STAT3,and regorafenib treatment specifically downregulated the phosphorylation of STAT3 in CK19+ cells.Interference with STAT3 downregulated the m RNA level of MRPs in CK19+ cells.And STAT3 inhibitor combined with regorafenib significantly enhanced the apoptosis of CK19+ cells.These data indicate that mitochondrial function plays a key role in maintaining the distinct phenotype of CK19+ HCC.4.We successfully established 30 HCC PDX models,with an established rate of 39.47%(30/76).The histopathology and CK19 expression of PDX tumors were consistent with the corresponding patients’ tumors.In the CK19+ PDXs group after regorafenib treatment,one HCC achieved tumor response,three HCCs showed stability,and only one HCC showed tumor nonresponse,the tumor control rate of regorafenib was 80%.Whereas in the CK19-PDXs group,all five HCCs showed tumor nonresponse to regorafenib,the tumor control rate of regorafenib was 0%.The Ki-67 positive rate of CK19+ PDX tumors was significantly lower than that of CK19-PDX tumors(29.7 ± 16.4% vs.45.0 ± 7.2%,P = 0.013)after regorafenib treatment.The nanoparticles loaded with regorafenib enhanced the anti-tumor effect of regorafenib in CK19+ PDX models.These suggest that CK19+ HCC can benefit from regorafenib treatment.Conclusion: CK19-positive HCC,as a special subtype,is associated with poor prognosis and malignant phenotype.Mitochondrial respiration was significantly increased in CK19-positive HCC,which could be downregulated by regorafenib.Regorafenib may preferentially inhibit CK19-positive HCC by disturbing mitochondrial respiratory and further promoting apoptosis through STAT3 signaling.CK19+ HCC can benefit from regorafenib treatment,which paves a pathway for elaborating the therapy-guided molecular subtypes of HCC.