| Chapter I.Mechanisms of IL-8 and Integrin ?v?3 in HCC InvasionBackgroundHepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death worldwide.During the first hospital visit,most patients have developed to advanced stage due to insidious onset,and miss the best opportunity to receive optimal therapy.With the in-depth studies on the pathogenesis of HCC,molecular-targeted therapies represented by sorafenib have achieved encouraging results,enrich the therapeutic approaches and provide new ideas for research regarding HCC.However,HCC is still a malignancy with high cancer-related mortality worldwide and poses a serious threat to human health.The main reason is that HCC is prone to vascular invasion and metastasis.Tumor invasion and metastasis is a complex,multi-step dynamic process.In each step,interaction between tumor cells and tumor microenvironment,activation of various tumor metastasis-related genes and molecules as well as formation of tumor blood vessels play a synergistic role in the whole process of metastasis.Therefore,A better understanding of the molecular mechanisms underlying tumor invasion and metastasis as well as development of molecular-targeted therapy is of great significance for improving the prognosis of HCC.Cytokines such as Interleukin-8(IL-8)are needed for mediating the interaction between tumor cells and tumor microenvironment.IL-8,also known as CXCL8,is a member of ELR+-CXC subfamily,and is rarely detectable in physiological states but rapidly secreted by inflammatory cells or tumor cells under pathological conditions.IL-8 has two G protein-coupled receptors,CXCR1 and CXCR2,which are widely distributed on the membranes of inflammatory cells,tumor cells,and tumor-associated stromal cells.IL-8 could bind to CXCR1/2,subsequently activate different downstream signaling pathways such as PI3K/Akt and MAPK,and finally mediate various biological behaviors.IL-8 plays an important role in tumor angiogenesis,proliferation,invasion,metastasis and drug resistance of many tumors,such as breast cancer,lung cancer and colon cancer.Previous studies reported that over-expression of IL-8 in HCC tissues was associated with higher rates of intra/extrahepatic metastasis and postoperative recurrence,and poorer prognosis.Further studies have shown that IL-8 is able to induce epithelial-mesenchymal transformation of HCC cells and promotes migration and invasion of HCC cells.In addition,elevated serum IL-8 is valuable for diagnosis and prognosis of HCC.Integrins are transmembrane heterodimeric molecules composed of ? and ?subunits.Due to various combinations of ? and ? subunits,at least 24 distinct integrins have been described.Integrins are able to perceive changes in tumor microenvironment and trigger a series of cellular responses by forming physical connections inside and outside the cell,thus allowing bidirectional integration' signals to control cell adhesion,migration,proliferation,survival and differentiation.They not only play an important role in normal physiological development,but also regulate a variety of cellular functions essential for cancer progression.Integrin ?v?3,which has been detected in various types of tumors including carcinoma of breast,prostate and pancreas,and melanoma,plays a critical role in tumor cell proliferation,migration,invasion,metastasis,and angiogenesis.The expression of integrin ?v?3 closely associates with disease progression and prognosis in a variety of tumors.As respect to HCC,integrin?v?3 has also been reported to be overexpressed in carcinoma tissue and mediates the invasion,metastasis and chemoresistance of HCC cells.Both IL-8 and integrin ?v?3 have been reported to overexpression in HCC tissues and play important roles in HCC invasion.However,both the correlation between IL-8 and integrin ?v?3 in HCC and the underlying mechanisms of IL-8 and integrin ?v?3 in HCC invasion remain largely unknown.Further study regarding this issue is needed.Objective1.To investigate the expression of IL-8 and integrin ?v?3 in HCC tissues,study the correlation between IL-8 and integrin ?v?3 expression in HCC tissues,and explore the correlations between the expression of IL-8 and integrin ?v?3 and various clinicopathological factors;2.To explore the mechanisms of IL-8 and integrin ?v?3 in HCC invasion.Methods1.The expression of IL-8 and integrin ?v?3 in 170 HCC tissues[135 conventional HCC and 35 sarcomatoid HCC(a highly aggressive EMT phenotype with tumor cells being spindle shaped)and 56 adjacent non-tumor tissues was detected by immunohisto-chemistry.The expression of IL-8 or integrin ?v?3 in tumor tissues and adjacent non-tumor tissues were compared respectively.Spearman's rank correlation test was used for correlation analysis between IL-8 and integrin ?v?3 expression.Moreover,the correlations between the expression of IL-8 and integrin ?v?3 and various clinicopathological factors were analyzed.2.The expression of IL-8,integrin av and integrin ?3 in HCC cells was detected by quantitative real-time PCR and Western blot.3.CCK8 assay was employed to detect the proliferation of HCC cells pretreated with IL-8 knockdown or exogenous IL-8.Transwell assay was employed to detect the invasiveness of HCC cells pretreated with IL-8 knockdown or exogenous IL-8.4.To study the effect of IL-8 on the expression of integrin ?v?3,quantitative real-time PCR and Western blot were used to detect the expression of integrin ?v?3 of HCC cells pretreated with IL-8 knockdown or exogenous IL-8.The integrin ?3 siRNA and over-expressed plasmid were used for reverse validation.5.To evaluate the role of CXCR1/2 in IL-8-mediated integrin ?3 expression and HCC invasion.Transwell assay and Western blot were used to detect the invasiveness,the expression of integrin ?3 of HCC cells pretreated with CXCR1/2 siRNA.6.To evaluate the role of PI3K/Akt pathway in IL-8-mediated integrin ?3 expression and HCC invasion.Transwell assay and Western blot were used to detect the invasiveness,the expression of integrin ?3 and the activation of PI3K/Akt pathway of HCC cells pretreated with IL-8 knockdown or exogenous IL-8.Results1.Among 170 HCC tissues,the median(Q1,Q3)IHC score of IL-8 was 4(2,5),and there was no significant difference between sarcomatoid HCC and conventional HCC(p=0.137).There were 89 cases(52.4%)with high expression of IL-8,including 69 conventional HCCs and 20 sarcomatoid HCCs,and the two pathological subtypes showed no significant difference in the rate of high expression(51.1%vs 57.1%,p=0.524).It was observed that the IHC score of IL-8 was significantly higher in HCC tissues compared with adjacent non-tumor tissues(p<0.001).IL-8 expression was significantly associated with both tumor thrombus and AJCC staging(all p<0.05).In 170 HCC tissues,the median(Q1,Q3)IHC score of integrin ?v?3 was 3(1,4),and there was no significant difference between sarcomatoid HCC and conventional HCC(p=0.100).There were 72 cases(42.4%)with high expression of integrin ?v?3,including 55 conventional HCCs and 17 sarcomatoid HCCs,and the two pathological subtypes showed no significant difference in the rate of high expression(40.7%vs 48.6%,p=0.403).It was observed that the IHC score of integrin ?v?3 was significantly higher in HCC tissues compared with adjacent non-tumor tissues(p<0.001).Integrin?v?3 expression was associated with tumor thrombus(p=0.03).There was a positive association between IL-8 and integrin ?v?3 expression(R=0.24,p=0.0014).Moreover,the combination high expression of IL-8 and integrin ?v?3 was correlated with tumor thrombus,adjacent organ invasion,AJCC staging and Edmondson-Steiner grade(all p<0.05),and had a tendency to correlate with lymph node metastasis(p=0.062).2.IL-8,integrin av and integrin ?3 were overexpressed in highly metastatic HCC cell lines compared with low metastatic cell lines.3.Both down-regulating the expression of endogenous IL-8 and incubating HCC cells with exogenous IL-8 had no significant effect on the proliferation of HCC cells.IL-8 siRNA transfection reduced HCC cell invasion,while exogenous IL-8 induced HCC cell invasion in a concentration-dependent manner.4.IL-8 siRNA transfection reduced integrin ?3 but not integrin av expression,while exogenous IL-8 up-regulates the expression of integrin ?3 but not integrin ?v in a concentration-dependent manner.Up-regulating integrin ?3 expression reversed the inhibitory effect of IL-8 siRNA on HCC cell invasion,while down-regulating the expression of integrin ?3 inhibited HCC cell invasion induced by exogenous IL-8.5.IL-8 induced HCC cell invasion and integrin ?3 expression could be significantly inhibited by transfection with CXCR1 siRNA or CXCR2 siRNA.6.When we stimulated HCC cells with exogenous IL-8,cell invasion and the levels of integrin ?3,p-PI3K,and p-Akt increased,which could be effectively inhibited by adding PI3K inhibitor LY294002.Conclusions1.In HCC tissues,there was a positive association between IL-8 and integrin ?v?3 expression.The combination high expression of IL-8 and integrin ?v?3 was correlated with tumor thrombus,adjacent organ invasion,AJCC staging and Edmondson-Steiner grade,and had a tendency to correlate with lymph node metastasis.2.Our results suggest that IL-8 induces HCC invasion by up-regulating integrin ?3,and both CXCR1/2 and PI3K/Akt pathway are involved in IL-8 induced HCC invasion by up-regulating integrin ?3.The IL-8/CXCR1/CXCR2/PI3K/Akt/integrin ?3 axis may serve as a potential treatment target for patients with HCCs.CHAPTER II.Clinicopathological Characteristics and Outcomes of Sarcomatoid HCCBackgroundThe most common histological pattern of hepatocellular carcinoma(HCC)is trabecular.In addition,there are some special histological subtypes of HCC,such as sarcomatoid HCC,fibrolamellar HCC and Scirrhous HCC.They differ from "conventional HCC"not only in cell morphology and tissue structure,but also in pathogenesis,gene mutation spectrum and prognosis.However,these histological subtypes of HCC are largely unknown due to their rarity.To our knowledge,current guidelines are devoid of recommendations based on HCC subtype.Therefore,comprehensively studying the clinical characteristics,treatment,prognosis and molecular mechanisms of these special subtypes of HCC,managing HCC patients according to histological classification,and taking individualized diagnosis and treatment,will benefit more patients,especially in the era of precision medicine.Sarcomatoid HCC is a highly invasive subtype of HCC,and characterized by high rates of organ invasion and metastasis,early recurrence and a poor prognosis.Sarcomatoid HCC accounted for approximately 2%among surgically resected HCC cases and 3.9%-9.4%among autopsied HCC patients.However,due to the large number of HCC cases and the fact that more than half of the global HCCs occur in China,the total number of HCC patients of this subtype is not small.To date,few data are available regarding sarcomatoid HCC.Therefore,it is of great significance to deepen the research on this subtype of HCC.Sarcomatoid HCCs contain variable proportions of sarcomatous and carcinomatous components,wherein the sarcomatous component usually consists of spindle-shaped cells that form interlacing bundles and show a partial storiform pattern and the carcinomatous component commonly comprises poorly differentiated[Edmondson-Steiner(ES)grade ? or ?]conventional HCC cells.The histogenesis of sarcomatous tissue in cancers,including HCC,has not yet been elucidated.There are several hypotheses such as collision theory,totipotent stem cell theory and conversion theory.The most widely accepted theory is the conversion theory,which postulates that the sarcomatous element derives from the carcinoma during tumor evolution.Several recent studies indicate that epithelial-mesenchymal transition is an important mechanism underlying the sarcomatous change,which further confirms the conversion theory.Previous literatures usually chosen conventional HCC as a control to study sarcomatoid HCC,and the results showed that sarcomatoid HCC was more aggressive and had a worse prognosis than conventional HCC.Previous studies have also demonstrated that sarcomatoid HCC were more poorly differentiated than conventional HCC.As we know,histological differentiation is an important prognostic factor for HCC patients,the poorer differentiation,the worse prognosis.However,previous studies did not consider the impact of histological differentiation on tumor characteristics and prognosis,and did not further stratify conventional HCC into low-grade(ES ?-?)and high-grade(ES ?-?)HCC.Sarcomatoid HCC contains both carcinomatous and sarcomatous components,but it's unclear which component plays a relatively important role for its aggressiveness.Up to now,only Maeda et al have done research on this issue.They studied 13 cases of sarcomatoid HCC treated by surgical resection and found that most portal venous invasions and metastases had sarcomatous components,which were postulated to be responsible for metastasis.However,in Maeda's study,5(38%)patients underwent preoperative treatment,such as TACE,and their analysis included an autopsied case with extensive postoperative metastases composed of sarcomatoid components;therefore,the results might be biased.In view of the deficiencies of previous studies,we will conduct further studies on sarcomatoid HCC.ObjectiveTo study the clinicopathological characteristics and outcomes of sarcomatoid HCC,and to further explore the histogenesis of sarcomatoid HCC.Methods We retrospectively reviewed the pathological records of surgically resected HCCs between January 2007 and December 2017 in Shandong Provincial Hospital Affiliated to Shandong University.A total of 196 HCCs(including 41 sarcomatoid HCCs and 155 high-grade HCC)were included in final analysis.Subsequently,we collected clinical information,laboratory test results,tumor-specific characteristics and survival data of the included patients.Numerical data are presented as the median(range)or mean ąSD.Differences between groups were compared using Pearson's chi-square test or the two-tailed Fisher's exact test for categorical data and the Mann-Whitney U test for numerical data.OS and RFS were determined using the Kaplan-Meier method,and differences between groups were assessed by the log-rank test.To further evaluate the impact of histological subtype on prognosis,univariate analyses of prognostic factors were performed using univariate Cox regression analysis.Moreover,the imaging data were reviewed by two radiologists,while the composition of invasive and metastatic sarcomatoid HCCs were evaluated by two pathologists.Results1.The age at diagnosis,sex composition,etiology of hepatopathy,liver cirrhosis,Child-Pugh classification,laboratory test results such as serum AFP and bilirubin,tumor location,tumor number,venous invasions,MVI,satellite nodules,spontaneous rupture,BCLC stage and recurrence pattern were comparable between the two groups(all p>0.05).Sarcomatoid HCC had a higher postoperative recurrence than high-grade HCC(82.9%vs 69.7%),but there was no significant difference between the two groups(p=0.091).Sarcomatoid HCC was more frequently diagnosed at an advanced stage with larger tumor and higher rates of nonspecific symptom,adjacent organ invasion,lymph node metastasis,tumor necrosis and lack of encapsulation than high-grade HCC(all p<0.05).Compared with high-grade HCC patients,sarcomatoid HCC patients are less likely to have elevated serum alpha-fetoprotein levels(5.8 vs 348.0 ng/ml,p<0.001)and typical dynamic imaging features of HCC(44.4%vs 72.7%,p=0.001).2.The sarcomatoid HCC patients had a shorter median OS than the high-grade HCC patients(10.5 vs 48.1 months,p<0.0001).The 1-,3-,and 5-year OS rates were 48.8%,17.3%,and 11.5%for the sarcomatoid group and 85.2%,53.4%,and 41.1%for the high-grade group,respectively.Moreover,the sarcomatoid HCC group had a shorter median RFS than the high-grade HCC group(5.6 vs 16.4 months,p<0.0001).The postoperative RFS rates were 49.7%and 83.1%at 6 months,20.9%and 60.2%at 1 year,and 6.3%and 31.8%at 3 years for the sarcomatoid and high-grade groups,respectively.Even after stratification by AJCC stage or differentiation grade of the carcinomatous component,the sarcomatoid HCC patients still had worse OS and shorter RFS than the high-grade HCC patients in each subgroup.Cox regression analysis was used to verify whether the sarcomatoid type is an independent prognostic factor for HCC patients.After controlling for confounding factors,sarcomatoid subtype was identified as an independent predictor of poorer OS and RFS in the multivariable analysis(all p<0.05).3.The histological composition of lymph node metastases,macrovascular invasions,bile duct invasions and multiple liver tumor lesions of sarcomatoid HCCs were analyzed.Among 33 lymph node metastases,26(78.8%)metastases contained purely carcinomatous components,2(6.1%)were purely sarcomatous,and 5(15.1%)had mixed carcinomatous and sarcomatous components.Six invasions were evaluated by pathological examination,and only 2(33.3%)had sarcomatous components.Among 10 patients with multinodular HCC,only 2(20.0%)had concurrent sarcomatoid HCC,whereas the others had simultaneous sarcomatoid and conventional HCC.In addition,among 6 patients with satellite nodules,sarcomatous changes were found in only 2 patients(33.3%).4.The sarcomatoid HCC patients were divided into three subgroups according to the proportion of sarcomatous components in the tumor:1)mixed subgroup ?50%(n=14 patients),2)mixed subgroup>50%(n=16),and 3)pure subgroup(n=11).The OS and RFS were similar among these subgroups(all p>0.05).Conclusions1.The majority of patients with sarcomatoid HCC had a history of chronic viral hepatitis and liver cirrhosis,Regular ultrasound screening of these high-risk patients could help detect tumors at an early stage and reduce the risk of death.2.Sarcomatoid HCC is more frequently diagnosed at an advanced stage with high rate of nonspecific symptom.Sarcomatoid HCC patients are less likely to have typical dynamic imaging features of HCC and elevated serum alpha-fetoprotein levels.3.Sarcomatoid HCC is a highly invasive subtype of HCC,which is prone to adjacent organ invasion,lymph node metastasis,tumor necrosis and lack of encapsulation.4.Sarcomatoid subtype is identified as an independent predictor of poorer OS and RFS.5.The carcinomatous components might exist in a "presarcomatoid" state,which mediate the highly aggressiveness of sarcomatoid HCC. |
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