in spite of improvements in surveillance and clinical treatment strategies , the overall survival ( os ) of hepatocellular carcinoma ( hcc ) patients is unsatisfactory due to the high incidence of tumor recurrence and metastasis even after hepatectomy and lack of effective adjuvant therapy . identification of reliable biomarkers that can be used in prediction of tumor recurrence or prognosis of hcc patients ensures more effective clinical management after curative hepatectomy . cell division cycle and apoptosis regulator 1 ( ccar1 ) was originally identified as a peri - nuclear phospho - protein reguired for apoptosis signaling by chemotherapy drugs adriamycin and etoposide independent of the p53 status of the human breast cancer cells . ccar1 plays a dynamic role in regulation of cell growth and apoptosis by serving as a cofactor of steroid / thyroid nuclear receptors , -catenin , and p53 in a variety of cell types including different cancer cells . kim et al .   reported that ccar1 promoted growth of mcf-7 human breast cancer cells in response to estradiol treatment . a recent study showed that ccar1 was required for growth of prostate cancer cells in part by functioning as a coactivator of androgen receptor transcription . another study reported that ccar1 was a binding partner of -catenin and mediated anchorage independent growth of colon cancer cells . however , whether ccar1 protein is overexpressed in hcc and the prognostic significance of ccar1 protein expression in hcc have not been reported . in 167 hcc patients with long - term follow - up , ccar1 protein expression was examined by immunohistochemistry to determine whether this protein can serve as a prognostic predictor . hcc tissues from 167 patients who underwent curative hepatectomy from july 2000 to may 2006 at samsung medical center , seoul , korea were analyzed . curative resection was defined as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors , as indicated by a computed tomography scan 1 month after surgery . tumor stages were classified according to both the american joint committee on cancer ( ajcc ) staging system   and barcelona clinic liver cancer ( bclc ) staging classification . intrahepatic metastasis and multicentric occurrence were defined according to the previously reported criteria . using 2 years as the cut off , all patients were followed up every 3 months after surgery and serum -fetoprotein levels were assessed and three phase dynamic computed tomography scans or magnetic resonance imaging were performed . the median follow - up period was 119.1 months ( range , 24.0 to 151.4 months ) for survivors . recurrence - free survival ( rfs ) was defined from the date of surgery until the date of tumor recurrence , metastasis , or last follow - up . os was defined from the date of surgery until the date of death or last follow - up . hcc - related death was defined as : ( 1 ) tumor occupying more than 80% of the liver , ( 2 ) portal venous tumor thrombus proximal to the second bifurcation , ( 3 ) obstructive jaundice due to the tumor , ( 4 ) distant metastases , or ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . hepatic failure was defined as the inability of the liver to perform its normal synthetic and metabolic function as part of normal physiology . epitope retrieval was performed with 0.01 mol / l citrate buffer at ph 6.0 for 30 minutes in a pressure cooker . the sections were incubated with rabbit polyclonal antibody to ccar1 ( hpa007856 , 1:100 , sigma - aldrich inc . , st . louis , mo ) for 60 minutes at room temperature . to validate the concordance between tissue microarrays and whole tumor sections , ccar1 expression was also detected for 40 corresponding whole tumor sections randomly chosen from the 167 cases . in negative controls , positive controls ( human normal kidney ) showed nuclear ccar1 expression in epithelial cells of convoluted tubules . immunoreactivity was evaluated independently by two blinded pathologists ( c .- k.p . and s.y.h . ) . nearly homogeneous nuclear immunostaining with moderate staining intensity was observed . to determine ccar1 protein expression , the percentage of tumor cells with positive staining was scored from 0% to 100% and each sample was rated from 0 to 4 ( 0 , < 5% ; 1 , 5%-25% ; 2 , 26%-50% ; 3 , 51%-75% ; and 4 , > duplicate tissue cores for each tumor showed high levels of homogeneity for the proportion of tumor cells with positive staining . chicago , il ) . the chi - square test or fisher exact test was used to examine possible correlations between ccar1 expression and clinicopathologic factors . survival curves were constructed using the kaplan - meier method , and differences in survival were evaluated using a log - rank test . univariate and multivariate analyses of survival were performed using the cox proportional hazards regression model . factors with prognostic significance in the univariate analysis were included in the subsequent multivariate analysis . hcc tissues from 167 patients who underwent curative hepatectomy from july 2000 to may 2006 at samsung medical center , seoul , korea were analyzed . curative resection was defined as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors , as indicated by a computed tomography scan 1 month after surgery . tumor stages were classified according to both the american joint committee on cancer ( ajcc ) staging system   and barcelona clinic liver cancer ( bclc ) staging classification . intrahepatic metastasis and multicentric occurrence were defined according to the previously reported criteria . using 2 years as the cut off , all patients were followed up every 3 months after surgery and serum -fetoprotein levels were assessed and three phase dynamic computed tomography scans or magnetic resonance imaging were performed . the median follow - up period was 119.1 months ( range , 24.0 to 151.4 months ) for survivors . recurrence - free survival ( rfs ) was defined from the date of surgery until the date of tumor recurrence , metastasis , or last follow - up . os was defined from the date of surgery until the date of death or last follow - up . hcc - related death was defined as : ( 1 ) tumor occupying more than 80% of the liver , ( 2 ) portal venous tumor thrombus proximal to the second bifurcation , ( 3 ) obstructive jaundice due to the tumor , ( 4 ) distant metastases , or ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . hepatic failure was defined as the inability of the liver to perform its normal synthetic and metabolic function as part of normal physiology . epitope retrieval was performed with 0.01 mol / l citrate buffer at ph 6.0 for 30 minutes in a pressure cooker . the sections were incubated with rabbit polyclonal antibody to ccar1 ( hpa007856 , 1:100 , sigma - aldrich inc . , st . louis , mo ) for 60 minutes at room temperature . to validate the concordance between tissue microarrays and whole tumor sections , ccar1 expression was also detected for 40 corresponding whole tumor sections randomly chosen from the 167 cases . in negative controls , positive controls ( human normal kidney ) showed nuclear ccar1 expression in epithelial cells of convoluted tubules . immunoreactivity was evaluated independently by two blinded pathologists ( c .- k.p . and s.y.h . ) . nearly homogeneous nuclear immunostaining with moderate staining intensity was observed . to determine ccar1 protein expression , the percentage of tumor cells with positive staining was scored from 0% to 100% and each sample was rated from 0 to 4 ( 0 , < 5% ; 1 , 5%-25% ; 2 , 26%-50% ; 3 , 51%-75% ; and 4 , > duplicate tissue cores for each tumor showed high levels of homogeneity for the proportion of tumor cells with positive staining . chicago , il ) . the chi - square test or fisher exact test was used to examine possible correlations between ccar1 expression and clinicopathologic factors . survival curves were constructed using the kaplan - meier method , and differences in survival were evaluated using a log - rank test . univariate and multivariate analyses of survival were performed using the cox proportional hazards regression model . factors with prognostic significance in the univariate analysis were included in the subsequent multivariate analysis . a p - value of < 0.05 was considered to indicate statistical significance . the study included 139 men and 28 women with an average age of 52.5 years ( range , 17 to 76 years ) . the adjacent non - tumor liver had liver cirrhosis in 88 cases ( 52.7% ) ( table 1 ) . tumor recurrence was detected in 122 patients ( 73.1% ) , early recurrence in 98 patients ( 58.7% ) , and late recurrence in 24 patients ( 14.4% ) . eight of these 14 deaths were due to hepatic failure , five were due to non - hepatic causes , and one was due to unknown cause . in hcc , immunoreactivity for ccar1 was observed in the nucleus of tumor cells with moderate staining intensity . weak ccar1 immunoreactivity was observed in the nucleus of bile duct epithelial cells , kupffer cells , and lymphocytes . ccar1 immunoreactivity was graded as low ( 0%-50% stained tumor cells regardless of staining intensity ) or high expression ( > 50% stained tumor cells ) . high ccar1 immunoreactivity was observed in 149 of the 167 hcc cases ( 89.2% ) ( fig . high ccar1 expression showed significant correlation with microvascular invasion ( p=0.002 ) , intrahepatic metastasis ( p=0.015 ) , and higher ajcc t stage ( p=0.017 ) . high ccar1 expression showed significant correlation with early recurrence ( p=0.001 ) , but not with late recurrence ( p=0.121 ) ( table 1 ) . in univariate analysis , early recurrence showed significant correlation with microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher ajcc t stage ( p < 0.001 ) , higher bclc stage ( p=0.002 ) , lower albumin level ( p=0.049 ) , viral etiology ( p=0.001 ) , and high ccar1 expression ( p=0.002 ) . to avoid potential bias , because ajcc t stage and bclc stage were associated with vascular invasion , multiple analyses with these variables were not performed . the significance of serum -fetoprotein level was not evaluated because of missing data ( n=161 ) . in multivariate analysis , intrahepatic metastasis ( p=0.010 ) and viral etiology ( p=0.019 ) were independent predictors of early recurrence ( table 2 ) . the rfs and os rates for 167 hcc patients were 35.7% and 71.7% at 3 years , 32.0% and 60.7% at 5 years , 27.1% and 51.1% at 7 years , and 26.0% and 46.6% at 9 years , respectively . in univariate analysis , larger tumor size , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t stage , higher bclc stage , and lower albumin level showed unfavorable effects on both rfs and os . high ccar1 expression showed unfavorable effects on both rfs and os ( table 3 ) . the 5-year rfs rate of the high ccar1 expression group was significantly lower than that of the low ccar1 expression group ( 27.1% vs. 72.2% ) ( p=0.002 ) ( fig . the mean rfs was 42.5 months for the high ccar1 expression group and 100.9 months for the low ccar1 expression group . in subgroup analysis , among patients with -fetoprotein  20 ng / ml ( n=54 ) and patients with ajcc t stage 1 ( n=62 ) , significant differences in rfs were observed between high ccar1 expression groups ( n=41 and n=50 , respectively ) and low ccar1 expression groups ( p=0.015 and p=0.004 , respectively ) ( fig . 2b and c ) . in further analysis , among patients with tumor size  5.0 cm ( n=96 ) , no significant difference in rfs was observed between high ccar1 expression group ( n=86 ) and low ccar1 expression group ( p=0.189 ) ( fig . . the 5-year os rate of the high ccar1 expression group was significantly lower than that of the low ccar1 expression group ( 58.0% vs. 83.3% ) ( p=0.015 ) ( fig . the mean os was 86.9 months for the high ccar1 expression group and 126.1 months for the low ccar1 expression group . in subgroup analysis , among patients with -fetoprotein  20 ng / ml ( n=54 ) , significant difference in os was observed between high ccar1 expression group ( n=41 ) and low ccar1 expression group ( p=0.046 ) ( fig . 3b ) . in further analysis , among patients with ajcc t stage 1 ( n=62 ) , the high ccar1 expression group ( n=50 ) tended to show an unfavorable effect on os ( p=0.089 ) ( fig . 5.0 cm ( n=96 ) , no significant difference in os was observed between high ccar1 expression group ( n=86 ) and low ccar1 expression group ( p=0.448 ) ( fig . intrahepatic metastasis and lower albumin level were independent predictors of both shorter rfs and shorter os . high ccar1 expression tended to be an independent predictor of shorter rfs ( p=0.054 ) , but not shorter os ( p=0.348 ) . patients with high ccar1 expression were more likely to suffer from recurrence than those with low ccar1 expression ( hazard ratio , 2.184 ) ( table 4 ) . the study included 139 men and 28 women with an average age of 52.5 years ( range , 17 to 76 years ) . the adjacent non - tumor liver had liver cirrhosis in 88 cases ( 52.7% ) ( table 1 ) . tumor recurrence was detected in 122 patients ( 73.1% ) , early recurrence in 98 patients ( 58.7% ) , and late recurrence in 24 patients ( 14.4% ) . eight of these 14 deaths were due to hepatic failure , five were due to non - hepatic causes , and one was due to unknown cause . in hcc , immunoreactivity for ccar1 was observed in the nucleus of tumor cells with moderate staining intensity . weak ccar1 immunoreactivity was observed in the nucleus of bile duct epithelial cells , kupffer cells , and lymphocytes . ccar1 immunoreactivity was graded as low ( 0%-50% stained tumor cells regardless of staining intensity ) or high expression ( > 50% stained tumor cells ) . high ccar1 immunoreactivity was observed in 149 of the 167 hcc cases ( 89.2% ) ( fig . 1 ) . high ccar1 expression showed significant correlation with microvascular invasion ( p=0.002 ) , intrahepatic metastasis ( p=0.015 ) , and higher ajcc t stage ( p=0.017 ) . high ccar1 expression showed significant correlation with early recurrence ( p=0.001 ) , but not with late recurrence ( p=0.121 ) ( table 1 ) . in univariate analysis , early recurrence showed significant correlation with microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher bclc stage ( p=0.002 ) , lower albumin level ( p=0.049 ) , viral etiology ( p=0.001 ) , and high ccar1 expression ( p=0.002 ) . to avoid potential bias , because ajcc t stage and bclc stage were associated with vascular invasion , multiple analyses with these variables were not performed . the significance of serum -fetoprotein level was not evaluated because of missing data ( n=161 ) . in multivariate analysis , intrahepatic metastasis ( p=0.010 ) and viral etiology ( p=0.019 ) were independent predictors of early recurrence ( table 2 ) . the rfs and os rates for 167 hcc patients were 35.7% and 71.7% at 3 years , 32.0% and 60.7% at 5 years , 27.1% and 51.1% at 7 years , and 26.0% and 46.6% at 9 years , respectively . in univariate analysis , larger tumor size , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t stage , higher bclc stage , and lower albumin level showed unfavorable effects on both rfs and os . high ccar1 expression showed unfavorable effects on both rfs and os ( table 3 ) . the 5-year rfs rate of the high ccar1 expression group was significantly lower than that of the low ccar1 expression group ( 27.1% vs. 72.2% ) ( p=0.002 ) ( fig . the mean rfs was 42.5 months for the high ccar1 expression group and 100.9 months for the low ccar1 expression group . in subgroup analysis , among patients with -fetoprotein  20 ng / ml ( n=54 ) and patients with ajcc t stage 1 ( n=62 ) , significant differences in rfs were observed between high ccar1 expression groups ( n=41 and n=50 , respectively ) and low ccar1 expression groups ( p=0.015 and p=0.004 , respectively ) ( fig . 2b and c ) . in further analysis , among patients with tumor size  5.0 cm ( n=96 ) , no significant difference in rfs was observed between high ccar1 expression group ( n=86 ) and low ccar1 expression group ( p=0.189 ) ( fig . the 5-year os rate of the high ccar1 expression group was significantly lower than that of the low ccar1 expression group ( 58.0% vs. 83.3% ) ( p=0.015 ) ( fig . the mean os was 86.9 months for the high ccar1 expression group and 126.1 months for the low ccar1 expression group . in subgroup analysis , among patients with -fetoprotein  20 ng / ml ( n=54 ) , significant difference in os was observed between high ccar1 expression group ( n=41 ) and low ccar1 expression group ( p=0.046 ) ( fig . 3b ) . in further analysis , among patients with ajcc t stage 1 ( n=62 ) , the high ccar1 expression group ( n=50 ) tended to show an unfavorable effect on os ( p=0.089 ) ( fig . 5.0 cm ( n=96 ) , no significant difference in os was observed between high ccar1 expression group ( n=86 ) and low ccar1 expression group ( p=0.448 ) ( fig . 3d ) . in multivariate analysis , intrahepatic metastasis and lower albumin level were independent predictors of both shorter rfs and shorter os . high ccar1 expression tended to be an independent predictor of shorter rfs ( p=0.054 ) , but not shorter os ( p=0.348 ) . patients with high ccar1 expression were more likely to suffer from recurrence than those with low ccar1 expression ( hazard ratio , 2.184 ) ( table 4 ) . ccar1 plays important roles in regulation of cancer cell growth in part by recruiting multiple mediators and confers optimal conformation for transcriptional functions of nuclear receptors [ 7 - 9 ] . apoptosis induction by ccar1 involves its binding with 14 - 3 - 3 protein and down - regulation of cell growth and cell cycle regulators c - myc , topoisomerase ii , cyclin b , and p21 rac1 . diminished expression of ccar1 in human breast cancers involved methylation - dependent gene silencing associated with hypermethylation of ccar1 promoter . ccar1 functional mimetics ( cfms ) are novel small molecule inhibitors of ccar1 interaction with anaphase promoting complex / cyclosome . cfms are potent inhibitors of a variety of cancer cells including drug ( adriamycin or tamoxifen)resistant breast cancer cells but not immortalized breast epithelial cells in part by activating apoptosis signaling and diminishing the levels of key cell cycle regulatory proteins . this study demonstrates , for the first time , that high ccar1 protein expression in hcc might be a marker of shorter rfs after curative hepatectomy . high ccar1 expression showed correlation with microvascular invasion , intrahepatic metastasis , and higher ajcc t stage , suggesting the involvement of ccar1 in the progression of hcc . in the current study , tumor recurrence was detected in 35 of 54 patients ( 64.8% ) with normal serum -fetoprotein levels and in 36 of 62 patients ( 58.1% ) with ajcc t stage 1 . identification of subpopulations with poor prognosis for early stage hcc patients is critical to the optimization of personalized treatment . ccar1 protein expression may be helpful in prediction of recurrence risk in patients with normal serum -fetoprotein levels and early stage hcc after curative hepatectomy . a nanolipid formulation of cfm-4 showed superior bioavailability and inhibited growth of xenografted triple - negative breast cancer and non - small cell lung cancer in nude mice , when administered orally . the absence of ccar1 protein expression in the normal liver enhances the potential of ccar1 as a treatment target in hcc . current treatment options for hccs are limited , thus discovery and development of molecules based on ccar1 signaling could yield novel tools for treatment of hccs . for the first time , the current study demonstrated that ccar1 protein could be a potential biomarker predicting rfs in hcc patients after curative hepatectomy . in addition , ccar1 had prognostic values in hcc patients with normal serum -fetoprotein levels or early stage hcc . patients with high ccar1 expression are considered candidates for adjuvant treatment after surgical resection . further study is needed to determine the underlying molecular mechanism of ccar1 in the development and progression of hcc .