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Combined hepatocellular-cholangiocarcinoma with a ductal plate malformation pattern is an extremely rare entity with unelucidated pathogenesis. We present the case of a 60-year-old male patient who underwent a sectionectomy for pre-operative diagnosis of hepatocellular carcinoma based on clinical and image findings. Gross examination of the specimen revealed a well-defined tumor with cystic change measuring 6.7 × 6.2 cm. Microscopically, the lesion had classical features of hepatocellular carcinoma and intrahepatic cholangiocarcinoma exhibited neoplastic glands with irregular-sized dilated lumens, resembling a ductal plate malformation. Postoperative diagnosis was combined hepatocellular-cholangiocarcinoma with ductal plate malformation pattern. Next-generation sequencing revealed genomic alteration in 15 genes: CDKN2A , CHD4 , CYP2D6 , ERBB3 , KIR3DL1 , KRAS , MDM2 , PIM1 , STAT6 , TPMT amplification, FANCD2 , FAT1 , FLT4 , RASA1 , and TP53 point mutation. This is the first case report of molecular alteration in combined hepatocellular-cholangiocarcinoma with ductal plate malformation pattern., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: To evaluate outcomes and risk factors associated with hypervascularization in hypovascular, hepatobiliary phase (HBP) hypointense nodules (HHNs) without T2 hyperintensity on gadoxetic acid-enhanced magnetic resonance (MR) images in chronic liver disease patients., Materials and Methods: 222 HHNs were analyzed. Multivariate analysis with a Cox proportional hazard regression model was used., Results: 41 nodules became hypervascular hepatocellular carcinoma (HCC). History of HCC, hyperintensity on T1WI or DWI, and higher growth rate were risk factors for hypervascularization (P < .05)., Conclusion: History of HCC, hyperintensity on T1WI or DWI, and higher growth rate were associated with nodular progression to hypervascular HCC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background Gadoxetic acid is being widely used for detection and characterization of hepatic nodules. However, there are no data regarding intra-individual comparison of imaging features of hepatocellular carcinoma (HCC) on dynamic computed tomography (CT), gadopentetate dimeglumine-enhanced magnetic resonance imaging (Gd-DTPA-MRI), and gadoxetic acid-enhanced MRI (Gd-EOB-MRI). Purpose To evaluate typical imaging features of HCC and capsule appearance with dynamic CT, Gd-DTPA-MRI, and Gd-EOB-MRI. Material and Methods We retrospectively reviewed 56 HCCs in 49 patients. Lesion attenuation/signal intensity was graded using a five-point scale based on dynamic phase and hepatobiliary phase (HBP) imaging. Subjective washout and capsule appearance were evaluated on portal venous phase (PVP) or delayed/transitional phase (DP/TP) imaging. The tumor-to-liver contrast ratio (TLCR) was calculated. Results Gd-DTPA-MRI and Gd-EOB-MRI was graded higher than CT on arterial phase ( P < 0.001). Gd-EOB-MRI was graded lower than Gd-DTPA-MRI on PVP and DP/TP ( P < 0.05). The detection rate of subjective washout and capsule appearance did not differ among the three imaging studies on either PVP or DP/TP. TLCR of Gd-EOB-MRI was lower than CT on PVP ( P = 0.004) and was lower than Gd-DTPA-MRI on DP/TP ( P = 0.001). Conclusion Arterial phase hyperenhancement and washout appearance of HCC were well demonstrated in Gd-EOB-MRI. The detection of capsule appearance using Gd-EOB-MRI was not inferior to Gd-DTPA-MRI or CT.

Recently, the roles of FAM83H in tumorigenesis have been interested and increased expression of FAM83H and MYC in hepatocellular carcinoma (HCC) have been reported. Therefore, we investigated the expression and role of FAM83H in 163 human HCCs and further investigated the relationship between FAM83H and oncogene MYC. The expression of FAM83H is elevated in liver cancer cells, and nuclear expression of FAM83H predicted shorter survival of HCC patients. In HLE and HepG2 HCC cells, knock-down of FAM83H inhibited proliferation and invasive activity of HCC cells. FAM83H induced expression of cyclin-D1, cyclin-E1, snail and MMP2 and inhibited the expression of P53 and P27. In hepatic tumor cells derived from Tet-O-MYC mice, the expression of mRNA and protein of FAM83H were dependent on MYC expression. Moreover, a chromatin immunoprecipitation assay demonstrated that MYC binds to the promotor of FAM83H and that MYC promotes the transcription of FAM83H, which was supported by the results of a dual-luciferase reporter assay. In conclusion, we present an oncogenic role of FAM83H in liver cancer, which is closely associated with the oncogene MYC. In addition, our results suggest FAM83H expression as a poor prognostic indicator of HCC patients.

Phosphorylation of proteins on serine/threonine residues that precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis-trans isomerase PIN1. PIN1-mediated prolyl-isomerization induces cell cycle arrest and growth inhibition through the regulation of target proteins, including TP53. We examined whether PIN1 acts in a different manner according to TP53 gene status in hepatocellular carcinoma (HCC). We investigated the expression of PIN1 and TP53 proteins in 119 HCC tissue samples. We also analyzed PIN1 expression in combination with TP53 gene mutation and its correlation with the clinical outcome. In addition, we used synthetic small interfering RNA to silence PIN1 gene expression in TP53 wild-type and TP53 mutant HCC cell lines, and then evaluated cell proliferation, migration and invasion. Expression of PIN1 was strongly associated with expression of TP53 protein or TP53 mutation of HCC samples. PIN1 and TP53 expression in TP53 mutant HCC cell lines was higher than that in TP53 wild-type HCC cell lines. Silencing of PIN1 in HLE cells containing mutant TP53 significantly decreased cell proliferation, migration and invasion. In contrast to PIN1 silencing in HLE cells, PIN1 silencing in HepG2 cells containing functional wild-type TP53 resulted in enhanced tumor cell proliferation. HCC patients bearing PIN1 expression with wild-type TP53 were predicted to demonstrate favorable relapse-free survival. Our results suggest that PIN1 plays a role in cancer cell proliferation, migration and invasion in a different manner according to the TP53 gene mutation status in HCC. In particular, interaction of PIN1 with mutant TP53 can act as a tumor promoter and increase its oncogenic activities in HCC.

Regulated intramembrane proteolysis of epithelial cell adhesion molecule (EpCAM) results in shedding of the extracellular domain (EpEX) and release of the intra-cellular domain (EpICD) into the cytoplasm. Released EpICD associates with FHL2, β-catenin and Lef-1 to form a nuclear complex and triggers oncogenic signaling. This study was conducted to examine the nuclear expression of EpICD in hepatocellular carcinoma (HCC) and to assess the role of EpICD in HCC. EpICD immunoexpression was examined in 100 cases of HCC using tissue microarrays and correlated with clinicopathological parameters. We also examined the role of EpICD in HCC using EpICD cDNA transfected HCC cell line and EpCAM silenced HCC cell line by small interfering RNA (siRNA). Nuclear expression of EpICD was observed in 19 of 100 (19%) cases. Nuclear expression of EpICD significantly correlated with nuclear expression of β-catenin, and Ki-67 labeling index. In addition, nuclear expression of EpICD was associated with higher histologic grade and advanced T category. Forced overexpression of EpICD in the HCC cell significantly increased the cell proliferation, migration and invasion. The overexpression of EpICD also increased the expression levels of the active form of β-catenin and c-myc and cyclin D1. In contrast, downregulation of EpCAM by siRNA decreased the cell proliferation, migration, invasion and the expression of active form of β-catenin, c-myc and cyclin D1. Our present data suggest that EpICD plays important roles in HCC progression by modulating expression of target genes of EpCAM.

The epithelial to mesenchymal transition (EMT) is a crucial process in tumor progression. EMT of tumor cells not only causes increased metastasis, but also contributes to drug resistance. Serum response factor (SRF) is a transcription factor that plays a central role in carcinogenesis and tumor progression in several types of cancers. We investigated the effect of EMT-related SRF, focusing on its promotion of chemoresistance against sorafenib in hepatocellular carcinoma (HCC). We examined SRF and Snail expression in 146 cases of HCCs by immunohistochemistry. We also examined the chemoresistance effect of SRF in HCC cells by transfecting HLE cells with SRF cDNA and SH-J1 cells with SRF antisense cDNA. Expression of SRF and Snail were detected in 37.6% (55 of 146 cases) and in 12.3% (18 of 146 cases) of the HCCs, respectively. None of the tumor-free liver tissues showed SRF or Snail expression. SRF expression was closely correlated with the expression of Snail (p<0.001) and expression of both SRF and Snail showed significant correlation with the high histological grade (p=0.015 and 0.003, respectively). Overexpression of SRF in HLE cells led to increased expression of mesenchymal markers, as well as increased cell growth and colony formation. Overexpression of SRF also led to a significant reduction in the cytotoxic effect of sorafenib in HLE cells. Conversely, inhibition of SRF expression in the SH-J1 cells significantly enhanced the apoptotic effects of sorafenib, along with the reduced expression of mesenchymal markers and restored the expression of E-cadherin. These results suggest that SRF is critical for HCC to acquire a mesenchymal phenotype, which leads to resistance against a sorafenib-mediated apoptotic effect.

Epithelial cell adhesion molecule (EpCAM) has been proposed as a marker for cancer stem cells in human hepatocellular carcinoma (HCC). However, the function and clinical significance of EpCAM in HCC is largely unknown. We examined EpCAM expression and localization in 28 dysplastic nodules (DNs) and their corresponding cirrhotic nodules, 79 HCC tissue sections and 132 HCC tissue microarray cores by immunohistochemistry and determined the relationship to clinicopathologic findings. We also examined the role of EpCAM in HCC using synthetic small interfering RNA to silence EpCAM gene expression in Huh-7 cells. EpCAM expression was very rare in DNs but dominantly appeared in a distinctly nodular type of small HCC. Expression of EpCAM was observed in 39% (31/79) of HCC tissue sections and in 34.1% (45/132) of tissue microarray sections. EpCAM expression in HCC was significantly associated with high tumor grade and serum α-fetoprotein level. Silencing EpCAM gene expression significantly decreased the proliferative activity and invasiveness of HCC cells. EpCAM expression was an independent prognostic factor for survival in patients with T1 HCC. The data indicate that EpCAM expression occurs at distinct nodular stage of HCC and could play an important role in HCC progression.

The increased expression of SIRT1 has recently been identified in numerous human tumors and a possible correlation with c-Myc oncogene has been proposed. However, it remains unclear whether SIRT1 functions as an oncogene or tumor suppressor. We sought to elucidate the role of SIRT1 in liver cancer under the influence of c-Myc and to determine the prognostic significance of SIRT1 and c-Myc expression in human hepatocellular carcinoma. The effect of either over-expression or knock down of SIRT1 on cell proliferation and survival was evaluated in both mouse and human liver cancer cells. Nicotinamide, an inhibitor of SIRT1, was also evaluated for its effects on liver tumorigenesis. The prognostic significance of the immunohistochemical detection of SIRT1 and c-Myc was evaluated in 154 hepatocellular carcinoma patients. SIRT1 and c-Myc regulate each other via a positive feedback loop and act synergistically to promote hepatocellular proliferation in both mice and human liver tumor cells. Tumor growth was significantly inhibited by nicotinamide in vivo and in vitro. In human hepatocellular carcinoma, SIRT1 expression positively correlated with c-Myc, Ki67 and p53 expression, as well as high á-fetoprotein level. Moreover, the expression of SIRT1, c-Myc and p53 were independent prognostic indicators of hepatocellular carcinoma. In conclusion, this study demonstrates that SIRT1 expression supports liver tumorigenesis and is closely correlated with oncogenic c-MYC expression. In addition, both SIRT1 and c-Myc may be useful prognostic indicators of hepatocellular carcinoma and SIRT1 targeted therapy may be beneficial in the treatment of hepatocellular carcinoma.

Serum response factor (SRF) regulates tran-scription of immediate early genes and triggers proliferation, migration and differentiation in several types of cells. Matrix metalloproteinases (MMPs), a group of zinc-dependent endopeptidases, play a crucial role in tumor invasion and metastasis. However, expression of SRF and its association with MMPs in hepatocellular carcinoma (HCC) have not been elucidated. We examined the expression levels of SRF, MMP-2 and MMP-9 in HCC tissues using Western blot assay. We also examined the effect of SRF on MMP expression and enzyme activity in HCC by transfection of SRF cDNA in HLE cells. Protein expression of SRF, MMP-2 and MMP-9 showed a significant increase in HCC tissues, compared with those of corresponding non-tumor tissues. High SRF expressing HCC tissues showed higher levels of expression of MMP-2 and MMP-9, compared with low SRF expressing HCC tissues. In addition, overexpression of SRF in HLE cells led to increased levels of expression of mRNA and protein, as well as increased enzyme activity of MMP-2 and MMP-9. Overexpression of SRF also led to significantly enhanced migration of HLE cells. These results suggest that overexpression of SRF in HCC may play an important role in tumor cell migration and invasion through upregulation of MMP-2 and MMP-9.

Silent mating type information regulation 2 homolog 1 (SIRT1) is a multifaceted, nicotinamide adenine dinucleotide-dependent protein deacetylase with involvement in a wide variety of cellular processes ranging from cancer to aging. Expression of SIRT1 was evaluated in 90 cases of hepatocellular carcinoma (HCC) and five HCC cell lines. The relationship between the mutation status of p53 and expression of SIRT1 was also investigated in 10 fresh HCC tissues. Synthetic small interfering RNA was used to silence SIRT1 gene expression by RNA interference (RNAi), and cell growth and cell cycle progression were assessed. Expression of SIRT1 was significantly elevated in the HCC tissues when compared to that of non-tumor tissues (p<0.001). Overexpression of SIRT1 and p53 was observed in 56% (50 of 90) and in 30% (27 of 90) of the HCCs, respectively. Expression of SIRT1 showed significant correlation with gender (p=0.023), serum AFP levels (p=0.030), viral infection (p=0.005) and p53 expression (p<0.021). Western blot analysis found no correlation between p53 mutation and expression levels of SIRT1. SIRT1 silencing was found to induce cell growth arrest in HCC cells. These results suggest an association of SIRT1 expression with HCC development and that SIRT1 plays a role in cancer cell growth.

Combined hepatocellular carcinoma and cholangiocarcinoma (combined HCC-CC) is a rare subtype of primary liver cancer. We investigated the histopathologic features of transitional or intermediate areas in 21 combined HCC-CCs and immunophenotypes using different hepatic progenitor cell markers (CK7, CK19, c-kit, NCAM, and EpCAM). Major histologic findings of transitional or intermediate areas of 21 combined HCC-CCs included strands/trabeculae of small, uniform, oval-shaped cells with scant cytoplasm and hyperchromatic nuclei embedded within an abundant stroma, small cells with an antler-like anastomosing pattern, and solid nests of intermediate hepatocyte-like cells surrounded by small cells in periphery, in order of frequency. The intermediate area of one tumor was composed predominantly of spindle cells arranged in short fascicles. Immunophenotype of tumor cells with intermediate morphology suggested a progenitor cell origin for this tumor. Clinical findings of combined HCC-CC showed a closer resemblance with those of HCC than those of CC. In univariate analysis, tumor size, TNM stage, and serum alpha-fetoprotein levels showed a significant association with poor patient survival. Serum alpha-fetoprotein level was an independent prognostic indicator in multivariate analysis. In conclusion, an awareness of the clinicopathologic features, specifically the various morphologic features of intermediate areas in this tumor, is essential for prevention of potential misdiagnosis as another tumor.

Serum response factor (SRF) regulates transcription of the immediate early genes and triggers proliferation, migration and differentiation in several types of cells. We examined the role of SRF in HCC by transfecting the SRF cDNA in HLE cells and the SRF anti-sense cDNA in sarcomatoid HCC cells. The overexpression of SRF in the HLE cells significantly increased the cell growth and proliferation. Overexpression of SRF increased actin polymerization of the HCC cells and induced morphologic changes. The mesenchymal markers vimentin, N-cadherin and RhoA were highly expressed in the SRF-transfected HLE cells. Furthermore, the overexpression of SRF in the HLE cells increased the expression levels of the active form of the beta-catenin and Wnt/beta-catenin target genes, such as c-myc and cyclin D1. The overexpression of SRF significantly enhanced the cell migration and invasiveness of HCC cells. Conversely, inhibition of the SRF expression in the sarcomatoid SH-J1 cells by the SRF anti-sense cDNA significantly decreased migration and invasion through the attenuated expression of mesenchymal markers and the proteins involved in the Wnt/beta-catenin pathway. These results indicate that the overexpression of SRF in HCC cells modulates the Wnt/beta-catenin pathway, and this plays an important role in HCC progression.

Spontaneous regression of hepatocellular carcinoma (HCC) is extremely rare. Among the numerous proposed mechanisms of spontaneous regression for HCC, immunological factors, which have yet to be fully understood, may have the most important roles in this rare phenomenon. A regressing HCC with lymphoid stroma is reported. A hepatic mass was detected in a 57-year-old man by abdominal computed tomography during a follow-up health check. The resected tumor was 2.7 x 2.4cm(2) in size, and was composed of two distinct nodules with a complete rim of fibrous tissue separating the tumor from the adjacent liver parenchyma. Microscopically, one nodule was replaced by a remarkable infiltration of inflammatory cells with insignificant amounts of vital tumor cells. The other nodule demonstrated a small portion of well- to moderately-differentiated hepatocellular carcinoma cells forming 2-3 thick trabeculae with massive inflammatory infiltrates. Infiltrating inflammatory cells were composed of T cells (CD4+>CD8+), macrophages, B cells, and other cells in order of prevalence. Epstein-Barr virus was not detected. After 60 months of follow-up, the patient is in good health without evidence of tumor recurrence.

The acquisition of a migratory and invasive phenotype by cells of epithelial origin is associated with a gain of mesenchymal characteristics concomitant with a loss of the epithelial phenotype, a phenomenon referred to as epithelial-mesenchymal transition (EMT). Vimentin, a cytoplasmic intermediate filament, is characteristic of mesenchymal cells and is usually not expressed in epithelial cells. Increased expression of vimentin in carcinomas correlates with parameters of malignant potential such as tumor grade and tumor invasion. Serum response factor (SRF) regulates transcription of immediate early genes and triggers proliferation, migration and differentiation in several types of cells. However, the role of SRF in hepatocellular carcinoma (HCC) has not been explored. The aims of this study were to evaluate the expression of SRF and to assess a functional role of SRF in HCC. First, we examined the expression of SRF in 55 human specimens of HCC and four different HCC cell lines, including a sarcomatoid HCC cell line. We also examined the role of SRF in HCC by transfection of an SRF expression plasmid into a HCC cell line. SRF was expressed in 13 of 55 cases of HCC. SRF was predominantly expressed in HCC cells, with intense labeling in the nucleus. No staining was observed in hepatocytes of normal and cirrhotic liver outside the tumor. SRF was significantly up-regulated in high grade HCC, especially in sarcomatoid HCC. Overexpression of SRF in hepatocellular carcinoma cells accelerates migration and invasion with subsequent acquisition of mesenchymal phenotype by expression of a mesenchymal marker (vimentin) and activation of immediate early genes. We propose for the first time that the expression of SRF in HCC cells associated with EMT may play an important role in HCC progression. Thus, functional antagonism of SRF will provide an additional therapeutic approach by controlling tumor cell invasion and metastasis.

Increased expression of vimentin in carcinomas correlates with parameters of malignant potential such as tumor grade and tumor metastasis. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been intensively evaluated as a potential target for the inhibition of cell growth and metastasis in cancer cells. In the present study, we examined whether PPARgamma is a possible target molecule for the prevention of cell growth and invasion by treatment with agonists (troglitazone, rosiglitazone) and antagonists (T0070907, GW9662) in four different hepatocellular carcinoma (HCC) cell lines. We also evaluated the effects of the PPARgamma agonists and antagonists on tumor cell migration and invasion. The expression level of PPARgamma protein was higher in the sarcomatoid SH-J1 and poorly differentiated HLE cell lines than that in the well-differentiated HCC cell lines (HepG2 and Huh-7). Expression of vimentin was high in the SH-J1 HCC cell line and minimally detected in the HLE cell line. Treatment with low doses of the PPARgamma antagonists inhibited cell growth and colony formation of all four of the HCC cell lines. Vimentin in the high-grade HCC cells was cleaved by the treatment with the PPARgamma antagonists. Furthermore, treatment with the PPARgamma antagonists also strongly inhibited migration and invasion of the SH-J1 and HLE cells. However, treatment with low doses of the agonists had no effect on vimentin expression, migration, and invasion of the high-grade HCC cells but cell growth was inhibited by treatment with high concentrations of the agonists. Our results indicate that treatment with a PPARgamma antagonist may prevent cell growth and invasion of high-grade HCC cells. Our findings also suggest that PPARgamma antagonists inhibit cell growth and invasion through vimentin disarrangement in high-grade HCC.

Background/aims: Hepatocellular carcinoma (HCC) is becoming one of the common malignant tumors worldwide, and it is characterized by its high vascularity. Caveolin is the major structural protein in caveolae, which are small omega-shaped invaginations within the plasma membrane. Caveolin has been implicated in mitogenic signaling, oncogenesis and angiogenesis. The expression of caveolin-1 and -2 in HCC and its potential relationship with angiogenesis has not been examined., Methods: Paraffin sections of 35 HCC specimens were immunostained with caveolin-1, caveolin-2, alpha-smooth muscle actin, and CD34 antibodies. In addition, the expression of caveolin-1 and -2 mRNA in HCC was examined. The relationship between the radiological findings and the number of unpaired arteries and microvessel density (MVD) was also investigated., Results: Caveolin-1 and -2 were expressed in the sinusoidal endothelial cells in 20 out of 35, and 18 out of 35 HCC specimens, respectively. Caveolin-1 and -2 were also expressed in the smooth muscle cells of the unpaired arteries in 26 out of 35, and 18 out of 35 HCC specimens, respectively. Increased expression of caveolin-1 and -2 mRNA was detected in 26.7% and 33.3% of the tumor specimens, respectively, compared with the corresponding non-tumorous adjacent liver tissues. There was a significant correlation between expression of caveolin-1, -2 in the smooth muscle cells of unpaired arteries and the number of unpaired arteries. The number of unpaired arteries in HCCs was found to be associated with the degree of contrast enhancement in the arterial phase imaging. However, it did not correlate with the degree of MVD., Conclusions: These findings suggest that the expression of caveolin-1, -2 is associated with the formation of unpaired arteries in HCC. In addition, there is a correlation between the degree of contrast enhancement of the HCC in the arterial phase image and the number of unpaired arteries.

Hepatocellular carcinoma (HCC) is becoming one of the common malignant tumors worldwide and is characterized by high vascularity. Angiogenesis (formation of new microvessels) is critical for growth and progression of various human solid tumors. Betacellulin (BTC) is a member of the epidermal growth factor (EGF) family, and its signal action is mediated through EGF receptors (EGFR). In this study, to understand the role of BTC in relation to EGFR in HCC, we examined localization, expression, and involvement in angiogenesis of BTC and EGFR. The results revealed that expression of BTC, EGFR, and tumor growth factor-alpha messenger RNA in HCC was increased by 80%, 60%, and 40%, respectively, as compared with those in the nontumorous tissues. Increased expression of BTC protein was observed in 31 (61%) of 51 HCC specimens, and the level of tumor growth factor-alpha protein was higher in 17 (33%) of 51 HCC specimens than in nonmalignant hepatocytes. Betacellulin was predominantly expressed in HCC cells, whereas EGFR was observed in sinusoidal endothelial cells of HCC in 25 tumors (49%). Betacellulin was secreted in all 4 examined HCC cell lines. The HCC specimens showing positive EGFR expression in tumor endothelial cells had a significantly higher microvessel density than those without EGFR expression (P < .005). A strong correlation was found between BTC expression in cancer cells and EGFR expression in tumor endothelial cells (P < .001). These findings suggest that overexpression of BTC by HCC cells and EGFR by tumor endothelial cells enhance vascularity in a paracrine manner.

The morbidity and mortality experienced by cancer patients is mainly due to the invasion and metastasis of the primary tumor. Recently, a potential metastasis-associated gene and its product, the metastatic tumor antigen 1 (MTA1), were identified; this gene has been found to be overexpressed in a variety of cancers. MTA1 is also known as a potent co-repressor of estrogen receptor element transcription in breast cancer cells. The expression of MTA1 in hepatocellular carcinoma (HCC) and its potential relationship to metastasis and to estrogen receptor alpha (ER-alpha) expression has not been examined, forming the basis for this study. Paraffin sections of 45 HCC specimens, 4 different HCC cell lines, and normal hepatocyte cell line (h NHeps) were immunostained with MTA1 and ER-alpha antibodies. In addition, we examined, by Western blotting, the MTA1 and ER-alpha expression levels in 4 human HCC lines (HepG2 [wild p53], HLE, HLF, and HuH-7 [mutant p53]). MTA1 was overexpressed in HCC cells versus nonmalignant hepatocytes in 31 of 45 HCC specimens (69%). Its expression was predominantly localized to the nucleus or cytoplasm of HCC cells. Nineteen of 20 HCC (95%) specimens with vascular invasion displayed strong MTA1 expression. Overexpression of MTA1 also significantly correlated with large tumor size. The cytoplasmic and nuclear immunoreactivity for ER-alpha was present in HCC specimens in 46% and 12%, respectively. Expression of MTA1 inversely correlated with the nuclear localization of ER-alpha. There was no marked difference in MTA1 and ER-alpha expression levels between HCC cell line expressing wild-type p53 and cell line with mutated p53 HCC. In conclusion, these findings indicate that overexpression of MTA1 is associated with HCC growth and vascular invasion. Nuclear translocation of ER-alpha inversely correlated with MTA1 expression, suggesting negative regulatory mechanisms.

Background/aims: Tumor angiogenesis, a major requirement for tumor growth and metastasis, is regulated by pro- and anti-angiogenic factors. Hepatocellular carcinoma (HCC) has become a common malignant tumor worldwide. It is characterized by a high vascularity., Methods: We studied the immunohistochemical expression of angiostatin, vascular endothelial cell growth factor (VEGF), matrix metalloproteinase (MMP)-9 and MMP-12, and the relationship between these results and the microvessel density (MVD) in 48 HCC specimens. To determine whether HCC cells express angiostatin per se, we examined the expression of angiostatin, MMP-9 and MMP-12 by Western blotting in four HCC cell lines., Results: Expression of angiostatin and MMP-12 (but not MMP-9) were strongly correlated with decreased MVD in HCCs (P=0.006, P=0.038, respectively). VEGF positive tumors showed a significantly higher MVD than VEGF negative tumors (P=0.01). We divided the 48 cases into the following four groups: group A, angiostatin (+), MMP-9 or -12 (+), and VEGF (-); group B, angiostatin (-) and VEGF (-); group C, angiostatin (+), MMP-9 or -12 (+), and VEGF (+); group D, angiostatin (-) and VEGF (+). There was a significant correlation with MVD among these groups (P<0.001). Angiostatin was detected by Western blotting in 2 out of 4 HCC cell lines and was associated with plasminogen and MMP expression., Conclusions: These results indicate that angiogenesis in HCC is a complex process involving multiple factors including angiostatin, VEGF, and MMP. Our results suggest that angiostatin is generated by MMP-mediated proteolysis of plasminogen in HCC cells.

Background/aims: The mitogenic signaling pathway of epidermal growth factor receptor (EGFR) is activated in a variety of cancers, including hepatocellular carcinoma (HCC). The recently cloned EGFR-related protein (ERRP) has been proposed to be a negative regulator of EGFR. The expression of ERRP in non-malignant liver cells and in HCC, and its relation to cell proliferation, have not been examined., Methods: Paraffin sections of formalin-fixed HCC specimens of 51 HCCs and 10 normal liver specimens were immunostained with anti-ERRP and anti-p53 antibodies. To determine the relationship between ERRP expression and cell proliferation, we performed double staining for ERRP and proliferating cell nuclear antigen (PCNA) in the same HCC specimens., Results: In normal liver, ERRP was expressed in 100% of specimens, while in non-malignant liver tissue from HCC, ERRP expression was present in 50% of the specimens (p < 0.001). In contrast, ERRP expression was present only in 14% of the HCC specimens. The expression of ERRP in cancer cells was inversely correlated with proliferative activity and tumor size (p < 0.001, p < 0.05, respectively). No significant correlation was found between p53 expression and the expression of ERRP., Conclusions: Human HCC, which has been shown to be associated with increased activation of EGFR, exhibits a substantial reduction in ERRP expression. The inverse relationship between ERRP expression, proliferative activity of tumor cells and tumor size suggests that in some HCC, ERRP is a negative regulator of HCC growth.

Survivin is a recently described anti-apoptosis protein and regulator of cell division. Its expression and localization in hepatocellular carcinoma (HCC) and in normal liver tissue has not been fully elucidated. We examined the expression of survivin, Fas, proliferating cell nuclear antigen (PCNA), and apoptosis in 47 specimens of hepatocellular carcinoma (HCC) and surrounding nonmalignant hepatic tissues. To further determine the relationship between survivin expression and cell proliferation and apoptosis, we performed double immunostaining for survivin and PCNA TUNEL staining in the same HCC specimens. Positive immunostaining for survivin was present in 35 of 47 (74%) HCCs. Twenty-two of 35 survivin-positive HCCs (63%) showed punctate nuclear staining in HCC cells, and the remaining 13 showed predominant cytoplasmic staining. In contrast, nonmalignant hepatocytes showed only cytoplasmic staining. HCC cells had significantly higher PCNA-labeling and apoptotic indices compared with the case of nonmalignant hepatic tissue (P<0.001). Furthermore, nucleus-positive HCC specimens for survivin showed the highest PCNA labeling index. The nuclear localization of survivin in HCC cells correlated with tumor cell de-differentiation with the exception of the HepG2 cell line. Survivin expression was inversely associated with apoptosis and was strongly associated with Fas expression (P=0.01). All 4 HCC cell lines examined showed survivin expression and punctate nuclear localization. Our results indicate that survivin is localized to the cytoplasm in quiescent nonmalignant liver cells to suppress apoptosis and translocates into the nucleus in HCC cells. In conclusion, translocation of survivin from the cytoplasm to the nucleus may constitute an important regulatory mechanism for cell proliferation and differentiation in HCC.

Hepatocellular carcinoma (HCC) is becoming one of the most common malignant tumors worldwide and is characterized by a high vascularity. Angiogenesis, formation of new microvessels, is critical for the growth and progression of various human solid tumors. Vascular endothelial growth factor (VEGF) and angiopoietins (Ang1 and Ang2) are endothelial cell-specific vasculogenic and angiogenic growth factors, but their expression and roles in HCC have not been extensively explored. The aim of this study was to determine the expression and cellular localization of VEGF, Ang1, and Ang2 in specimens of resected human HCC using in situ hybridization and immunohistochemical staining and to examine their relationship to microvessel density (MVD) and tumor size. We also investigated whether mutation of p53 protein might affect the expression of the above angiogenic growth factors. VEGF and Ang2 were strongly expressed and localized predominantly to cancer cells, whereas Ang1 was detected in supportive cells of large blood vessels, stromal cells, endothelial cells, and tumor cells. Expression of the VEGF protein and the Ang2 (but not Ang1) mRNA were strongly correlated with MVD (P <.05, P =.001) and tumor size (P <.05). There was also a strong correlation between VEGF protein and Ang2 mRNA expression (P <.001). However, no significant correlation was found between overexpression of p53 and the expression of VEGF, angiopoietins, or MVD. These findings suggest that overproduction of the angiogenic growth factors VEGF and Ang2 by HCC cells may increase vascularity and tumor growth in a paracrine manner. Our findings also suggest that interaction between VEGF and Ang2 may play a critical role in tumor angiogenesis in HCC.

We first established a sarcomatoid hepatocellular carcinoma cell line, designated as SH-J1, and applied comparative genomic hybridization and fluorescence in situ hybridization (FISH) with chromosome painting probes for the characterization of the chromosomal rearrangements. In the SH-J1 cell line, the pleomorphic spindle cells were arranged in bundles of interlacing patterns and were positive in immunohistochemical staining with hepatocyte-related markers. By G-banding and FISH, the chromosomal gains were detected at 6p and 17, whereas losses were observed at 3p21-pter, 3q27-qter, 4, 6q, 13pter-q11, 16, 18, 19p13, and Y.